Secretory Component Research Articles

The efficacy of combining feG and galantide in mild caerulein-induced acute pancreatitis in mice

We have previously shown that galantide ameliorates mild acute pancreatitis (AP), and the salivary tripeptide analogue, feG, ameliorates severe AP in mice. In this study, we compared the efficacy of combining galantide and feG with that of the individual agents in treating mild AP induced in mice with 7-hourly caerulein injections. Galantide was co-administered with each caerulein injection commencing with the first injection. feG was co-administered with the first injection of caerulein as a single intraperitoneal injection. Combination of the agents was also administered. Control animals received galantide, feG, or saline alone. Pancreata were harvested for histological examination and estimation of myeloperoxidase (MPO) activity. Plasma enzyme activities were measured. Galantide significantly reduced AP-induced hyperenzymemia by 41–49%. The combination of galantide and feG significantly reduced AP-induced hyperenzymemia by 39–40%, whereas feG alone was without effect. Plasma enzyme activity in the control groups was comparable with pre-treatment activity. Galantide, feG, and their combination significantly reduced MPO activity by 83, 44 and 74% respectively, and % abnormal acinar cells by 32, 29 and 36% respectively. This study demonstrates for the first time the beneficial effect of feG in mild caerulein-induced AP. Moreover the data indicate that the hyperenzymemia in mild caerulein-induced AP at 12 h possibly reflect a larger secretory component as compared to enzyme release due to neutrophil-mediated acinar cell damage. The effects of the treatment with both peptides indicate a possible role for galantide in modulating neutrophil chemotaxis/activation and supports the hypothesis that galantide may influence neurogenic inflammation in AP.

GlycoSpectrumScan: Fishing Glycopeptides from MS Spectra of Protease Digests of Human Colostrum sIgA

With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked, <or=5 O-linked sites), IgA2 (4 N-linked sites) and J-chain (1 N-linked site) is described. GlycoSpectrumScan is freely available at www.glycospectrumscan.org .

Progress in understanding the pathogenesis of IgA nephropathy: new perspectives for the near future?

Progress in understanding the pathogenesis of IgA nephropathy has shown that probably there is no a single IgA nephropathy with the same pathogenic mechanism, clinical course and response to therapy. The evidence currently available suggests the existence of at least two possible mechanisms of IgA deposition in the renal mesangium. In a small percentage of patients, mesangial deposition of IgA1 colocalizes with secretory component, indicating that the deposited IgA1 in glomeruli originates completely or partly in the mucose-associated lymphoid tissue. This deposition pattern has been associated with activation of complement by the lectin pathway and has been associated with a worse prognosis, although this last statement needs to be confirmed in long-term studies. The mechanisms responsible for secretory IgA deposition are not known. In the majority of patients with IgA nephropathy secretory component is not detectable in the mesangium. In these cases, the presence of elevated circulating levels of galactose-deficient IgA, produced by bone marrow plasma cells would be a predisposing factor but not sufficient to induce nephropathy. To produce kidney disease, galactose-deficient IgA1 must be deposited in the renal mesangium, and once there, either by interaction with specific receptors (CD71?), by direct activation of complement or by being the target of an IgG autoimmune response anti-IgA, induce activation, proliferation and increased mesangial matrix synthesis and eventually cell injury. In parallel, galactose-deficient IgA, through interaction with the RR Fc alpha/gamma, may activate circulating lymphocytes and monocytes and enhance their response to chemoattractants produced by the mesangial cell, causing, thus, the inflammatory infiltrate to initiate and maintain the interstitial injury. In the next few years, advances recently added to the knowledge of the pathogenesis of nephropathy IgA1 could provide new variables that allow walking in the direction of having a classification of patients based not only in clinical and morphological criteria but also having a greater pathogenic basis.

Mucosal Immunity: Induction, Dissemination, and Effector Functions

Prevention of infections by vaccination remains a compelling goal to improve public health. Most infections involve the mucosae, but the development of vaccines against many of these pathogens has yet to be successful. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion - a term coined for non-inflammatory antibody shielding of internal body surfaces - mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA) - produced by local plasma cells stimulated by antigens that target the mucosae. SIgA was early shown to be complexed with an epithelial glycoprotein - the secretory component (SC). In 1974, a common SC-dependent transport of pIgA and pentameric IgM was proposed. From the basolateral surface, pIg-SC complexes are taken up by endocytosis and finally extruded into the lumen. Membrane SC is now referred to as polymeric Ig receptor (pIgR). In 1980, it was shown to be synthesized as a larger transmembrane protein - first cloned from rabbit and then from human. Mice deficient for pIgR showed that this is the only receptor responsible for epithelial transport of IgA and IgM. In the gut, induction of B cells occurs in gut-associated lymphoid tissue, particularly the Peyer's patches, but also in mesenteric lymph nodes. Plasma cell differentiation is accomplished in the lamina propria to which the memory/effector cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue - but by different homing receptors. Such compartmentalization is a challenge for development of mucosal vaccines.

Relationship between Epstein-Barr virus infection and nasopharyngeal carcinoma pathogenesis

Nasopharyngeal carcinoma (NPC) is one of the common cancers among Chinese living in South China, Taiwan, Singapore, and several other countries or regions in distinct areas. The etiological factors have not been clearly identified yet. So far, no major gene related with hereditary factor has been identified in NPC carcinogenesis; however, some environmental factors, such as consumption of salted fish and long-term exposure to sulfuric-acid vapor, have been tentatively linked to NPC induction, while research has proposed that there is a close association between Epstein-Barr virus (EBV) and NPC pathogenesis. To investigate the relationship between NPC and EBV, we have established ten NPC cell lines. After extensive investigation, we conclude that EBV may establish an infection only in nasopharyngeal neoplastic cells, not in metaplastic epithelial cells, through the IgA receptor (secretory component protein)-mediated endocytosis. Our observations indicate that EBV plays an important role in enhancement of NPC progression, but is involved in neither the initiation nor the promotion of NPC pathogenesis.

Interaction of Copper and Human Salivary Proteins

Interaction of taste molecules with saliva is the first step in the flavor perception process. Saliva is assumed to influence copper-induced sensation by controlling the copper solubility or causing astringency via binding of proteins with copper. This study was performed to identify the nature of copper-protein interactions in relation to the sensory perception of copper. Saliva was treated with CuSO4 x 5H2O at levels of 0, 2.5, 5, 10, 20, or 40 mg/L, and changes in salivary proteins were analyzed using high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein peaks that showed changes in HPLC were characterized with SDS-PAGE. HPLC analysis revealed that copper treatment up to 40 mg/L decreased several proteins, including the dominant peak, by 70%. This peak was composed of alpha-amylase, a secretory component, and basic proline-rich proteins. SDS-PAGE results showed that salivary proteins of molecular weight 29 kDa and 33 kDa precipitated when copper was added at concentrations > or =10 mg/L. This study provides biochemical information for understanding perception mechanisms of copper sensation.

Influenza influences ion channels

Influenza influences ion channels

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

The Relative Roles of Bacteria and Host Inflammatory Cells in SIgA Degradation

Secretory Immunoglobulin (Ig) A, the most important Ig for lung defense, is highly dependent on its molecular structure for its immune activity. Proteolytic cleavage of SIgA may occur in the airways and render the SIgA molecule inactive. Previous studies have attributed IgA cleavage to neutrophils (polymorphonucleur cells [PMNs]) and other host immunoinflammatory cells in the airways or bacterial pathogens. The resultant IgA degradation leads to airway inflammation and subsequent pneumonia. The aim of this study was to discern the relative roles of host inflammatory cells and bacteria in SIgA cleavage in vitro. SIgA was cocultured with PMNs, PMNs activated with fMLP, monocytes, monocytes pretreated with lipopolysaccharide (LPS), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas, or Acinetobacter alone or with monocyte culture supernatants and PMNs. SIgA cleavage resulted in two fractions, a <75 kDa fraction (secretory component fraction) and an intact SIgA fraction by size exclusion ultrafiltration. This was quantified by ELISA and confirmed by SDS-PAGE. Neither naïve nor activated PMNs showed any cleavage of SIgA. Unstimulated monocytes or LPS stimulated monocytes also failed to demonstrate fragmentation of SIgA. Only modest SIgA cleavage was noted when both PMNs and monocytes were stimulated before coculture with SIgA but marked SIgA proteolysis was noted with the addition of Pseudomonas or Acinetobacter but not MRSA. Bacterial pathogens, but not activated immunoinflammatory cells, were responsible for SIgA degradation in this study. This was evident only with Pseudomonas and Acinetobacter. This may be a virulence factor for pneumonia with these pathogens in vivo.

Immunoglobulins in human seminal plasma.

Single radial immunodiffusion has been used to evaluate immunoglobulins and secretory piece (SP) in human seminal plasma. Samples were collected from 90 healthy volunteers, 202 subjects submitted to vasectomy for contraceptive purposes, tested at various intervals after surgery, and 725 patients grouped according to selected andrological disorders. Results may be summarized as follows. In normal subjects IgG and IgA were constantly present (mean values +/- SD: 8.14 +/- 2.82 mg/dl; 1.91 +/- 1.03 mg/dl, respectively) while IgM were detected in trace amounts (from 0.7 to 3.3 mg/dl) in 10% of subjects, and negligible or absent in the remaining subjects. In vasectomized subjects IgG and IgA showed a significant increase only in the first 3 months after vasectomy, probably due to surgery. In andrological patients Ig showed an increase in cases with antisperm antibodies (A b) and in those with infections of genital tract and positive semen culture. On the basis of these findings and the secretory piece assay data the importance appears to be stressed of the local immunocompetent system at least in some andrological diseases.

Structural requirements for the interaction of human IgM and IgA with the human Fcα/μ receptor

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcalpha/mu receptor (hFcalpha/muR). Ligand polymerization status was crucial for the interaction, because hFcalpha/muR binding did not occur with monomeric Ab of either class. hFcalpha/muR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcalpha/muR binding. IgM binding required contributions from both Cmu3 and Cmu4 Fc domains, whereas for dIgA, an exposed loop in the Calpha3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcalphaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFcalpha/muR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFcalpha/muR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFcalpha/muR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.

Airway Concentrations of Angiopoietin-1 and Endostatin in Ventilated Extremely Premature Infants Are Decreased After Funisitis and Unbalanced With Bronchopulmonary Dysplasia/Death

A systemic inflammatory response of the fetus, reflected by histologic funisitis, is a risk factor for bronchopulmonary dysplasia (BPD). Impaired pulmonary angiogenesis accompanied by simplification and rarification of alveoli is a histologic hallmark of BPD. Angiopoietin-1 mediates vascular development, maturation, and stabilization. Endostatin mainly acts as an angiostatic factor. We hypothesized that funisitis was associated with changes of endostatin and angiopoietin-1 concentrations in the airways and that an imbalance between the factors might be associated with BPD or death. We measured concentrations of angiopoietin-1 and endostatin by enzyme-linked immunosorbent assay in tracheobronchial aspirate fluid samples of 42 ventilated preterm infants during postnatal days 1 through 15. The secretory component for IgA served as reference protein. A standardized histologic examination was used to distinguish three groups: chorioamnionitis, funisitis, and controls without inflammation. Concentrations of the mediators steadily decreased. Funisitis was associated with lower concentrations of both proteins, which might impair their physiologic activities in pulmonary angiogenesis. An increase of the ratio angiopoietin-1/endostatin until day 7 of life indicated a shift of the mediators potentially favoring angiogenesis. However, infants, who developed BPD or died, had a decreased ratio on days 1, 3, and 15, suggesting an imbalance toward inhibition of pulmonary angiogenesis.

Identification of glycoprotein receptors within the human salivary proteome for the lectin‐like BabA and SabA adhesins of Helicobacter pylori by fluorescence‐based 2‐D bacterial overlay

Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.

Functional and structural characterisation of human colostrum free secretory component

Secretory component (SC) in association with polymeric IgA (pIgA) forms secretory IgA (SIgA), the major antibody active at mucosal surfaces. SC also exists in a free form in secretions, with innate neutralizing properties against important pathogens. IgA-bound SC and free secretory component (FSC) are both produced by proteolytic cleavage of the polymeric Ig receptor whose function is to transport IgA and IgM across mucosal epithelia. Although the proteases have not been characterised and the site(s) of cleavage of the polymeric Ig receptor has been debated, it has been assumed that bound and free SC are produced by cleavage at the same site. Here we show by SDS-PAGE analyses that FSC is slightly smaller than SIgA1- or SIgA2-bound SC when purified simultaneously. The FSC preparation was functionally active, shown by binding to dimeric and polymeric IgA, and by its ability to trigger a respiratory burst by binding to ‘SC receptors’ on eosinophils. We also show that FSC from different human secretions have different molecular sizes. The solution structure of FSC from colostrum was studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of 4.25S is close to that for recombinant FSC. The X-ray scattering curve showed that FSC adopts a compact structure in solution which corresponds well to the J-shaped domain arrangement determined previously for recombinant FSC which terminates at residue Arg585. The smaller sizes of the FSC forms are attributable to variable cleavages of the C-terminal linker region, and may result from the absence of dimeric IgA. The FSC modelling accounts for the lack of effect of the C-terminal linker on the known functions of FSC.

The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses

Secretory IgA (SIgA) is the most prevalent human antibody and is central to mucosal immunity. It exists as two subclasses, SIgA1 and SIgA2, where SIgA2 has a shorter hinge joining the Fab and Fc regions. Both forms of SIgA are predominantly dimeric and contain an additional protein called the secretory component (SC) that is attached during the secretory process and is believed to protect SIgA in harsh mucosal conditions. Here we locate the five SC domains relative to dimeric IgA2 within SIgA2 using constrained scattering modeling. The x-ray and sedimentation parameters showed that SIgA2 has an extended solution structure. The constrained modeling of SIgA2 was initiated using two IgA2 monomers that were positioned according to our best fit solution structure for dimeric IgA1. SC was best located along the convex edge of the Fc-Fc region. The best fit models showed that SIgA2 is significantly nonplanar in its structure, in distinction to our previous near planar SIgA1 structure. Both the shorter IgA2 hinges and the presence of SC appear to displace the four Fab regions out of the Fc plane in SIgA2. This may explain the noncovalent binding of SC in some SIgA2 molecules. This nonplanar structure is predicted to result in specific immune properties for SIgA2 and SIgA1. It may explain differences observed between the SIgA1 and SIgA2 subclasses in terms of their interactions with antigens, susceptibility to proteases, effects on receptors, and distribution in different tissues. The different structures account for the prevalence of both forms in mucosal secretions.

Intranasal administration of peptide antigens of HIV with mucosal adjuvant CpG ODN coentrapped in microparticles enhances the mucosal and systemic immune responses

The mucosal immune system acts as a first line of defense against infection caused by luminal pathogens. Because HIV is transmitted primarily via mucosal associated tissues, particularly with sexual transmission, understanding antiviral immunity present at these sites is important. As most of the peptide antigens show poor immunogenicity when immunized alone but after incorporating the same peptide antigens along with adjuvant CpG ODN in microparticles has shown enhanced immunogenicity in the murine model. In the present study we have investigated the immunomodulatory effects of two adjuvants, CpG 1826 and CpG 2006 (Class B, Also known as type K) to the four peptide antigens of HIV such as envelope glycoproteins gp41 Leucine Zipper, gp41 fusion domain and gp120-C2 as well as regulatory protein (Nef) in microparticles, exploring nasal route with single immunization schedule. Peptide (s) alone in the microparticles elicited low peptide specific IgG and IgA peak titres in the sera, whereas the inclusion of CpG ODN with peptides in microparticles significantly enhanced peptide specific IgG and IgA peak titres and such responses were sustained for longer durations. Similarly higher SIgA response was achieved in the mucosal washes with CpG encapsulated in microparticles. Such presence of SIgA in washes was further correlated with the presence of secretory component (SC) in the respective washes. Both adjuvants induced excellent peptide specific IgG and IgA immune responses. Thus the overall study highlighted the importance of CpG ODNs as a mucosal adjuvant for weaker peptide antigens and thus can explore for developing peptide based vaccine against HIV.

Location of secretory component on the Fc edge of dimeric IgA1 reveals insight into the role of secretory IgA1 in mucosal immunity

Secretory immunoglobulin A (SIgA) is the most prevalent antibody in the human body and a first line of defense in mucosal immunity. We located secretory component (SC) relative to dimeric IgA1 (dIgA1) within the SIgA1 structure using the constrained modeling of solution scattering and analytical ultracentrifugation data. The extended solution structure of dIgA1 is largely preserved within SIgA1. From conformational searches of SC locations, the best-fit SC models within SIgA1 show that SC is extended along the outermost convex edge of the Fc dimer in dIgA1. The topology of our SIgA1 structure reveals that it is able to bind to one FcαRI receptor molecule. SC binding to the Fc dimer confers protection to SIgA1 by the masking of proteolytically susceptible surface sites from bacterial proteases in the harsh environment of the mucosa. The models support a “zipper-like” unfolding of SC upon dIgA1 in the formation and transportation of SIgA1 into the mucosa.

Biological inferences from IgM binding characteristics of recombinant human secretory component mutants

The polymeric immunoglobulin receptor (pIgR) or membrane secretory component (SC) selectively transports polymeric IgA and IgM across secretory epithelial cells to mucosal surfaces. The ligand binding ectodomain consists of five homologous Ig-like domains with domain I being an absolute requirement for binding. The role of DII to V in IgM binding remains unknown. Here, using in vitro refolded non-glycosylated recombinant domain deletion mutants of human SC, we show by biological and biophysical binding assays that DII to V are required for high affinity binding to IgM. Competitive binding analysis, by whole cell ELISA, showed that DII–V significantly increase the affinity of recombinant SC for IgM ( K i = 2.42 nM) as opposed to recombinant DI only ( K i = 44.8 nM). Lastly, we provide qualitative data highlighting the complexity of measuring the IgM/SC interaction using surface plasmon resonance spectroscopy.

Characterization of the Lassa virus GP1 ectodomain shedding: implications for improved diagnostic platforms.

BackgroundThere is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, detection of early markers of Lassa virus (LASV) infection may improve diagnosis and ultimately successful treatment with antivirals. Characterization of LASV GP1 ectodomain shedding is an important step toward developing sensitive diagnostics to detect circulating levels of this viral glycoprotein in infected patient sera.ResultsSecretion of GP1 from mammalian cells expressing a native LASV GPC gene was not mediated by proteolytic cleavage, as determined by treatment with a panel of matrix metalloprotease (MMP) inhibitors. The shedding of GP1 was also not the result of over-expression of GPC under the control of a strong intron-A containing CMV promoter, as the soluble component could be immunoprecipitated from supernatants of cells expressing low levels of GPC under the control of an intronless promoter. Cells transfected with GPC retained surface membrane-associated expression of GP1 as determined by immunofluorescence assay, in addition to secreting the glycoprotein.Secreted GP1 derived from GPC expression has a higher content of high mannose N-linked glycosylation than sGP1 expressed independently from the GP2 portion of the protein. Neither GP1 isoform contains sialylated N-glycans, O-linked carbohydrate chains, or galactose-β(1-4)-N-acetylglucosamine commonly present in complex and hybrid N-glycan structures.ConclusionThese results demonstrate the non-proteolytic secretory nature of GP1 shedding during expression of the arenaviral glycoprotein complex. This phenomenon parallels shedding of a secretory glycoprotein component in filovirus replication. The glycosylation pattern of soluble GP1 resulting from expression of GPC was different from that of a soluble GP1 construct (sGP1-RRAA-FLAG), highlighting the intricately orchestrated post translational processing of the LASV glycoprotein complex.

Clinical research progress in secretory component structure and function

Clinical research progress in secretory component structure and function