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BACKGROUND
thioredoxins are widely distributed in nature from prokaryotes to eukaryotes.
these proteins, which belong to the oxidoreductase thiol:disulfide superfamily <cit> , are characterized by the active site signature sequence wcxxc.
this sequence motif constitutes the redox center mediating the isomerization of specific disulfide bridges on trx target proteins <cit> .
in yeasts and mammals, the cytoplasmic trx redox system is complemented by a second trx system within mitochondria.
in plants, the system is more intricate due to the presence of chloroplastic trxs that are strongly associated with the regulation of chloroplast metabolism and function <cit> .
in mammals and yeast, only two and three trx-encoding genes, respectively, have been identified so far.
in contrast, about <dig> genes encoding trxs are contained in arabidopsis thaliana genome, recently reviewed in <cit> .
trxs were initially described as reductants of ribonucleotide reductase during dna synthesis <cit> .
later, these proteins were shown to take part in a variety of important physiological processes, for example as electron donors for several biosynthetic oxidoreductases <cit> or as protectants against oxidative damage by reduction of the disulphide bridges within many proteins.
interestingly, trxs and trx-related proteins are being found to be involved in several sexual plant reproduction processes as well, as reviewed in <cit> .
the functional diversity of trxs correlates with their wide distribution in nature and with the large variability in their primary structures <cit> .
their features and functions have been recently reviewed <cit> .
plant trxs can be divided into eight types based on their sequence <cit> .
types f, m, x, y, and z are localized in chloroplasts, type o is found in mitochondria, and type s is associated with the endoplasmic reticulum <cit> .
information about the subcellular localization of type h , the largest group of this protein family, is limited since this group includes proteins located in the cytosol as well as in mitochondria and even secreted to the apoplast <cit> .
plant trxs are also involved in highly specialized biological processes, including self-incompatibility in brassica <cit> .
two trxs h proteins, thl <dig> and thl <dig> interact with the c-terminal domain of the s-locus receptor kinase , which is the female determinant in the sporophytic si system in brassica <cit> .
the formation of the srk-thl complex occurs during self-compatible pollinations and it has been proposed that it prevents the srk dimerization and self-phosphorylation; the last event is essential to the activation of the pollen rejection response <cit> .
moreover, suppression of thl <dig> and thl <dig> in transgenic plants has shown that both trxs are required for full pollen acceptance <cit> .
trxs h also may play a role in the gametophytic s-rnase-based si system in nicotiana alata since natrxh reduces in vitro to the s-rnase, the female s-determinant <cit> .
moreover, the natrxh transcript is more abundant in si species than in self-compatible ones from nicotiana spp.
<cit> .
in general, evidence indicating the involvement of trxs and, in general, thiol/disulphide containing proteins within plant sexual reproduction processes is increasing, meaning that redox regulation plays a pivotal role in regulating these signalling mechanisms <cit> .
trx h group is subdivided into three subgroups <cit> .
subgroup <dig> includes trxs with an n-terminal extension.
some evidence suggests a role for this extension in trx intracellular trafficking.
in populus tremula, the n-terminus of pttrxh <dig> functions as a mitochondrial targeting signal <cit> .
as with other subgroup <dig> members, n. alata natrxh contains extensions toward its c- and n-termini, but their functions have not been investigated.
notably, natrxh does not possess a canonical signal peptide at its n-terminus but is secreted onto the extracellular matrix of the style <cit> .
therefore, either or both the n- or c-terminus could be involved in natrxh secretion and/or mediate the protein-protein interaction of natrxh with its target proteins.
here, we show that natrxh secretion depends on an inner segment within its n-terminal extension.
this segment, nβ, guides secretion of natrxh through the er and golgi.
in addition, pull-down assays indicate that the c-terminal extension participates in the interaction with s-rnase.
likewise, in silico structure modeling predicts both the n- and c-terminal extensions to be solvent exposed and to fold into stable secondary structure elements.
the model is consistent with an active role of both extensions in tertiary structure stabilization, with little or no effect on natrxh reductase activity.
RESULTS
natrxh localizes to the extracellular matrix of the transmitting tissue in n. alata styles or associates with secretory pathway elements
previously, we demonstrated that natrxh co-localizes to the extracellular matrix of the stylar transmitting tissue in n. alata along with the s-rnase <cit> .
although it lacks a canonical signal peptide, natrxh contains sufficient information to guide its secretion, raising the possibility that this protein could follow a non-classical secretion pathway, as suggested by the secretome <dig> algorithm <cit> .
immuno-gold labelling and electron microscopy data were consistent with an natrxh localization at the ecm of the same n. alata stylar tissue .
notably, a semi-quantitative analysis, counting all observed particles from five different micrographs at a 12 k resolution, revealed gold particles to be associated with structures related to the secretory system .
this association is consistent with the immune detection of both natrxh and the vatpase in the microsomal fraction of a protein crude extract from n. alata styles .
in figure 1b, d, e, and f, gold particles are observed in association with vesicles, some of which reach the plasma membrane.
these images are suggestive of membrane fusion leading to the extracellular release of the vesicle content, including natrxh , which also was found at the ecm, labelled as cell wall .
figure 1c and d are representative micrographs where natrxh was found in association either with the er or the golgi.
in contrast to the secretome <dig> algorithm prediction <cit> , our data show at least a fraction of natrxh travelling through the er and golgi secretory pathway en route to its final apoplastic localization in the styles of n. alata.
however, as previously mentioned, natrxh lacks a canonical signal peptide, and the localization found through immuno-gold and electron microscopy provides cellular confirmation of secretion.
natrxh n- and c-terminal extensions
as previously reported <cit> and shown in figure <dig> natrxh is secreted in n. alata styles.
contrary to the secretome <dig> algorithm, which predicts a non-classical secretion signal for natrxh, the hidden markov algorithm <cit> predicts a cleavage site between residues ala- <dig> and ala- <dig> albeit with a low probability <cit> .
multiple alignment of several trxs h from subgroup <dig> showed that the natrxh n-terminal extension sequence is at least <dig> residues long and its c-terminal extension comprises residues e- <dig> to q- <dig> .based on the above predictions, we divided the n-terminus of natrxh in two motifs: nα and nβ .
the c-terminal extension was defined starting at e- <dig> .
the nβ region is crucial for natrxh secretion
to test if either extension is responsible for natrxh secretion, we generated natrxh deletion mutants lacking different sequence segments, fused to green fluorescent protein , and then transiently expressed them in onion epidermal cells.
first, we showed that the full-length natrxh fused to gfp is observable in the extracellular space of onion epidermal cells , as reported in n. benthamiana and a. thaliana <cit> .
the same was observed for the stylar ecm protein p <dig> <cit> fused to gfp .
we observed the same pattern when the nα motif is deleted from the n-terminus of natrxh and, therefore, concluded the nα domain is not required for targeting natrxh to the apoplast.
however, when natrxhΔnαβ, which lacks both the nα and nβ motifs, was expressed as a gfp fusion protein, fluorescence was localized inside the cells, indicating that secretion was abolished .
when the c-terminus was deleted from natrxh , gfp fluorescence was localized to the apoplast .
these data show that the n-terminal extension carries all the information for natrxh secretion.
however, in contrast to an orthodox n-terminal signal peptide, the first <dig> amino acids are not required, as the inner nβ domain promotes secretion in the absence of the nα segment.
to test this hypothesis, we generated an natrxh protein mutant with the nα domain adjacent to the trx core, deleting the nβ domain , and then expressed it as a gfp fusion protein.
transient expression of natrxhΔnβ: gfp is shown in figures 2b- <dig> and b- <dig> gfp fluorescence can be observed within the cytosol.
furthermore, fusion to gfp of the nβ domain alone leads to extracellular localization of the gfp signal, which resembles the distribution found for full-length natrxh .
together, these outcomes provide strong evidence that the nβ domain is both essential and sufficient for natrxh secretion.
natrxh uses the endomembrane system to reach the apoplast
while clearly sufficient to function as a secretion signal, the nβ domain may guide natrxh secretion through an unorthodox secretion pathway.
this possibility is suggested by the nβ domain’s unusual position within the primary structure and the absence of a long hydrophobic amino acid region .
to evaluate if nβ-led secretion proceeds via the er, we looked for the presence of natrxh in the er using two natrxh fusion proteins, natrxh:gfp and nβ: gfp, both of which exhibit the er retention signal kdel <cit> .
as a control, we also fused p <dig> to gfp.
p <dig> is a known secreted protein from n. alata <cit> with a typical signal peptide that is expected to follow the classical er/golgi pathway.
the gfp signal from all gfp fusion proteins exhibits a typical er distribution pattern surrounding the nucleus.
the reticulate fluorescent pattern observed with both fusion proteins and, interestingly, with the nβ: gfp as well , contrasts with the blurred pattern of the nucleus .
these data are consistent with the passage of natrxh through the er on its way out of the cell .evidence for participation of the golgi network in natrxh secretion was obtained from treatment of onion epidermal cells with the fungal toxin brefeldin a .
bfa blocks vesicle formation at the golgi network, which prevents secretion of nap11:gfp, nathx:gfp, and nβ:gfp .
additional evidence that natrxh is secreted through vesicles is natrxh association with a membrane fraction .
taken together, these results show that the nβ domain is a hydrophilic novel internal signal able to promote natrxh secretion via the er/golgi.
the n-terminal region of natrxh accounts for structural stability but not for its reductase activity
to evaluate whether the n-terminal extension, the c-terminal extension, or both extensions participate in natrxh reductase activity, we overexpressed four natrxh mutants as gst fusion proteins in escherichia coli.
the natrxhΔnα, natrxhΔnαβ, and natrxhΔcoo proteins were recovered from the soluble phase from bacterial sonicates , as reported for the full natrxh <cit> .
notably, natrxhΔnβ is only detected at the insoluble phase , suggesting that the protein does not fold correctly; therefore, its activity as a disulphide reductase could not be tested.
when compared to full-length natrxh, the natrxh variants show no differences in their ability to reduce insulin disulfide bonds using dithiothreitol as an electron donor <cit> .
this result demonstrates that the n-terminal extension functions in natrxh trafficking and, like the c-terminus, does not participate in natrxh’s ability to reduce target proteins.
n. alata s-rnase interacts in vitro with natrxh by its c-terminal region
we previously reported the in vitro interaction of natrxh with the pistil s-determinant s-rnase from n. alata.
the interaction takes place regardless of the natrxh redox state <cit> .
to test whether the n-terminal or c-terminal region accounts for this specific protein-protein interaction, we prepared gst:natrxh-, gst:natrxhΔnα-, gst:natrxhΔnαβ-, and gst:natrxhΔcoo-affi-gel affinity columns and passed through them extracellular stylar protein extracts from n. alata s105s <dig>
figure 6a shows that the s105-rnase was retained in the natrxh-gst-affi-gel matrix, as reported by juárez-díaz et al.
<cit> .
notably, we observed a similar binding behaviour when crude style extracts from n. alata s105s <dig> were passed through the natrxhΔnα and natrxhΔnαβ matrices .
noteworthy, when the protein extracts are passed through the affinity column with natrxhΔcoo, the s105-rnase is not retained .
these data show that the natrxh c-terminus contributes to the interaction with the s105-rnase.
the nβ domain plays a structural role in natrxh
natrxh is predicted to interact with other trafficking-related proteins to be secreted.
thus, the nβ domain is likely to be exposed at the molecular surface to facilitate such interactions.
to support this hypothesis, we constructed a model of natrxh using a combination of homology modeling and molecular dynamic simulations.
we used modeller 9v <dig> <cit> for the homology modelling and gromacs <dig> .
<dig> <cit> for the md simulations.
while the closest homologue of natrxh with a known 3d-structure is the hordeum vulgare h <dig> trx , the n. alata protein possesses extensions toward its n- and c-termini, which has no homologues in the protein data bank <cit> .
we obtained a predicted conformation for these extensions by performing two rounds of md simulations.
the structure shown in figure 7e is a representative conformation, drawn with visual molecular dynamics molecular viewer <cit> ; mobile regions are shown in orange, blue and green.
at the end of the second run, the n- and c-termini folded to form a “beta sheet hat” separated from the trx core and opposite the putative reactive site loop .
the beta sheet was fully formed after 20 ns and remained stable thereafter.
only four segments in the protein showed significant fluctuation in the final model: the first <dig> and the last <dig> residues, the loop where the reactive cysteine residues reside , and a loop connecting the core to the n-side of the “beta sheet hat” .
the model was rated from very good to fairly good by atomic non-local environment assessment <cit> and proq <cit> .
with the rd.hmm protocol <cit> , we used the coordinates of the backbone atoms of the model to retrieve the n. alata natrxh amino acid sequence from the ncbi nr protein database <cit> with a statistical significance substantially higher than the one for the h. vulgare sequence .
in contrast, the 2iwt crystal as well as some of the initial models from modeller, when subjected to the rd.hmm protocol, scored the sequence of the barley protein and several other trx h proteins with high probability, while the n. alata amino acid sequence was recovered with an e value above <dig> .
according to its quality and appropriateness scores , the model appears to be reasonably close to the reduced form of the n. alata natrxh 3d structure.
the appropriateness score is worth noting because the rd.hmm is known to be very sensitive, which may result in false negatives .
however, no false positives have been found yet.
interestingly, in all models produced, the n-terminus remains accessible to the solvent, especially the region corresponding to residues <dig> to <dig> which coincides with the nβ domain.
in addition, the final conformations of the n- and c-termini anchors and the n-terminal extension to the hat forces the poorly ordered loop of amino acids from residues <dig> to <dig> to remain on the protein surface.
since this region seems to be sufficient for natrxh secretion, its anchorage may facilitate the recognition of this sequence by some unidentified component of the secretion pathway.
to assess the potential of the natrxh extensions to interact with other proteins, we compared them to intrinsically disordered regions .
the amino acid sequences known as intrinsically disordered proteins or idrs, among other names, are proteins or partial regions of proteins that lack stable and well-defined 3d structures under physiological conditions in vitro <cit> .
we identified the idrs using disembl <cit> ; server at <cit> , which relies on three criteria to assign an amino acid sequence as disordered: loops/coils, hot loops, and remark <dig> the loops/coils definition identified residues <dig> to <dig> <dig> to <dig> <dig> to <dig> and <dig> to <dig> as idrs ; hot loops reported segments <dig> to <dig> and <dig> to <dig> as idrs ; and remark <dig> defined the first 28 n-terminal residues as the only idr in natrxh .
according to disembl, the natrxh extensions are idrs, and all three criteria agree with md simulations in the prediction of the nβ region as a poorly structured protein segment.
discussion
here, we demonstrated that the nβ domain is required for natrxh secretion.
additionally, we provided evidence on natrxh targeting to the apoplast via the er/golgi regardless of the absence of a distinguishable hydrophobic signal peptide.
finally, we also present data that suggest that the c-terminal region of natrxh is an important mediator of the natrxh:s-rnase interaction.
natrxh is transported through vesicles toward the apoplast
the immune assays we performed clearly show that natrxh is mainly localized to membranous bodies, primarily vesicles, which correlate with the finding of natrxh in the microsomal fraction.
these data strongly indicate that natrxh is carried to the extracellular space by means of a vesicle-dependent secretion pathway.
the electron microscopy data clearly place natrxh inside vesicles , although some gold particles were observed to be associated with er and other unidentified membranous systems, we cannot affirm that these vesicles come from the er, the golgi, or both.
although natrxh lacks a canonical signal peptide, its association with vesicles correlates well with its extracellular localization.
a possible secretion mechanism for proteins of this kind relies on their direct interaction with secretion vesicles without previous association to the er/golgi <cit> .
in mammalian cells and yeast, some proteins are secreted through an er/golgi-independent pathway.
such is the case of insulin degrading enzymes <cit> , interleukins il-1b and il- <dig> <cit> , and some yeast proteins lacking a signal peptide <cit> .
natrxh has an internal and hydrophilic secretion signal and is secreted via the er/golgi
the symplastic localization of the natrxhΔnα mutant shows that the first 17 n-terminal residues are not essential for natrxh secretion.
instead, the internal amino acid sequence a17eaesgsssep <dig> , despite lacking the characteristic hydrophobicity shown on classical signal peptides <cit> , is essential and sufficient for natrxh secretion, as observed by the cytoplasmic localization of the natrxhΔnβ mutant .
this motif is also sufficient to direct the nβ gfp-tagged to the extracellular space .
most proteins secreted through the er/golgi pathway are translated in ribosomes attached to the er membrane and possess a signal peptide localized at the n-terminus <cit> .
one important property of such signal peptides is their hydrophobicity <cit> .
this feature is essential for recognition of the nascent peptide by the signal receptor particle <cit> .
although our data indicate that natrxh passes through the er and golgi en route to the apoplast —as shown with the kdel constructs , the blocking of natrxh secretion by bfa , and the presence of natrxh in the microsomal fraction — we do not know how natrxh is transported into the er and how the golgi participates in its secretion.
although several possible scenarios are feasible, we currently have no evidence to favor any of them.
two examples that support secretion of proteins without a conventional signal peptide and using the endomembrane system are the proteins il-1β and acba <cit> .
il-1β joins secretory lysosomes and is released when those lysosomes fuse with the plasma membrane <cit> .
il-1β also can be captured directly into multivesicular bodies or be sequestered by autophagosomes and fuse with multivesicular bodies <cit> .
non-classical secretion of cytoplasmic plant proteins has also been documented, as reviewed in <cit> .
it has been demonstrated that proteins without signal peptide, such as celery mannitol dehydrogenase in a. thaliana, traffic to the apoplast while bypassing the classical er/golgi secretion pathway <cit> .
another example is the hygromycin phosphotransferase in a. thaliana, which is secreted through a golgi-independent route mediated by the golgi-localized synaptotagmin <dig> <cit> .
however, this is unlikely to be the case for natrxh since our data clearly showed that it goes through both er and golgi for its secretion .
another possible route that natrxh could follow to the apoplast is through specialized vesicles, such as the exosome-like nanovesicles described in olea europea pollen tubes, called pollensomes <cit> .
some of the pollensomes are proposed to be er- and golgi-derived vesicles based on the fact that ole e <dig> from o. europea was found to be within these pollensomes <cit> .
regarding natrxh, we observed that some of it was contained in cytoplasmic vesicles and some of them were observed fused to the plasma membrane .
in the apoplast, natrxh was never found associated to any exosome-like structure, as described for pollensomes <cit> .
an additional possibility is that natrxh could associate to endomembrane systems through lipidic modifications.
actually, traverso et al.
<cit> found that natrxh is in vitro myristoylated at gly- <dig> suggesting that natrxh may be a membrane-associated protein in planta.
based on this, it was speculated that it could be the manner about how natrxh is transported to the apoplast <cit> .
however, this scenario appears to be unlikely to occur because our deletion analysis outcomes indicated that the first <dig> amino acids are not essential for natrxh secretion, instead it was the inner domain, the nβ motif, the one that directly led its secretion .
the nβ motif is apparently exclusive to plant trxs.
besides natrxh, a similar motif has been found in only two soybean thioredoxins that, notably, are associated with the plasma membrane.
both soybean trxs have an n-terminal extension <cit> that includes a region with a high similarity index to the nβ sequence .
our cell biology data along with our molecular assays by transient expression of different versions of natrxh fused to gfp indicate that natrxh secretion is due to its nβ motif and that the protein follows a secretion pathway that requires the er, the golgi apparatus, and secretion vesicles.
how natrxh interacts with these secretory elements is not known since the natrxh n-terminus does not have any of the typical signal peptide biochemical properties.
however, the absence of an orthodox signal peptide in natrxh reveals the existence of an alternative secretion mechanism that uses, to some extent, the er/golgi secretory pathway.
the accurate mechanism that leads natrxh secretion needs to be clarified and future research will be of great interest in order to unravel possible novel plant trafficking routes.
natrxh:s-rnase interaction
protein and mrna levels of natrxh are higher in the styles of si plants than in self-compatible plants, and s-rnase interacts with natrxh in vitro.
these facts have been used to classify natrxh as a pistil modifier gene that accounts for pollen rejection in n. alata <cit> .this work contributes to our understanding of the molecular mechanism mediating the natrxh: s-rnase interaction.
the pull-down experiments show that the natrxh c-terminal extension is essential for its interaction with s-rnase .
however, this region does not affect natrxh secretion or trx activity .
therefore, it appears that natrxh is able to fold correctly in the absence of the c-terminal domain or at least fold well enough to sustain its native-like reductase activity.
in n. alata, several proteins are directly involved in pollen rejection.
in this species, s-rnase degrades the pollen tube rna and determines sexual incompatibility on the female side.
the natrxh:s-rnase interaction could be relevant to the si response.
natrxh likely stabilizes s-rnase or inhibits its ribonuclease activity in the pollen tube.
indeed, oxley and bacic <cit> showed that s-rnase ribonuclease activity is affected by redox state in vitro.
however, the redox state of natrxh does not impair its interaction with s-rnase <cit> .
s-rnase forms complexes with other stylar proteins <cit> .
while 120 k is known to be essential for si in n. alata <cit> , the precise function of these protein complexes in si is still unclear.
it is possible that natrxh participates as an associating factor to transport such as s-rnase, 120 k, natts or p <dig> to the pollen tube or, alternatively, to release these proteins from s-rnase complexes once inside the pollen tube.
both scenarios may be possible since a redox change by natrxh could play an important role for modifying s-rnase and stylar protein complexes.
it has been determined that one of the targets of natrxh is actually s-rnase <cit> .
therefore, further research is needed to determine if natrxh is a modifier factor in n. alata si by altering the s-rnase redox state in planta.
although the data presented here are consistent with a role of natrxh in pollen rejection in si nicotiana species, loss of function assays would provide direct evidence of this role.
finally, homology modeling to predict the 3d structure of trx h revealed high sequence similarity between the h. vulgare and n. alata trxh proteins, including conservation of the reactive site loop.
the quality of the predicted model indicates similarity at the structural level too.
the barley trx h protein plays an important regulatory role during seed germination <cit> , and one of its targets is the barley α-amylase/subtilisin inhibitor, a homologue of the si modifier n. alata nastep protein recently described by our group <cit> .
both nastep and h. vulgare inhibitor proteins share extensive structural similarity; the loop equivalent to the one mediating the interaction of the barley proteins is present in nastep but is longer and has four cysteine residues instead of two <cit> .
an interaction of natrxh and nastep at some point along the physiological events regulating pollen rejection in nicotiana is a possibility worth future consideration.
CONCLUSIONS
thioredoxins type h clustered within the subgroup <dig> contain non-conserved extensions towards the n- and/or the c-termini, which function is still unclear.
in this work, we showed that the n-terminal extension of natrxh, a subgroup <dig> trx h from n. alata, contained the sufficient information to lead its secretion towards the apoplast.
interestingly, this extension contains two distinguishable motifs, called nα and nβ, divided by the hidden markov algorithm <cit> prediction of a cleavage site on the ala- <dig> position.
while the nα domain appeared unlikely to have any function on natrxh secretion, the nβ was the responsible for its particular subcellular localization.
the nβ domain is the only sequence necessary for natrxh secretion.
transient expression experiments in epidermal onion cells of the nβ-gfp fusion protein revealed its localization in the apoplast, as occurred with the full natrxh protein fused to gfp and with the natrxhΔnα and natrxhΔcoo mutants as well.
these data corroborated the nβ function as a novel signal peptide since its primary structure position and hydrophilic profile do not follow the typical biochemical features of the classical transport sequences.
the nβ biochemical features suggested an er/golgi independent secretion pathway.
however, natrxh was detected in a microsomal fraction and, furthermore, was immune-detected mainly associated to classical secretory elements when observed within the cells in n. alata styles, providing evidences that the natrxh could be secreted through the classical er/golgi pathway or at least, it uses the elements of this route.
this hypothesis was also tested by fusing the er retention signal kdel to natrxh-gfp and treating the cells, when expressing the fusion protein natrxh-gfp, with bfa, separately.
in the first case, the natrxh-gfp was found associated to the er and in the latter, the natrxh-gfp secretion was abolished, confirming that the natrxh actually goes through these organelles in order to be secreted.
furthermore, it was also found that the nβ domain played an important structural role on the natrxh tertiary structure stability since the natrxhΔnβ, which only lacks the nβ domain, was detected in inclusion bodies when overexpressed in e. coli cells and not in the soluble one as the wild type and the other natrxh versions did .
regarding the c-terminus, it was found to be essential for the natrxh-s-rnase in vitro interaction, since the s-rnase was unable to bind to a natrxhΔcoo-containing column.
finally, the in silico analysis showed that the natrxh n- and c-termini are solvent exposed, suggesting a protein-protein interaction role.
while this function appears to be essential for the s-rnase interaction, it also provided evidences on the nβ domain, which should interact either with a non-identified secretory element or interact in a different manner as the classical secreted proteins do with srp.
interestingly, the n-terminal extension clearly showed two structural motifs, which coincide with the nα and nβ domains tested in this work.
| 29,468 | thioredoxins are widely distributed in nature from prokaryotes to eukaryotes. these proteins, which belong to the oxidoreductase thiol:disulfide superfamily, are characterized by the active site signature sequence wcxxc. this sequence motif constitutes the redox center mediating the isomerization of specific disulfide bridges on trx target proteins. in yeasts and mammals, the cytoplasmic trx redox system is complemented by a second trx system within mitochondria. in plants, the system is more intricate due to the presence of chloroplastic trxs that are strongly associated with the regulation of chloroplast metabolism and function. in mammals and yeast, only two and three trx-encoding genes, respectively, have been identified so far. in contrast, about genes encoding trxs are contained in arabidopsis thaliana genome, recently reviewed in. trxs were initially described as reductants of ribonucleotide reductase during dna synthesis. later, these proteins were shown to take part in a variety of important physiological processes, for example as electron donors for several biosynthetic oxidoreductases or as protectants against oxidative damage by reduction of the disulphide bridges within many proteins. interestingly, trxs and trx-related proteins are being found to be involved in several sexual plant reproduction processes as well, as reviewed in. the functional diversity of trxs correlates with their wide distribution in nature and with the large variability in their primary structures. their features and functions have been recently reviewed. plant trxs can be divided into eight types based on their sequence. types f, m, x, y, and z are localized in chloroplasts, type o is found in mitochondria, and type s is associated with the endoplasmic reticulum. information about the subcellular localization of type h, the largest group of this protein family, is limited since this group includes proteins located in the cytosol as well as in mitochondria and even secreted to the apoplast. plant trxs are also involved in highly specialized biological processes, including self-incompatibility in brassica. two trxs h proteins, thl and thl interact with the c-terminal domain of the s-locus receptor kinase, which is the female determinant in the sporophytic si system in brassica. the formation of the srk-thl complex occurs during self-compatible pollinations and it has been proposed that it prevents the srk dimerization and self-phosphorylation; the last event is essential to the activation of the pollen rejection response. moreover, suppression of thl and thl in transgenic plants has shown that both trxs are required for full pollen acceptance. trxs h also may play a role in the gametophytic s-rnase-based si system in nicotiana alata since natrxh reduces in vitro to the s-rnase, the female s-determinant. moreover, the natrxh transcript is more abundant in si species than in self-compatible ones from nicotiana spp.. in general, evidence indicating the involvement of trxs and, in general, thiol/disulphide containing proteins within plant sexual reproduction processes is increasing, meaning that redox regulation plays a pivotal role in regulating these signalling mechanisms. trx h group is subdivided into three subgroups. subgroup includes trxs with an n-terminal extension. some evidence suggests a role for this extension in trx intracellular trafficking. in populus tremula, the n-terminus of pttrxh functions as a mitochondrial targeting signal. as with other subgroup members, n. alata natrxh contains extensions toward its c- and n-termini, but their functions have not been investigated. notably, natrxh does not possess a canonical signal peptide at its n-terminus but is secreted onto the extracellular matrix of the style. therefore, either or both the n- or c-terminus could be involved in natrxh secretion and/or mediate the protein-protein interaction of natrxh with its target proteins. here, we show that natrxh secretion depends on an inner segment within its n-terminal extension. this segment, nβ, guides secretion of natrxh through the er and golgi. in addition, pull-down assays indicate that the c-terminal extension participates in the interaction with s-rnase. likewise, in silico structure modeling predicts both the n- and c-terminal extensions to be solvent exposed and to fold into stable secondary structure elements. the model is consistent with an active role of both extensions in tertiary structure stabilization, with little or no effect on natrxh reductase activity. natrxh localizes to the extracellular matrix of the transmitting tissue in n. alata styles or associates with secretory pathway elements previously, we demonstrated that natrxh co-localizes to the extracellular matrix of the stylar transmitting tissue in n. alata along with the s-rnase. although it lacks a canonical signal peptide, natrxh contains sufficient information to guide its secretion, raising the possibility that this protein could follow a non-classical secretion pathway, as suggested by the secretome algorithm. immuno-gold labelling and electron microscopy data were consistent with an natrxh localization at the ecm of the same n. alata stylar tissue. notably, a semi-quantitative analysis, counting all observed particles from five different micrographs at a 12 k resolution, revealed gold particles to be associated with structures related to the secretory system. this association is consistent with the immune detection of both natrxh and the vatpase in the microsomal fraction of a protein crude extract from n. alata styles. in figure 1b, d, e, and f, gold particles are observed in association with vesicles, some of which reach the plasma membrane. these images are suggestive of membrane fusion leading to the extracellular release of the vesicle content, including natrxh, which also was found at the ecm, labelled as cell wall. figure 1c and d are representative micrographs where natrxh was found in association either with the er or the golgi. in contrast to the secretome algorithm prediction, our data show at least a fraction of natrxh travelling through the er and golgi secretory pathway en route to its final apoplastic localization in the styles of n. alata. however, as previously mentioned, natrxh lacks a canonical signal peptide, and the localization found through immuno-gold and electron microscopy provides cellular confirmation of secretion. natrxh n- and c-terminal extensions as previously reported and shown in figure natrxh is secreted in n. alata styles. contrary to the secretome algorithm, which predicts a non-classical secretion signal for natrxh, the hidden markov algorithm predicts a cleavage site between residues ala- and ala- albeit with a low probability. multiple alignment of several trxs h from subgroup showed that the natrxh n-terminal extension sequence is at least residues long and its c-terminal extension comprises residues e- to q-.based on the above predictions, we divided the n-terminus of natrxh in two motifs: nα and nβ. the c-terminal extension was defined starting at e-. the nβ region is crucial for natrxh secretion to test if either extension is responsible for natrxh secretion, we generated natrxh deletion mutants lacking different sequence segments, fused to green fluorescent protein, and then transiently expressed them in onion epidermal cells. first, we showed that the full-length natrxh fused to gfp is observable in the extracellular space of onion epidermal cells, as reported in n. benthamiana and a. thaliana. the same was observed for the stylar ecm protein p fused to gfp. we observed the same pattern when the nα motif is deleted from the n-terminus of natrxh and, therefore, concluded the nα domain is not required for targeting natrxh to the apoplast. however, when natrxhΔnαβ, which lacks both the nα and nβ motifs, was expressed as a gfp fusion protein, fluorescence was localized inside the cells, indicating that secretion was abolished. when the c-terminus was deleted from natrxh, gfp fluorescence was localized to the apoplast. these data show that the n-terminal extension carries all the information for natrxh secretion. however, in contrast to an orthodox n-terminal signal peptide, the first amino acids are not required, as the inner nβ domain promotes secretion in the absence of the nα segment. to test this hypothesis, we generated an natrxh protein mutant with the nα domain adjacent to the trx core, deleting the nβ domain, and then expressed it as a gfp fusion protein. transient expression of natrxhΔnβ: gfp is shown in figures 2b- and b- gfp fluorescence can be observed within the cytosol. furthermore, fusion to gfp of the nβ domain alone leads to extracellular localization of the gfp signal, which resembles the distribution found for full-length natrxh. together, these outcomes provide strong evidence that the nβ domain is both essential and sufficient for natrxh secretion. natrxh uses the endomembrane system to reach the apoplast while clearly sufficient to function as a secretion signal, the nβ domain may guide natrxh secretion through an unorthodox secretion pathway. this possibility is suggested by the nβ domain’s unusual position within the primary structure and the absence of a long hydrophobic amino acid region. to evaluate if nβ-led secretion proceeds via the er, we looked for the presence of natrxh in the er using two natrxh fusion proteins, natrxh:gfp and nβ: gfp, both of which exhibit the er retention signal kdel. as a control, we also fused p to gfp. p is a known secreted protein from n. alata with a typical signal peptide that is expected to follow the classical er/golgi pathway. the gfp signal from all gfp fusion proteins exhibits a typical er distribution pattern surrounding the nucleus. the reticulate fluorescent pattern observed with both fusion proteins and, interestingly, with the nβ: gfp as well, contrasts with the blurred pattern of the nucleus. these data are consistent with the passage of natrxh through the er on its way out of the cell.evidence for participation of the golgi network in natrxh secretion was obtained from treatment of onion epidermal cells with the fungal toxin brefeldin a. bfa blocks vesicle formation at the golgi network, which prevents secretion of nap11:gfp, nathx:gfp, and nβ:gfp. additional evidence that natrxh is secreted through vesicles is natrxh association with a membrane fraction. taken together, these results show that the nβ domain is a hydrophilic novel internal signal able to promote natrxh secretion via the er/golgi. the n-terminal region of natrxh accounts for structural stability but not for its reductase activity to evaluate whether the n-terminal extension, the c-terminal extension, or both extensions participate in natrxh reductase activity, we overexpressed four natrxh mutants as gst fusion proteins in escherichia coli. the natrxhΔnα, natrxhΔnαβ, and natrxhΔcoo proteins were recovered from the soluble phase from bacterial sonicates, as reported for the full natrxh. notably, natrxhΔnβ is only detected at the insoluble phase, suggesting that the protein does not fold correctly; therefore, its activity as a disulphide reductase could not be tested. when compared to full-length natrxh, the natrxh variants show no differences in their ability to reduce insulin disulfide bonds using dithiothreitol as an electron donor. this result demonstrates that the n-terminal extension functions in natrxh trafficking and, like the c-terminus, does not participate in natrxh’s ability to reduce target proteins. n. alata s-rnase interacts in vitro with natrxh by its c-terminal region we previously reported the in vitro interaction of natrxh with the pistil s-determinant s-rnase from n. alata. the interaction takes place regardless of the natrxh redox state. to test whether the n-terminal or c-terminal region accounts for this specific protein-protein interaction, we prepared gst:natrxh-, gst:natrxhΔnα-, gst:natrxhΔnαβ-, and gst:natrxhΔcoo-affi-gel affinity columns and passed through them extracellular stylar protein extracts from n. alata s105s figure 6a shows that the s105-rnase was retained in the natrxh-gst-affi-gel matrix, as reported by juárez-díaz et al.. notably, we observed a similar binding behaviour when crude style extracts from n. alata s105s were passed through the natrxhΔnα and natrxhΔnαβ matrices. noteworthy, when the protein extracts are passed through the affinity column with natrxhΔcoo, the s105-rnase is not retained. these data show that the natrxh c-terminus contributes to the interaction with the s105-rnase. the nβ domain plays a structural role in natrxh natrxh is predicted to interact with other trafficking-related proteins to be secreted. thus, the nβ domain is likely to be exposed at the molecular surface to facilitate such interactions. to support this hypothesis, we constructed a model of natrxh using a combination of homology modeling and molecular dynamic simulations. we used modeller 9v for the homology modelling and gromacs. for the md simulations. while the closest homologue of natrxh with a known 3d-structure is the hordeum vulgare h trx, the n. alata protein possesses extensions toward its n- and c-termini, which has no homologues in the protein data bank. we obtained a predicted conformation for these extensions by performing two rounds of md simulations. the structure shown in figure 7e is a representative conformation, drawn with visual molecular dynamics molecular viewer; mobile regions are shown in orange, blue and green. at the end of the second run, the n- and c-termini folded to form a “beta sheet hat” separated from the trx core and opposite the putative reactive site loop. the beta sheet was fully formed after 20 ns and remained stable thereafter. only four segments in the protein showed significant fluctuation in the final model: the first and the last residues, the loop where the reactive cysteine residues reside, and a loop connecting the core to the n-side of the “beta sheet hat”. the model was rated from very good to fairly good by atomic non-local environment assessment and proq. with the rd.hmm protocol, we used the coordinates of the backbone atoms of the model to retrieve the n. alata natrxh amino acid sequence from the ncbi nr protein database with a statistical significance substantially higher than the one for the h. vulgare sequence. in contrast, the 2iwt crystal as well as some of the initial models from modeller, when subjected to the rd.hmm protocol, scored the sequence of the barley protein and several other trx h proteins with high probability, while the n. alata amino acid sequence was recovered with an e value above. according to its quality and appropriateness scores, the model appears to be reasonably close to the reduced form of the n. alata natrxh 3d structure. the appropriateness score is worth noting because the rd.hmm is known to be very sensitive, which may result in false negatives. however, no false positives have been found yet. interestingly, in all models produced, the n-terminus remains accessible to the solvent, especially the region corresponding to residues to which coincides with the nβ domain. in addition, the final conformations of the n- and c-termini anchors and the n-terminal extension to the hat forces the poorly ordered loop of amino acids from residues to to remain on the protein surface. since this region seems to be sufficient for natrxh secretion, its anchorage may facilitate the recognition of this sequence by some unidentified component of the secretion pathway. to assess the potential of the natrxh extensions to interact with other proteins, we compared them to intrinsically disordered regions. the amino acid sequences known as intrinsically disordered proteins or idrs, among other names, are proteins or partial regions of proteins that lack stable and well-defined 3d structures under physiological conditions in vitro. we identified the idrs using disembl; server at, which relies on three criteria to assign an amino acid sequence as disordered: loops/coils, hot loops, and remark the loops/coils definition identified residues to to to and to as idrs; hot loops reported segments to and to as idrs; and remark defined the first 28 n-terminal residues as the only idr in natrxh. according to disembl, the natrxh extensions are idrs, and all three criteria agree with md simulations in the prediction of the nβ region as a poorly structured protein segment. discussion here, we demonstrated that the nβ domain is required for natrxh secretion. additionally, we provided evidence on natrxh targeting to the apoplast via the er/golgi regardless of the absence of a distinguishable hydrophobic signal peptide. finally, we also present data that suggest that the c-terminal region of natrxh is an important mediator of the natrxh:s-rnase interaction. natrxh is transported through vesicles toward the apoplast the immune assays we performed clearly show that natrxh is mainly localized to membranous bodies, primarily vesicles, which correlate with the finding of natrxh in the microsomal fraction. these data strongly indicate that natrxh is carried to the extracellular space by means of a vesicle-dependent secretion pathway. the electron microscopy data clearly place natrxh inside vesicles, although some gold particles were observed to be associated with er and other unidentified membranous systems, we cannot affirm that these vesicles come from the er, the golgi, or both. although natrxh lacks a canonical signal peptide, its association with vesicles correlates well with its extracellular localization. a possible secretion mechanism for proteins of this kind relies on their direct interaction with secretion vesicles without previous association to the er/golgi. in mammalian cells and yeast, some proteins are secreted through an er/golgi-independent pathway. such is the case of insulin degrading enzymes, interleukins il-1b and il-, and some yeast proteins lacking a signal peptide. natrxh has an internal and hydrophilic secretion signal and is secreted via the er/golgi the symplastic localization of the natrxhΔnα mutant shows that the first 17 n-terminal residues are not essential for natrxh secretion. instead, the internal amino acid sequence a17eaesgsssep, despite lacking the characteristic hydrophobicity shown on classical signal peptides, is essential and sufficient for natrxh secretion, as observed by the cytoplasmic localization of the natrxhΔnβ mutant. this motif is also sufficient to direct the nβ gfp-tagged to the extracellular space. most proteins secreted through the er/golgi pathway are translated in ribosomes attached to the er membrane and possess a signal peptide localized at the n-terminus. one important property of such signal peptides is their hydrophobicity. this feature is essential for recognition of the nascent peptide by the signal receptor particle. although our data indicate that natrxh passes through the er and golgi en route to the apoplast —as shown with the kdel constructs, the blocking of natrxh secretion by bfa, and the presence of natrxh in the microsomal fraction — we do not know how natrxh is transported into the er and how the golgi participates in its secretion. although several possible scenarios are feasible, we currently have no evidence to favor any of them. two examples that support secretion of proteins without a conventional signal peptide and using the endomembrane system are the proteins il-1β and acba. il-1β joins secretory lysosomes and is released when those lysosomes fuse with the plasma membrane. il-1β also can be captured directly into multivesicular bodies or be sequestered by autophagosomes and fuse with multivesicular bodies. non-classical secretion of cytoplasmic plant proteins has also been documented, as reviewed in. it has been demonstrated that proteins without signal peptide, such as celery mannitol dehydrogenase in a. thaliana, traffic to the apoplast while bypassing the classical er/golgi secretion pathway. another example is the hygromycin phosphotransferase in a. thaliana, which is secreted through a golgi-independent route mediated by the golgi-localized synaptotagmin. however, this is unlikely to be the case for natrxh since our data clearly showed that it goes through both er and golgi for its secretion. another possible route that natrxh could follow to the apoplast is through specialized vesicles, such as the exosome-like nanovesicles described in olea europea pollen tubes, called pollensomes. some of the pollensomes are proposed to be er- and golgi-derived vesicles based on the fact that ole e from o. europea was found to be within these pollensomes. regarding natrxh, we observed that some of it was contained in cytoplasmic vesicles and some of them were observed fused to the plasma membrane. in the apoplast, natrxh was never found associated to any exosome-like structure, as described for pollensomes. an additional possibility is that natrxh could associate to endomembrane systems through lipidic modifications. actually, traverso et al. found that natrxh is in vitro myristoylated at gly- suggesting that natrxh may be a membrane-associated protein in planta. based on this, it was speculated that it could be the manner about how natrxh is transported to the apoplast. however, this scenario appears to be unlikely to occur because our deletion analysis outcomes indicated that the first amino acids are not essential for natrxh secretion, instead it was the inner domain, the nβ motif, the one that directly led its secretion. the nβ motif is apparently exclusive to plant trxs. besides natrxh, a similar motif has been found in only two soybean thioredoxins that, notably, are associated with the plasma membrane. both soybean trxs have an n-terminal extension that includes a region with a high similarity index to the nβ sequence. our cell biology data along with our molecular assays by transient expression of different versions of natrxh fused to gfp indicate that natrxh secretion is due to its nβ motif and that the protein follows a secretion pathway that requires the er, the golgi apparatus, and secretion vesicles. how natrxh interacts with these secretory elements is not known since the natrxh n-terminus does not have any of the typical signal peptide biochemical properties. however, the absence of an orthodox signal peptide in natrxh reveals the existence of an alternative secretion mechanism that uses, to some extent, the er/golgi secretory pathway. the accurate mechanism that leads natrxh secretion needs to be clarified and future research will be of great interest in order to unravel possible novel plant trafficking routes. natrxh:s-rnase interaction protein and mrna levels of natrxh are higher in the styles of si plants than in self-compatible plants, and s-rnase interacts with natrxh in vitro. these facts have been used to classify natrxh as a pistil modifier gene that accounts for pollen rejection in n. alata.this work contributes to our understanding of the molecular mechanism mediating the natrxh: s-rnase interaction. the pull-down experiments show that the natrxh c-terminal extension is essential for its interaction with s-rnase. however, this region does not affect natrxh secretion or trx activity. therefore, it appears that natrxh is able to fold correctly in the absence of the c-terminal domain or at least fold well enough to sustain its native-like reductase activity. in n. alata, several proteins are directly involved in pollen rejection. in this species, s-rnase degrades the pollen tube rna and determines sexual incompatibility on the female side. the natrxh:s-rnase interaction could be relevant to the si response. natrxh likely stabilizes s-rnase or inhibits its ribonuclease activity in the pollen tube. indeed, oxley and bacic showed that s-rnase ribonuclease activity is affected by redox state in vitro. however, the redox state of natrxh does not impair its interaction with s-rnase. s-rnase forms complexes with other stylar proteins. while 120 k is known to be essential for si in n. alata, the precise function of these protein complexes in si is still unclear. it is possible that natrxh participates as an associating factor to transport such as s-rnase, 120 k, natts or p to the pollen tube or, alternatively, to release these proteins from s-rnase complexes once inside the pollen tube. both scenarios may be possible since a redox change by natrxh could play an important role for modifying s-rnase and stylar protein complexes. it has been determined that one of the targets of natrxh is actually s-rnase. therefore, further research is needed to determine if natrxh is a modifier factor in n. alata si by altering the s-rnase redox state in planta. although the data presented here are consistent with a role of natrxh in pollen rejection in si nicotiana species, loss of function assays would provide direct evidence of this role. finally, homology modeling to predict the 3d structure of trx h revealed high sequence similarity between the h. vulgare and n. alata trxh proteins, including conservation of the reactive site loop. the quality of the predicted model indicates similarity at the structural level too. the barley trx h protein plays an important regulatory role during seed germination, and one of its targets is the barley α-amylase/subtilisin inhibitor, a homologue of the si modifier n. alata nastep protein recently described by our group. both nastep and h. vulgare inhibitor proteins share extensive structural similarity; the loop equivalent to the one mediating the interaction of the barley proteins is present in nastep but is longer and has four cysteine residues instead of two. an interaction of natrxh and nastep at some point along the physiological events regulating pollen rejection in nicotiana is a possibility worth future consideration. thioredoxins type h clustered within the subgroup contain non-conserved extensions towards the n- and/or the c-termini, which function is still unclear. in this work, we showed that the n-terminal extension of natrxh, a subgroup trx h from n. alata, contained the sufficient information to lead its secretion towards the apoplast. interestingly, this extension contains two distinguishable motifs, called nα and nβ, divided by the hidden markov algorithm prediction of a cleavage site on the ala- position. while the nα domain appeared unlikely to have any function on natrxh secretion, the nβ was the responsible for its particular subcellular localization. the nβ domain is the only sequence necessary for natrxh secretion. transient expression experiments in epidermal onion cells of the nβ-gfp fusion protein revealed its localization in the apoplast, as occurred with the full natrxh protein fused to gfp and with the natrxhΔnα and natrxhΔcoo mutants as well. these data corroborated the nβ function as a novel signal peptide since its primary structure position and hydrophilic profile do not follow the typical biochemical features of the classical transport sequences. the nβ biochemical features suggested an er/golgi independent secretion pathway. however, natrxh was detected in a microsomal fraction and, furthermore, was immune-detected mainly associated to classical secretory elements when observed within the cells in n. alata styles, providing evidences that the natrxh could be secreted through the classical er/golgi pathway or at least, it uses the elements of this route. this hypothesis was also tested by fusing the er retention signal kdel to natrxh-gfp and treating the cells, when expressing the fusion protein natrxh-gfp, with bfa, separately. in the first case, the natrxh-gfp was found associated to the er and in the latter, the natrxh-gfp secretion was abolished, confirming that the natrxh actually goes through these organelles in order to be secreted. furthermore, it was also found that the nβ domain played an important structural role on the natrxh tertiary structure stability since the natrxhΔnβ, which only lacks the nβ domain, was detected in inclusion bodies when overexpressed in e. coli cells and not in the soluble one as the wild type and the other natrxh versions did. regarding the c-terminus, it was found to be essential for the natrxh-s-rnase in vitro interaction, since the s-rnase was unable to bind to a natrxhΔcoo-containing column. finally, the in silico analysis showed that the natrxh n- and c-termini are solvent exposed, suggesting a protein-protein interaction role. while this function appears to be essential for the s-rnase interaction, it also provided evidences on the nβ domain, which should interact either with a non-identified secretory element or interact in a different manner as the classical secreted proteins do with srp. interestingly, the n-terminal extension clearly showed two structural motifs, which coincide with the nα and nβ domains tested in this work. | therefore, the nβ domain sequence is suggested to be a novel signal peptide. the lack of hydrophobicity as well as the location of the secretion signal within the natrxh primary structure, suggest an unorthodox secretion route for natrxh. natrxh contains n- and c-terminal extensions, a feature shared by thioredoxin h proteins of subgroup to ascertain the function of these extensions in natrxh secretion and protein-protein interaction, we performed a deletion analysis on natrxh and fused the resulting variants to gfp. we found an internal domain in the n-terminal extension, called nβ, that is essential for natrxh secretion but is not hydrophobic, a canonical feature of a signal peptide. natrxh interacts in vitro with s-rnase and co-localizes with it in the extracellular matrix of the stylar transmitting tissue. notably, we found that the fusion protein natrxh-gfp is retained in the endoplasmic reticulum and that treatment of natrxh-gfp-expressing cells with brefeldin a leads to its retention in the golgi, which indicates that natrxh uses, to some extent, the endoplasmic reticulum and golgi apparatus for secretion. although the biochemical features of the n-terminus suggested a non-classical secretion pathway, our results provided evidence that natrxh at least uses the endoplasmic reticulum, golgi apparatus and also vesicles for secretion. furthermore, we found that nβ contributes to natrxh tertiary structure stabilization and that the c-terminus functions in the protein-protein interaction with s-rnase. while the c-terminus directly participates in protein-protein interaction, particularly on its interaction with s-rnase in vitro; the n-terminal extension contains two structurally different motifs: nα and nβ. nβ, the inner domain, is essential and enough to target natrxh towards the apoplast. interestingly, when it was fused to gfp, this protein was also found in the cell wall of the onion cells. | natrxh, a thioredoxin type h, shows differential expression between self-incompatible and self-compatible nicotiana species. natrxh interacts in vitro with s-rnase and co-localizes with it in the extracellular matrix of the stylar transmitting tissue. natrxh contains n- and c-terminal extensions, a feature shared by thioredoxin h proteins of subgroup to ascertain the function of these extensions in natrxh secretion and protein-protein interaction, we performed a deletion analysis on natrxh and fused the resulting variants to gfp. we found an internal domain in the n-terminal extension, called nβ, that is essential for natrxh secretion but is not hydrophobic, a canonical feature of a signal peptide. the lack of hydrophobicity as well as the location of the secretion signal within the natrxh primary structure, suggest an unorthodox secretion route for natrxh. notably, we found that the fusion protein natrxh-gfp is retained in the endoplasmic reticulum and that treatment of natrxh-gfp-expressing cells with brefeldin a leads to its retention in the golgi, which indicates that natrxh uses, to some extent, the endoplasmic reticulum and golgi apparatus for secretion. furthermore, we found that nβ contributes to natrxh tertiary structure stabilization and that the c-terminus functions in the protein-protein interaction with s-rnase. the extensions contained in natrxh sequence have specific functions on the protein. while the c-terminus directly participates in protein-protein interaction, particularly on its interaction with s-rnase in vitro; the n-terminal extension contains two structurally different motifs: nα and nβ. nβ, the inner domain, is essential and enough to target natrxh towards the apoplast. interestingly, when it was fused to gfp, this protein was also found in the cell wall of the onion cells. although the biochemical features of the n-terminus suggested a non-classical secretion pathway, our results provided evidence that natrxh at least uses the endoplasmic reticulum, golgi apparatus and also vesicles for secretion. therefore, the nβ domain sequence is suggested to be a novel signal peptide. thioredoxinsecretionself-incompatibilitynicotiana alatagametophytics-rnase |
BACKGROUND
immunoglobulin a vasculitis , also known as henoch-schönlein purpura, is an autoimmune disease caused by the deposition of iga-dominant immune complexes in small vessels <cit> .
it is the most common cutaneous vasculitis in children, and its annual incidence is 13– <dig> per <dig> children under 17 years old <cit> .
igav usually develops following the upper respiratory infection of viruses, bacteria, parasites, or others; with the common ones being group a streptococci, mycoplasma, epstein-barr virus, varicella virus and others <cit> .
the clinical features of igav are characterized by a tetrad of non-thrombocytopenic palpable purpura , arthralgia/arthritis , bowel angina , and hematuria/proteinuria <cit> .
therapy for igav is mostly supportive and symptomatic, because the disease is usually benign and self-limited.
for patients with severe active symptoms in one or multiple organs, glucocorticoids are administered to improve the treatment effect.
complications are rare.
however, complications resulting from blood vessel lesions in different organ systems could sometimes be severe, of which, renal involvement is the most serious complication and the principle cause of mortality in igav patients .
although the pathogenesis of igav is not completely understood, it is clear that both the aberrant deposition of glycosylated iga in small vascular walls and the subsequent activation of an alternate complement pathway play a central role in igav development <cit> .
multiple immune cell types including cd4+ helper t cells, b cells and natural killer cells are implicated in the pathogenesis of igav <cit> .
furthermore, th1/th <dig> imbalance, the hyperactivity of th <dig> cells and the decline in the ratio of cd4+/cd8+ cells increase the synthesis and release of immunoglobulins in igav patients.
the increased frequency of peripheral th <dig> cells and serum il- <dig> levels were also observed in childhood igav <cit> .
follicular helper t cells are a subset of cd4+ th cells that are specialized in helping b cell responses to produce antigen-specific antibodies such as iga, ige, igg and igm in autoimmune diseases, infectious diseases, and tumors .
although no unique markers have been reported for tfh cells, they could be identified through a combination of markers closely related to their functions including chemokine receptor cxcr <dig> programmed death- <dig> , inducible costimulator , slam adapter protein , b and t lymphocyte attenuator , cd <dig> ligand and cytokine interleukin <dig> .
originally identified in germinal centers of secondary lymphoid organs and essential for germinal center formation, b-cell affinity maturation, class switch recombination, and the generation of plasma and memory b cells , tfh counterparts were recently detected in tonsils <cit> and blood circulations <cit> .
the expansion of circulating tfh cells has been reported in various autoimmune diseases <cit> , suggesting their pathogenic significance.
xie et al.
revealed that the frequency of circulating cd4+cxcr5+icos+ tfh cells in children with active igav was significantly higher than in healthy children <cit> .
wang et al.
also reported that the upregulation of circulating tfh cells and downregulation of circulating follicular regulatory t cells may contribute to the pathogenesis of igav in children <cit> .
at present, many questions remain to be addressed in regard to the expansion of circulating tfh cells in autoimmune diseases.
when tfh cells are characterized by common markers, are we obtaining a homogenous or heterogeneous population of tfh cells?
if tfh cells are composed of heterogeneous subpopulations, as suggested by other studies <cit> , is each subpopulation functionally equivalent in the pathogenesis of a specific autoimmune disease?
the answers to these questions would improve our understanding not only on tfh cells and their functions, but also on their specific contribution to autoimmune diseases, which would facilitate therapeutic development.
in order to address these questions, in this study, we focused on six phenotypic subpopulations of circulating tfh cells as defined by the expressions of distinct molecules, examined their associations with igav, specifically the different dominant symptoms presented in igav, and explored the correlations between these tfh subpopulations and key igav clinical parameters.
methods
patients
the experimental protocols were established following the declaration of helsinki and approved by the human ethics committee of jilin university .
written informed consent was obtained from all participants.
a total of <dig> patients with newly diagnosed active igav admitted to the inpatient care of the department of pediatrics, the first hospital of jilin university from september <dig> to september <dig> were recruited into this study.
all patients met the following criteria: children under 18 years, confirmed diagnosis of igav according to the european league against rheumatism/ pediatric rheumatology international trials organization/ pediatric rheumatology european society criteria <cit> , and patients without other autoimmune diseases.
the detailed eular/printo/pres diagnostic criteria are as follows: the presence of palpable purpura , together with at least one of following findings: diffuse abdominal pain ; histopathology characterized by typical leukocytoclastic vasculitis with predominant iga deposits or proliferative glomerulonephritis with predominant iga deposits; acute arthritis or arthralgia ; renal involvement manifested by proteinuria , and/or hematuria .
according to presenting symptoms, the <dig> patients in this study were further divided into five groups: skin type , abdominal type , kidney type , joint type and the mixed type .
due to the self-limited and benign course of igav, symptom-oriented and supportive therapies were administered to patients following admission.
for patients that presented severe gastrointestinal complications or proliferative glomerulonephritis, gc were administered.
following treatment, remission was defined as the satisfaction of the following two criteria: after 5– <dig> days of treatment, all skin purpura became obviously shallow or completely subsided, and no new rash appeared; children with intestinal wall edema, arthralgia, hematuria and/or proteinuria, and other related symptoms experienced a dramatic relief of symptoms.
as controls, <dig> age- and gender-matched healthy individuals were recruited into this study.
upon recruitment, the following clinical parameters were measured on all participants: routine blood test, serum immunoglobulin and complement level , serum c-reactive protein , urinary protein , and urinary rbc and white blood cell count .
cell isolation
fasting venous blood samples were collected from hc and igav patients upon admission, and after disease remission , respectively.
peripheral blood mononuclear cells were isolated from individual patients and hc by density-gradient centrifugation using ficoll-paque plus at 800 × g for 30 min at 25 °c.
flow cytometry analysis
freshly isolated pbmcs were cultured in 10 % fetal calf serum rpmi- <dig> in u-bottom 24-well tissue culture plates , stimulated with or without 50 ng/ml of phorbol myristate acetate plus 2 μg/ml of ionomycin for one hour, followed by treatment with brefeldin a for an additional five hours.
then, these cells were stained in duplicate with bv510-anti-cd <dig> apc-h7-anti-cd <dig> bb515-anti-cxcr <dig> pe-cy5-anti-cd45ra, pe-cf594-anti-cd <dig> and bv421-anti-cd <dig> at room temperature for 30 min.
subsequently, cells were fixed, permeabilized, and stained with pe-anti-il- <dig> .
the frequencies of distinct tfh cells were analyzed by multicolor flow cytometry , and data were processed using flowjo software .
measurement of plasma il- <dig> by cytometric bead array
plasma il- <dig> concentrations were determined by a cba human soluble protein master buffer kit according to the manufacturer’s instructions, analyzed using a flow cytometer , and quantified using the cellquest pro and cba software .
statistical analysis
overall variations among the different groups were analyzed by one-way anova.
all data were presented as median and range.
student’s unpaired or paired t-test was appropriately chosen between groups.
mann–whitney test was performed for nonparametric data between the two studied groups.
the relationship between variables was analyzed by pearson rank correlation test.
all statistical analyses were performed using spss version <dig> software.
a two-tailed p value < <dig> was considered statistically significant.
RESULTS
clinical characteristics of children with igav
the general demographic and clinical characteristics of all participants are summarized in table <dig> according to the presenting symptoms, eight patients presented with skin purpura , eight with gastrointestinal tract discomfort , five with microhematuria and/or mild proteinuria , three with arthralgia and/or arthritis , and three with two or more non-purpura symptoms .
preceding upper airway infections were recorded in <dig> patients, and <dig> patients were tested positive for mycoplasma infection.
upon recruitment, the wbc count , platelet , serum iga , ige and complement c <dig> levels were significantly higher in igav patients than in hc .table <dig> the demographic and clinical characteristics of participants
*p < <dig> , vs hc the values before treatment
detection of circulating tfh cells
in order to assess the significance of circulating tfh cells in igav, focus was given on the following tfh cells: cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5highicos+pd-1high, cd4+cxcr5+icos−pd-1+, and cxcr5+cd45ra−il-21+, which were identified by flow cytometry .
cd4+cxcr5+ tfh cells and its four subpopulations, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cd4+cxcr5+icos−pd-1+, were gated from cd3+cd4+ t cells ; while cxcr5+cd45ra−il-21+ tfh cells were independently gated from cd3+cd4+ t cells .fig.
<dig> detection of circulating tfh cells by flow cytometry.
pbmcs were isolated from igav patients and age- and gender-matched healthy controls , stained with fluorophore-conjugated antibody targeting indicated proteins, and analyzed by flow cytometry.
a the gating strategy to identify cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, and cd4+cxcr5+icos−pd-1+ tfh cells.
b the gating strategy to identify cxcr5 + cd45ra-il-21+ tfh cells
association of different phenotypic subpopulations of tfh cells and cytokine with igav symptoms and treatment options
next, we analyzed the status of different subpopulations of tfh cells and plasma tfh cytokine il- <dig> in igav, as well as their associations to igav symptoms and treatments.
the frequencies of circulating cd4+cxcr5+ , cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5highicos+pd-1high and cxcr5+cd45ra−il-21+ tfh cells, as well as plasma il- <dig> levels, were all significantly higher in igav patients than in hc ; while the frequency of circulating cd4+cxcr5+icos−pd-1+ was not significantly different between these two groups .fig.
<dig> association of different phenotypes of tfh cells with igav symptoms and treatment options.
the comparison of indicated tfh cells and plasma il- <dig> levels between igav patients and hc , between igav patients with different symptom types and hc .
*p < <dig> , **p < <dig> , compared with the hc group; ns, not significant, when compared with the hc group
further analysis on the association of circulating tfh cells or plasma il- <dig> levels with the presenting symptoms of igav revealed different patterns of association : compared to levels in hc, cd4+cxcr5+ tfh cells were significantly higher in patients with skin, kidney, joint and mixed types , but not in those with abdominal type ; cd4+cxcr5+icos+ tfh cells increased in patients with abdominal, kidney and mixed types , but not in those with skin or joint type ; both cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high tfh cells were dramatically elevated in patients with all symptom types; cxcr5+cd45ra−il-21+ tfh cells were significantly higher in patients with skin, abdominal, kidney and mixed types , but not in those with joint type .
interestingly, cd4+cxcr5+icos−pd-1+ tfh cells was significantly lower in patients with an abdominal type , but not in those with other types , compared with hc.
furthermore, plasma il- <dig> levels were significantly higher in patients with skin and abdominal types , but not in those with kidney, joint or mixed type , compared with hc.
when the association of different tfh cells with treatment options were analyzed among patients entering disease remission, no significant difference was detected in any of the tfh cells or plasma il- <dig> .
alterations of tfh cells and plasma il- <dig> following treatment
following admission, all patients received symptom-oriented and supportive therapies; and <dig> patients achieved disease remission.
among these patients, <dig> patients were examined for these subpopulations of tfh cells before treatment during the active stage of the disease, as well as after treatment during the remission stage .
with disease remission, the frequencies of circulating cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were significantly reduced from the corresponding value in the active stage .
no significant difference was detected in cd4+cxcr5+icos−pd-1+ cells following disease remission .
meanwhile, plasma il- <dig> levels also significantly decreased in the remission stage, when compared to the active stage .fig.
<dig> treatment-induced alterations of different subpopulations of tfh cells and plasma il- <dig> after proper treatment, disease remission was achieved in <dig> patients.
the frequency of the indicated tfh cells and plasma il- <dig> levels were compared between the active and remission stages of the disease
correlation between tfh cells and serum iga, c <dig> and plasma il-21
when the correlation between different tfh cells and different clinical parameters of igav were analyzed, it was found that circulating cxcr5+cd45ra−il-21+ , cd4+cxcr5+icos+ tfh cells , cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high , but not cd4+cxcr5+icos−pd-1+ tfh cells, were significantly and positively correlated with serum iga levels .
circulating levels of cd4+cxcr5+icos+ , cd4+cxcr5+icos+pd-1+ and cxcr5+cd45ra−il-21+ tfh cells were also significantly and positively correlated with plasma il- <dig> levels .
furthermore, circulating cxcr5+cd45ra−il-21+ tfh cells were the only cells significantly and negatively correlated with serum c <dig> levels .fig.
<dig> correlation between different phenotypic subpopulations of tfh cells and serum iga, complement c <dig> or plasma il- <dig> the correlation between the indicated tfh cells and serum iga , plasma il- <dig> and c <dig> was analyzed by pearson rank correlation test
discussion
in this study, we presented prime evidence that circulating cd4+cxcr5+ tfh cells are not homogenous, but rather a heterogeneous population of cells distinguishable by combinations of tfh phenotypic markers.
functionally, these phenotypic subpopulations are differentially regulated in igav patients presenting different patterns of association with the dominant symptoms of the disease, and un-equivalently correlated with key clinical igav parameters.
upon disease remission following treatment, these cells also responded differently.
this is the first study that revealed the differential contributions of tfh cells in igav pathogenesis and their alterations following disease progression.
consistent with their specialized functions to help b cells in antibody production, the aberrant expansion of tfh cells have been identified in autoimmune diseases including systemic lupus erythematosus , sjogren’s syndrome and juvenile dermatomyositis; which are all characterized by the production of pathogenic autoantibodies <cit> .
among the plethora of autoimmune diseases, igav is a common connective tissue disease associated with the vascular deposition of iga-dominant immunoglobulin complexes <cit> .
most recently, scientists began to investigate the potential involvement of tfh cells in igav, and two studies have both identified the expansion of circulating cd4+cxcr5+icos+ tfh cells in igav patients, compared with cells in healthy controls <cit> .
however, neither these two nor other studies explored the potential phenotypic subpopulations of tfh cells in igav or their association with different clinical features of igav.
consistent with these two studies, we have shown that the frequency of cd4+cxcr5+icos+ tfh cells in peripheral blood from igav patients were significantly higher than in healthy individuals.
in addition, we have revealed that the expansion was not unique to cd4+cxcr5+icos+ cells, since the frequencies of circulating cd4+cxcr5+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were also significantly higher in igav patients.
furthermore, correlation studies revealed that the frequencies of circulating cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were significantly and positively correlated with serum iga levels.
in contrast, the frequency of cd4+cxcr5+icos−pd-1+ tfh cells did not present any significant change in igav patients, compared to healthy individuals, which is consistent with the findings of xie et al.
<cit> .
besides, cd4+cxcr5+icos−pd-1+ tfh cells were not correlated with serum iga levels.
these data suggest that more than one phenotypic subpopulations of tfh cells, but not all, may contribute to the pathogenesis and progression of igav.
although not unique for tfh cells, common tfh cell markers are closely associated with the functions of these cells: chemokine receptor cxcr <dig> is important for b-cell homing to b cell follicles <cit> ; pd- <dig> supports the survival and selection of high-affinity plasma cells in the germinal center <cit> ; icos is essential for the maintenance and function of tfh in the germinal center <cit> ; il- <dig> critically regulates the growth, differentiation and class-switching of b cells <cit> .
in circulating tfh cells, the exact functions of each marker remains to be addressed; but they may not significantly differ from those in germinal center tfh cells.
all markers were identified in cd4+cxcr5+ tfh cells.
however, it is not known whether different combinations of these markers would generate distinct subpopulations of tfh cells; and more importantly, whether these subpopulations would be functionally different from each other.
in this study, we revealed that there are at least four different phenotypic subpopulations of circulating tfh cells: cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, cd4+cxcr5+icos−pd-1+ and cxcr5+cd45ra−il-21+.
even though they may not be completely exclusive from each other, they did present varied biological functions in igav.
cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were all significantly expanded in the circulation of igav; and their levels lowered dramatically following effective treatment and disease remission.
however, their frequencies varied with the dominant clinical symptoms presented in patients: cd4+cxcr5+icos+ cells were not significantly altered in patients with predominant skin or joint symptoms; cxcr5+cd45ra−il-21+ cells were not significantly altered in patients with predominant joint symptoms; both circulating cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high were significantly expanded in iga patients presenting with all dominant symptoms, in which cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high were most robustly expanded in patients with mixed symptoms, as well as in cxcr5+cd45ra−il-21+ cells in those with skin symptoms.
these findings imply that different subpopulations of tfh cells differentially regulate the development of organ-specific symptoms.
interestingly, although the frequency of circulating cd4+cxcr5+icos−pd-1+ tfh cells did not change dramatically in igav patients compared to healthy individuals, their level was significantly lower in patients presenting abdominal symptoms.
pd- <dig> and its ligands play a critical role in maintaining peripheral tolerance <cit> .
signaling through pd- <dig> attenuates the signaling downstream of t cell receptors and inhibits t cell expansion, cytokine production and cytolytic activity.
in addition, pd- <dig> signaling inhibits the aberrant activation of t cells and benefits the induction of regulatory t cells .
the non-elevation of cd4+cxcr5+icos−pd-1+ tfh cells in igav and its downregulation in patients presenting abdominal dominant symptoms may reflect a pathogenic mechanism during igav development, specifically the development of abdominal symptoms in igav; which would inhibit the potential immunosuppressive activity of tfh cells, and thus shift the balance toward enhanced autoimmunity.
the characteristic cytokine produced by tfh cells is il- <dig> which is a type i cytokine with pleiotropic immune activities including regulating germinal center b-cell responses, isotype switching and the generation of memory b cells .
both b and cd4+ t cells require il- <dig> signaling for generating long-term humoral immunity <cit> .
many cd4+ t cells can produce il- <dig> with the most abundant sources being tfh and th <dig> cells <cit> .
in this study, we found that plasma il- <dig> levels were significantly elevated in igav patients, particularly in patients with dominant skin and abdominal symptoms; but not in patients with joint, kidney or mixed symptoms, as compared with healthy individuals.
following disease remission, elevated plasma il- <dig> level was significantly reduced; suggesting that circulating il- <dig> levels are sensitive indicators for active igav.
furthermore, we identified significant and positive correlations between plasma il- <dig> levels with the frequency of circulating cxcr5+cd45ra−il-21+, cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icos+ tfh cells, implying that these three phenotypes of tfh cells may contribute to igav development through the secretion of il- <dig>
gc is a powerful anti-inflammatory drug, but we only use it for patients with severe symptoms.
it can inhibit inflammation by downregulating t and b cell function and reducing cytokine production.
in this study, we divided patients during the remission stage into the gc group and the non-gc group .
surprisingly, the difference in tfh cell subsets between these two groups was not significant.
these findings also supported the notion that igav is a common kind of self-limiting disease, and its recovery is mainly based on its own re-established immune homeostasis.
although gc can rapidly relieve severe active symptoms, a small dose of gc does not lead to immunosuppression, cushing’s syndrome, and other adverse reactions.
in addition, this result does not reflect the value of gc on immune regulation due to the lack of long-term follow-up and tracing studies; hence, we could not absolutely determine the value of gc therapy for igav, especially for the long-term prognosis of renal type.
the number of patients in this study is few, and there is a need to explore more patients, especially with different prognosis types, in future studies.
although the major conclusions drawn from this study are limited by the relatively small sample size, these provide seminal findings that would guide future, larger-scale studies.
furthermore, it is important to further characterize the tfh cell subsets from this study, both on phenotypes and functions; and compare them with tfh cell subsets defined from other studies such as th <dig> th <dig> and th <dig> subsets <cit> .
in summary, this study identified multiple phenotypic subpopulations of tfh cells, namely, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, cd4+cxcr5+icos−pd-1+ and cxcr5+cd45ra−il-21+; which are functionally important for the pathogenesis of igav.
the levels of these subpopulations in the peripheral circulation of igav in patients were significantly higher than in healthy individuals; they also correlate with igav clinical markers including circulating il- <dig> and iga levels, which decreased following disease remission.
in addition, these subsets presented differential associations with the organ-specific symptoms of igav.
therefore, these tfh cells not only serve as indicators of igav symptoms and progression, but also becomes therapeutic targets that enable the individualized or symptom-oriented treatment of igav.
CONCLUSIONS
igav is the most common cutaneous vasculitis in children, immune system disorders play a key role in its pathogenesis.
here, we found tfh cells may differentially contribute to the development of igav or predict disease progression.
these findings provide novel insights in the pathogenesis of igav, and this may be new targets for intervention of organ-specific igav.
abbreviations
cbacytometric bead array
hchealthy controls
igaviga vasculitis
lcvleukocytoclastic vasculitis
pbmcperipheral blood mononuclear cells
tfh cellsfollicular helper t cells
the authors thank the patients for their participation in this study.
this work was supported by the key laboratory of zoonoses research of the first hospital of jilin university.
funding
this work was supported by natural science foundation of jilin provincial science and technology department .
availability of data and materials
all data generated or analysed during this study are included in this published article.
authors’contributions
dl carried out the experiments, and analyzed, and interpreted the data.
jw collected clinical data.
cl and jl performed literature search.
sy and yj contributed to the conception and design of the study, the analysis and interpretation of the data, and drafting and revising the manuscript.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interest.
consent for publication
not applicable.
ethics approval and consent to participate
the experimental protocols were established following the declaration of helsinki and approved by the human ethics committee of jilin university .
written informed consent was obtained from all participants.
| 21,709 | immunoglobulin a vasculitis, also known as henoch-schönlein purpura, is an autoimmune disease caused by the deposition of iga-dominant immune complexes in small vessels. it is the most common cutaneous vasculitis in children, and its annual incidence is 13– per children under 17 years old. igav usually develops following the upper respiratory infection of viruses, bacteria, parasites, or others; with the common ones being group a streptococci, mycoplasma, epstein-barr virus, varicella virus and others. the clinical features of igav are characterized by a tetrad of non-thrombocytopenic palpable purpura, arthralgia/arthritis, bowel angina, and hematuria/proteinuria. therapy for igav is mostly supportive and symptomatic, because the disease is usually benign and self-limited. for patients with severe active symptoms in one or multiple organs, glucocorticoids are administered to improve the treatment effect. complications are rare. however, complications resulting from blood vessel lesions in different organ systems could sometimes be severe, of which, renal involvement is the most serious complication and the principle cause of mortality in igav patients. although the pathogenesis of igav is not completely understood, it is clear that both the aberrant deposition of glycosylated iga in small vascular walls and the subsequent activation of an alternate complement pathway play a central role in igav development. multiple immune cell types including cd4+ helper t cells, b cells and natural killer cells are implicated in the pathogenesis of igav. furthermore, th1/th imbalance, the hyperactivity of th cells and the decline in the ratio of cd4+/cd8+ cells increase the synthesis and release of immunoglobulins in igav patients. the increased frequency of peripheral th cells and serum il- levels were also observed in childhood igav. follicular helper t cells are a subset of cd4+ th cells that are specialized in helping b cell responses to produce antigen-specific antibodies such as iga, ige, igg and igm in autoimmune diseases, infectious diseases, and tumors. although no unique markers have been reported for tfh cells, they could be identified through a combination of markers closely related to their functions including chemokine receptor cxcr programmed death-, inducible costimulator, slam adapter protein, b and t lymphocyte attenuator, cd ligand and cytokine interleukin. originally identified in germinal centers of secondary lymphoid organs and essential for germinal center formation, b-cell affinity maturation, class switch recombination, and the generation of plasma and memory b cells, tfh counterparts were recently detected in tonsils and blood circulations. the expansion of circulating tfh cells has been reported in various autoimmune diseases, suggesting their pathogenic significance. xie et al. revealed that the frequency of circulating cd4+cxcr5+icos+ tfh cells in children with active igav was significantly higher than in healthy children. wang et al. also reported that the upregulation of circulating tfh cells and downregulation of circulating follicular regulatory t cells may contribute to the pathogenesis of igav in children. at present, many questions remain to be addressed in regard to the expansion of circulating tfh cells in autoimmune diseases. when tfh cells are characterized by common markers, are we obtaining a homogenous or heterogeneous population of tfh cells? if tfh cells are composed of heterogeneous subpopulations, as suggested by other studies, is each subpopulation functionally equivalent in the pathogenesis of a specific autoimmune disease? the answers to these questions would improve our understanding not only on tfh cells and their functions, but also on their specific contribution to autoimmune diseases, which would facilitate therapeutic development. in order to address these questions, in this study, we focused on six phenotypic subpopulations of circulating tfh cells as defined by the expressions of distinct molecules, examined their associations with igav, specifically the different dominant symptoms presented in igav, and explored the correlations between these tfh subpopulations and key igav clinical parameters. methods patients the experimental protocols were established following the declaration of helsinki and approved by the human ethics committee of jilin university. written informed consent was obtained from all participants. a total of patients with newly diagnosed active igav admitted to the inpatient care of the department of pediatrics, the first hospital of jilin university from september to september were recruited into this study. all patients met the following criteria: children under 18 years, confirmed diagnosis of igav according to the european league against rheumatism/ pediatric rheumatology international trials organization/ pediatric rheumatology european society criteria, and patients without other autoimmune diseases. the detailed eular/printo/pres diagnostic criteria are as follows: the presence of palpable purpura, together with at least one of following findings: diffuse abdominal pain; histopathology characterized by typical leukocytoclastic vasculitis with predominant iga deposits or proliferative glomerulonephritis with predominant iga deposits; acute arthritis or arthralgia; renal involvement manifested by proteinuria, and/or hematuria. according to presenting symptoms, the patients in this study were further divided into five groups: skin type, abdominal type, kidney type, joint type and the mixed type. due to the self-limited and benign course of igav, symptom-oriented and supportive therapies were administered to patients following admission. for patients that presented severe gastrointestinal complications or proliferative glomerulonephritis, gc were administered. following treatment, remission was defined as the satisfaction of the following two criteria: after 5– days of treatment, all skin purpura became obviously shallow or completely subsided, and no new rash appeared; children with intestinal wall edema, arthralgia, hematuria and/or proteinuria, and other related symptoms experienced a dramatic relief of symptoms. as controls, age- and gender-matched healthy individuals were recruited into this study. upon recruitment, the following clinical parameters were measured on all participants: routine blood test, serum immunoglobulin and complement level, serum c-reactive protein, urinary protein, and urinary rbc and white blood cell count. cell isolation fasting venous blood samples were collected from hc and igav patients upon admission, and after disease remission, respectively. peripheral blood mononuclear cells were isolated from individual patients and hc by density-gradient centrifugation using ficoll-paque plus at 800 × g for 30 min at 25 °c. flow cytometry analysis freshly isolated pbmcs were cultured in 10 % fetal calf serum rpmi- in u-bottom 24-well tissue culture plates, stimulated with or without 50 ng/ml of phorbol myristate acetate plus 2 μg/ml of ionomycin for one hour, followed by treatment with brefeldin a for an additional five hours. then, these cells were stained in duplicate with bv510-anti-cd apc-h7-anti-cd bb515-anti-cxcr pe-cy5-anti-cd45ra, pe-cf594-anti-cd and bv421-anti-cd at room temperature for 30 min. subsequently, cells were fixed, permeabilized, and stained with pe-anti-il-. the frequencies of distinct tfh cells were analyzed by multicolor flow cytometry, and data were processed using flowjo software. measurement of plasma il- by cytometric bead array plasma il- concentrations were determined by a cba human soluble protein master buffer kit according to the manufacturer’s instructions, analyzed using a flow cytometer, and quantified using the cellquest pro and cba software. statistical analysis overall variations among the different groups were analyzed by one-way anova. all data were presented as median and range. student’s unpaired or paired t-test was appropriately chosen between groups. mann–whitney test was performed for nonparametric data between the two studied groups. the relationship between variables was analyzed by pearson rank correlation test. all statistical analyses were performed using spss version software. a two-tailed p value < was considered statistically significant. clinical characteristics of children with igav the general demographic and clinical characteristics of all participants are summarized in table according to the presenting symptoms, eight patients presented with skin purpura, eight with gastrointestinal tract discomfort, five with microhematuria and/or mild proteinuria, three with arthralgia and/or arthritis, and three with two or more non-purpura symptoms. preceding upper airway infections were recorded in patients, and patients were tested positive for mycoplasma infection. upon recruitment, the wbc count, platelet, serum iga, ige and complement c levels were significantly higher in igav patients than in hc.table the demographic and clinical characteristics of participants *p < , vs hc the values before treatment detection of circulating tfh cells in order to assess the significance of circulating tfh cells in igav, focus was given on the following tfh cells: cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5highicos+pd-1high, cd4+cxcr5+icos−pd-1+, and cxcr5+cd45ra−il-21+, which were identified by flow cytometry. cd4+cxcr5+ tfh cells and its four subpopulations, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cd4+cxcr5+icos−pd-1+, were gated from cd3+cd4+ t cells; while cxcr5+cd45ra−il-21+ tfh cells were independently gated from cd3+cd4+ t cells.fig. detection of circulating tfh cells by flow cytometry. pbmcs were isolated from igav patients and age- and gender-matched healthy controls, stained with fluorophore-conjugated antibody targeting indicated proteins, and analyzed by flow cytometry. a the gating strategy to identify cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, and cd4+cxcr5+icos−pd-1+ tfh cells. b the gating strategy to identify cxcr5 + cd45ra-il-21+ tfh cells association of different phenotypic subpopulations of tfh cells and cytokine with igav symptoms and treatment options next, we analyzed the status of different subpopulations of tfh cells and plasma tfh cytokine il- in igav, as well as their associations to igav symptoms and treatments. the frequencies of circulating cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5highicos+pd-1high and cxcr5+cd45ra−il-21+ tfh cells, as well as plasma il- levels, were all significantly higher in igav patients than in hc; while the frequency of circulating cd4+cxcr5+icos−pd-1+ was not significantly different between these two groups.fig. association of different phenotypes of tfh cells with igav symptoms and treatment options. the comparison of indicated tfh cells and plasma il- levels between igav patients and hc, between igav patients with different symptom types and hc. *p < , **p < , compared with the hc group; ns, not significant, when compared with the hc group further analysis on the association of circulating tfh cells or plasma il- levels with the presenting symptoms of igav revealed different patterns of association: compared to levels in hc, cd4+cxcr5+ tfh cells were significantly higher in patients with skin, kidney, joint and mixed types, but not in those with abdominal type; cd4+cxcr5+icos+ tfh cells increased in patients with abdominal, kidney and mixed types, but not in those with skin or joint type; both cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high tfh cells were dramatically elevated in patients with all symptom types; cxcr5+cd45ra−il-21+ tfh cells were significantly higher in patients with skin, abdominal, kidney and mixed types, but not in those with joint type. interestingly, cd4+cxcr5+icos−pd-1+ tfh cells was significantly lower in patients with an abdominal type, but not in those with other types, compared with hc. furthermore, plasma il- levels were significantly higher in patients with skin and abdominal types, but not in those with kidney, joint or mixed type, compared with hc. when the association of different tfh cells with treatment options were analyzed among patients entering disease remission, no significant difference was detected in any of the tfh cells or plasma il-. alterations of tfh cells and plasma il- following treatment following admission, all patients received symptom-oriented and supportive therapies; and patients achieved disease remission. among these patients, patients were examined for these subpopulations of tfh cells before treatment during the active stage of the disease, as well as after treatment during the remission stage. with disease remission, the frequencies of circulating cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were significantly reduced from the corresponding value in the active stage. no significant difference was detected in cd4+cxcr5+icos−pd-1+ cells following disease remission. meanwhile, plasma il- levels also significantly decreased in the remission stage, when compared to the active stage.fig. treatment-induced alterations of different subpopulations of tfh cells and plasma il- after proper treatment, disease remission was achieved in patients. the frequency of the indicated tfh cells and plasma il- levels were compared between the active and remission stages of the disease correlation between tfh cells and serum iga, c and plasma il-21 when the correlation between different tfh cells and different clinical parameters of igav were analyzed, it was found that circulating cxcr5+cd45ra−il-21+, cd4+cxcr5+icos+ tfh cells, cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high, but not cd4+cxcr5+icos−pd-1+ tfh cells, were significantly and positively correlated with serum iga levels. circulating levels of cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+ and cxcr5+cd45ra−il-21+ tfh cells were also significantly and positively correlated with plasma il- levels. furthermore, circulating cxcr5+cd45ra−il-21+ tfh cells were the only cells significantly and negatively correlated with serum c levels.fig. correlation between different phenotypic subpopulations of tfh cells and serum iga, complement c or plasma il- the correlation between the indicated tfh cells and serum iga, plasma il- and c was analyzed by pearson rank correlation test discussion in this study, we presented prime evidence that circulating cd4+cxcr5+ tfh cells are not homogenous, but rather a heterogeneous population of cells distinguishable by combinations of tfh phenotypic markers. functionally, these phenotypic subpopulations are differentially regulated in igav patients presenting different patterns of association with the dominant symptoms of the disease, and un-equivalently correlated with key clinical igav parameters. upon disease remission following treatment, these cells also responded differently. this is the first study that revealed the differential contributions of tfh cells in igav pathogenesis and their alterations following disease progression. consistent with their specialized functions to help b cells in antibody production, the aberrant expansion of tfh cells have been identified in autoimmune diseases including systemic lupus erythematosus, sjogren’s syndrome and juvenile dermatomyositis; which are all characterized by the production of pathogenic autoantibodies. among the plethora of autoimmune diseases, igav is a common connective tissue disease associated with the vascular deposition of iga-dominant immunoglobulin complexes. most recently, scientists began to investigate the potential involvement of tfh cells in igav, and two studies have both identified the expansion of circulating cd4+cxcr5+icos+ tfh cells in igav patients, compared with cells in healthy controls. however, neither these two nor other studies explored the potential phenotypic subpopulations of tfh cells in igav or their association with different clinical features of igav. consistent with these two studies, we have shown that the frequency of cd4+cxcr5+icos+ tfh cells in peripheral blood from igav patients were significantly higher than in healthy individuals. in addition, we have revealed that the expansion was not unique to cd4+cxcr5+icos+ cells, since the frequencies of circulating cd4+cxcr5+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were also significantly higher in igav patients. furthermore, correlation studies revealed that the frequencies of circulating cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were significantly and positively correlated with serum iga levels. in contrast, the frequency of cd4+cxcr5+icos−pd-1+ tfh cells did not present any significant change in igav patients, compared to healthy individuals, which is consistent with the findings of xie et al.. besides, cd4+cxcr5+icos−pd-1+ tfh cells were not correlated with serum iga levels. these data suggest that more than one phenotypic subpopulations of tfh cells, but not all, may contribute to the pathogenesis and progression of igav. although not unique for tfh cells, common tfh cell markers are closely associated with the functions of these cells: chemokine receptor cxcr is important for b-cell homing to b cell follicles; pd- supports the survival and selection of high-affinity plasma cells in the germinal center; icos is essential for the maintenance and function of tfh in the germinal center; il- critically regulates the growth, differentiation and class-switching of b cells. in circulating tfh cells, the exact functions of each marker remains to be addressed; but they may not significantly differ from those in germinal center tfh cells. all markers were identified in cd4+cxcr5+ tfh cells. however, it is not known whether different combinations of these markers would generate distinct subpopulations of tfh cells; and more importantly, whether these subpopulations would be functionally different from each other. in this study, we revealed that there are at least four different phenotypic subpopulations of circulating tfh cells: cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, cd4+cxcr5+icos−pd-1+ and cxcr5+cd45ra−il-21+. even though they may not be completely exclusive from each other, they did present varied biological functions in igav. cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells were all significantly expanded in the circulation of igav; and their levels lowered dramatically following effective treatment and disease remission. however, their frequencies varied with the dominant clinical symptoms presented in patients: cd4+cxcr5+icos+ cells were not significantly altered in patients with predominant skin or joint symptoms; cxcr5+cd45ra−il-21+ cells were not significantly altered in patients with predominant joint symptoms; both circulating cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high were significantly expanded in iga patients presenting with all dominant symptoms, in which cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icoshighpd-1high were most robustly expanded in patients with mixed symptoms, as well as in cxcr5+cd45ra−il-21+ cells in those with skin symptoms. these findings imply that different subpopulations of tfh cells differentially regulate the development of organ-specific symptoms. interestingly, although the frequency of circulating cd4+cxcr5+icos−pd-1+ tfh cells did not change dramatically in igav patients compared to healthy individuals, their level was significantly lower in patients presenting abdominal symptoms. pd- and its ligands play a critical role in maintaining peripheral tolerance. signaling through pd- attenuates the signaling downstream of t cell receptors and inhibits t cell expansion, cytokine production and cytolytic activity. in addition, pd- signaling inhibits the aberrant activation of t cells and benefits the induction of regulatory t cells. the non-elevation of cd4+cxcr5+icos−pd-1+ tfh cells in igav and its downregulation in patients presenting abdominal dominant symptoms may reflect a pathogenic mechanism during igav development, specifically the development of abdominal symptoms in igav; which would inhibit the potential immunosuppressive activity of tfh cells, and thus shift the balance toward enhanced autoimmunity. the characteristic cytokine produced by tfh cells is il- which is a type i cytokine with pleiotropic immune activities including regulating germinal center b-cell responses, isotype switching and the generation of memory b cells. both b and cd4+ t cells require il- signaling for generating long-term humoral immunity. many cd4+ t cells can produce il- with the most abundant sources being tfh and th cells. in this study, we found that plasma il- levels were significantly elevated in igav patients, particularly in patients with dominant skin and abdominal symptoms; but not in patients with joint, kidney or mixed symptoms, as compared with healthy individuals. following disease remission, elevated plasma il- level was significantly reduced; suggesting that circulating il- levels are sensitive indicators for active igav. furthermore, we identified significant and positive correlations between plasma il- levels with the frequency of circulating cxcr5+cd45ra−il-21+, cd4+cxcr5+icos+pd-1+ and cd4+cxcr5+icos+ tfh cells, implying that these three phenotypes of tfh cells may contribute to igav development through the secretion of il- gc is a powerful anti-inflammatory drug, but we only use it for patients with severe symptoms. it can inhibit inflammation by downregulating t and b cell function and reducing cytokine production. in this study, we divided patients during the remission stage into the gc group and the non-gc group. surprisingly, the difference in tfh cell subsets between these two groups was not significant. these findings also supported the notion that igav is a common kind of self-limiting disease, and its recovery is mainly based on its own re-established immune homeostasis. although gc can rapidly relieve severe active symptoms, a small dose of gc does not lead to immunosuppression, cushing’s syndrome, and other adverse reactions. in addition, this result does not reflect the value of gc on immune regulation due to the lack of long-term follow-up and tracing studies; hence, we could not absolutely determine the value of gc therapy for igav, especially for the long-term prognosis of renal type. the number of patients in this study is few, and there is a need to explore more patients, especially with different prognosis types, in future studies. although the major conclusions drawn from this study are limited by the relatively small sample size, these provide seminal findings that would guide future, larger-scale studies. furthermore, it is important to further characterize the tfh cell subsets from this study, both on phenotypes and functions; and compare them with tfh cell subsets defined from other studies such as th th and th subsets. in summary, this study identified multiple phenotypic subpopulations of tfh cells, namely, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, cd4+cxcr5+icos−pd-1+ and cxcr5+cd45ra−il-21+; which are functionally important for the pathogenesis of igav. the levels of these subpopulations in the peripheral circulation of igav in patients were significantly higher than in healthy individuals; they also correlate with igav clinical markers including circulating il- and iga levels, which decreased following disease remission. in addition, these subsets presented differential associations with the organ-specific symptoms of igav. therefore, these tfh cells not only serve as indicators of igav symptoms and progression, but also becomes therapeutic targets that enable the individualized or symptom-oriented treatment of igav. igav is the most common cutaneous vasculitis in children, immune system disorders play a key role in its pathogenesis. here, we found tfh cells may differentially contribute to the development of igav or predict disease progression. these findings provide novel insights in the pathogenesis of igav, and this may be new targets for intervention of organ-specific igav. abbreviations cbacytometric bead array hchealthy controls igaviga vasculitis lcvleukocytoclastic vasculitis pbmcperipheral blood mononuclear cells tfh cellsfollicular helper t cells the authors thank the patients for their participation in this study. this work was supported by the key laboratory of zoonoses research of the first hospital of jilin university. funding this work was supported by natural science foundation of jilin provincial science and technology department. availability of data and materials all data generated or analysed during this study are included in this published article. authors’contributions dl carried out the experiments, and analyzed, and interpreted the data. jw collected clinical data. cl and jl performed literature search. sy and yj contributed to the conception and design of the study, the analysis and interpretation of the data, and drafting and revising the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interest. consent for publication not applicable. ethics approval and consent to participate the experimental protocols were established following the declaration of helsinki and approved by the human ethics committee of jilin university. written informed consent was obtained from all participants. | in this study, we examined the status of different subpopulations of tfh cells in peripheral circulation and their associations with various clinical characteristics of iga vasculitis. according to the phenotypic expressions of different molecules, focus was given on six subpopulations of tfh cells: cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, cd4+cxcr5+icos−pd-1+, and cxcr5+cd45ra−il-21+. significantly higher frequencies of cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells, as well as higher levels of plasma il- were detected in igav patients compared to hc. when the disease entered the remission stage following treatment, circulating levels of cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells, as well as plasma il- levels were reduced. among the six subpopulations of tfh cells, both cd4+cxcr5+icos+ and cxcr5+cd45ra−il-21+ significantly and positively correlated with serum iga and plasma il- levels, but only cxcr5+cd45ra−il-21+ significantly and negatively correlated with the serum c level. tfh cells may differentially contribute to the development of igav or predict disease progression. follicular helper t cellsiga vasculitisinterleukin 21symptomsremissionglucocorticoid natural science foundation of jilin provincial science and technology department20160101065jcyang sirui issue-copyright-statement© the author 2016 the frequencies of these six subpopulations and the circulating level of tfh-related cytokine interleukin were measured from patients with igav and healthy controls by flow cytometry and flow cytometric bead array, respectively. circulating follicular helper t cells are a heterogeneous population of cd4+ helper t cells that promotes pathogenic immune responses in autoimmune diseases. these findings provide novel insights in the pathogenesis of igav and may benefit treatment development targeting organ-specific presenting symptoms of igav. | circulating follicular helper t cells are a heterogeneous population of cd4+ helper t cells that promotes pathogenic immune responses in autoimmune diseases. in this study, we examined the status of different subpopulations of tfh cells in peripheral circulation and their associations with various clinical characteristics of iga vasculitis. methods according to the phenotypic expressions of different molecules, focus was given on six subpopulations of tfh cells: cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high, cd4+cxcr5+icos−pd-1+, and cxcr5+cd45ra−il-21+. the frequencies of these six subpopulations and the circulating level of tfh-related cytokine interleukin were measured from patients with igav and healthy controls by flow cytometry and flow cytometric bead array, respectively. significantly higher frequencies of cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells, as well as higher levels of plasma il- were detected in igav patients compared to hc. the level of each tfh subpopulation varied by the presenting symptoms of igav, but did not differ between patients treated or not treated with glucocorticoids. when the disease entered the remission stage following treatment, circulating levels of cd4+cxcr5+, cd4+cxcr5+icos+, cd4+cxcr5+icos+pd-1+, cd4+cxcr5+icoshighpd-1high and cxcr5+cd45ra−il-21+ tfh cells, as well as plasma il- levels were reduced. among the six subpopulations of tfh cells, both cd4+cxcr5+icos+ and cxcr5+cd45ra−il-21+ significantly and positively correlated with serum iga and plasma il- levels, but only cxcr5+cd45ra−il-21+ significantly and negatively correlated with the serum c level. tfh cells may differentially contribute to the development of igav or predict disease progression. these findings provide novel insights in the pathogenesis of igav and may benefit treatment development targeting organ-specific presenting symptoms of igav. keywords follicular helper t cellsiga vasculitisinterleukin 21symptomsremissionglucocorticoid natural science foundation of jilin provincial science and technology department20160101065jcyang sirui issue-copyright-statement© the author 2016 |
"BACKGROUND\nheterosis refers to the superior performance in biomass, yield, or other agronomic trai(...TRUNCATED) | 27,866 | " heterosis refers to the superior performance in biomass, yield, or other agronomic traits of hybri(...TRUNCATED) | "however, a comprehensive perspective on transcriptional variation and its correlation with heterosi(...TRUNCATED) | " hybridization is a prominent process in the evolution of crop plants that can give rise to gene ex(...TRUNCATED) |
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"BACKGROUND\nany group of organisms has a single true pedigree that extends back through time as an (...TRUNCATED) | 7,330 | " any group of organisms has a single true pedigree that extends back through time as an unbroken ch(...TRUNCATED) | "clade mtw contains haplotypes from the iberian peninsula and the western alps, clade mtc includes h(...TRUNCATED) | " the chamois, distributed over most of the medium to high altitude mountain ranges of southern eura(...TRUNCATED) |
"BACKGROUND\nphytohormones are central regulators of all aspects of plant life.\nthey modulate plant(...TRUNCATED) | 17,927 | " phytohormones are central regulators of all aspects of plant life. they modulate plant development(...TRUNCATED) | "our results suggest that max previously characterized for its role in regulation of polar auxin tra(...TRUNCATED) | " the arabidopsis thaliana f-box protein more axillary growth has previously been characterized for (...TRUNCATED) |
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"BACKGROUND\nrecently, mesenchymal stem cells were proven to be present in dental tissues, includin(...TRUNCATED) | 9,530 | " recently, mesenchymal stem cells were proven to be present in dental tissues, including dental pul(...TRUNCATED) | "we aimed to demonstrate that df stem cells from impacted molars and canines can be used to improve (...TRUNCATED) | " we aimed to demonstrate that df stem cells from impacted molars and canines can be used to improve(...TRUNCATED) |
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Dataset Card for "SumPubmed"
Original Dataset Description
- Repository: https://github.com/vgupta123/sumpubmed
- Paper: More Information Needed
Description of dataset processing
5 rows were dropped from the original dataset taken from KAGGLE as they were missing the respective 'shorter_abstract' entries.
The 'line_text' and 'filename_text' columns were left untouched while the remaining ones were processed to remove the '\n' (many repetitions of those present in the original dataset), '<dig>', '<cit>', 'BACKGROUND', 'RESULTS' and 'CONCLUSIONS' matching strings which were deemed not necessary for the purpose of summarization. Additionally, extra spaces were removed and spacing around punctuations was fixed.
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