Human plasma-derived polymeric IgA and IgM antibodies associate with secretory component to yield biologically active secretory-like antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

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.

Mass spectrometry of native rat amelogenins: primary transcripts, secretory isoforms, and C-terminal degradation.

Cloning technologies have established unambiguously that amelogenins always seem larger in molecular weight (Mr) by gel electrophoresis (SDS-PAGE) than by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in vivo. In this study, discrete protein fractions at 31-20 kDa (Mr(SDS)) were prepared from freeze-dried rat incisor enamel by techniques optimized for preserving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amelogenin. As expected, the molecular weights estimated by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-PAGE. Plots of Mr(SDS) vs. Mr(MS) for all fractions showed high linear correlation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform of rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major proteins forming the 25-, 24-, and 23-kDa fractions (Mr(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P169-A170, P164-L165, and F151-S152 C-terminal cleavage sites, respectively. Proteins in the 20-kDa fraction (Mr(SDS)) most closely matched by ESI-MS fragments of the R156 secretory isoform that were C-terminally-modified at the equivalent P164-L165 site.

Carbohydrate moieties in human secretory component.

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.

Expression, purification and biochemical characterization of recombinant murine secretory component: a novel tool in mucosal immunology.

Reconstitution of secretory IgA (S-IgA) by the association in vitro of secretory component (SC) and polymeric IgA (pIgA) obtained from hybridomas is a valuable tool in the study of the structure-function relationship in this particular class of antibody. Although dimeric IgA (dIgA) can be obtained and purified from hybridoma clones, SC remains tedious to isolate in sufficient amounts from colostral milk. Several murine models for the study of mucosal immunity are available, which could potentially benefit from the use of cognate IgA antibodies in various molecular forms, including dIgA and S-IgA. We report here on the establishment of two expression systems allowing the production of milligram amounts of pure recombinant murine SC (rmSC) with preserved murine pIgA-binding capability. The first system relies on the use of recombinant vaccinia virus to prompt infected HeLa cells to express the murine SC protein, whereas the second system is based on a stably transfected cell clone exhibiting murine glycosylation. The second source of rmSC will permit the study of the role of its sugar moieties in pathogen-host interactions, and the evaluation of its function in passive protection without risking adverse immune responses. The extensive biochemical characterization conducted in this study demonstrates that rmSC is a dependable and convenient alternative to the natural product, and indicates that the J chain is dispensable in the recognition of pIgA and SC in vitro, whereas it is required for proper pIgA-polymeric Ig receptor interaction in vivo.

Production of secretory component and pathogenesis of gastric cancer in Helicobacter pylori-infected stomach.

The purpose of this study was to examine the production of secretory component (SC) and immunoglobulin A (IgA) in the gastric mucosa with Helicobacter pylori infection and to investigate the influence of immunological reactions on various phases of infection (gastritis, intestinal metaplasia, gastric cancer). Production of SC and IgA was assessed by immunohistochemical staining in (1) endoscopic biopsy samples of H. pylori-eradicated cases (n = 25), and (2) surgically resected stomach tissues of H. pylori-positive gastric cancer cases, intestinal type (IGC, n = 25) and diffuse type (DGC, n = 25). Before eradication therapy, all samples showed positive staining of SC and IgA in epithelial cells, and IgA was also positive in plasma cells in the mucosal layer. H. pylori bacteria were positively stained for SC and IgA. After treatment, the degree of SC and IgA staining in epithelial cells was reduced with successful eradication; but with intestinal metaplasia, SC staining was positive regardless of the results of treatment. In nonmetaplastic mucosa, SC-positive cells were increased in the glandular neck zone to the surface mucosal layer; and the intensity of SC staining was increased in proportion to the degree of mucosal inflammation and IgA-positive cell aggregation. In intestinal metaplasia, SC was positive irrespective of the degree of inflammation. Most cancer foci also showed positive staining of SC, irrespective of histological type. Production of SC and IgA was thought to be a specific reaction against H. pylori infection, occurring from the early to the late stages and not limited to intestinal metaplasia. It was suggested that immunological reactions against H. pylori infection might generally be involved with the pathogenesis of intestinal metaplasia and both histological types of gastric cancer (IGC and DGC).

Binding of Clostridium difficile toxin A to human milk secretory component.

Toxigenic Clostridium difficile is isolated from a majority of healthy human infants. The exact mechanism of asymptomatic colonisation is unclear; however, previous studies in this laboratory have shown that components of both the immunoglobulin and non-immunoglobulin fractions of human milk bind to toxin A and prevent its interaction with hamster intestinal brush border membranes (BBMs). Secretory IgA (sIgA) is the primary immunoglobulin found in human milk. As sIgA resists digestion in the infant stomach and passes at high levels into the colon, its ability to bind toxin A was the subject of this investigation. Purified sIgA in concentrations at and below those found in human milk inhibited the binding of toxin A to purified BBM receptors. Heating sIgA to 100 degrees C for 5 min did not affect its inhibitory activity. IgM, IgG and serum IgA did not appreciably inhibit the binding of toxin A to BBM receptors. SDS-PAGE separated sIgA into three major bands: secretory component, heavy chains and light chains. Autoradiography with radiolabelled toxin A revealed that toxin A bound to the secretory component (SC) of sIgA. When the three purified subunits of sIgA were coated on to microtitration wells, SC bound significantly more toxin A than the heavy or light chains of sIgA. Purified SC also inhibited toxin binding to receptors in a dose-dependent fashion similar to sIgA. The heavy and light chains of sIgA did not inhibit toxin A receptor binding. Removing carbohydrates from sIgA and SC by enzymic digestion showed that toxin A binds much less to deglycosylated SC than to glycosylated SC. These data suggest that SC in human milk binds to toxin A and may function as a receptor analogue, protecting human infants against C. difficile-associated disease.

Simplified method for purification of colostrum to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid.

Many studies employ bronchoalveolar lavage fluid for assessment of biologically active substances secreted from the lung. However, investigators continue to search for a useful reference standard to correct for the inevitable but variable degree of dilution of this fluid. The glycoprotein, soluble secretory component of IgA, may serve as a valid reference protein. We report a simplified method for the purification of secretory component from colostrum. Soluble secretory component was isolated from human colostrum using serial centrifugation, size-exclusion fractionation and ion-exchange chromatography. Secretory component rich fractions were assayed by enzyme immunoassay. They were also evaluated for total amino acid content and distribution and sequence determination with satisfactory agreement with published results. We then demonstrated that soluble secretory component concentration in tracheal aspirate fluid did not correlate with either albumin or with total protein measured in the same samples. Therefore, we conclude that the secretory component of IgA serves as a useful reference marker because its use may avoid errors resulting from leakage of plasma proteins into epithelial lining fluid. Advantages of this method for establishing a standard for secretory component include ready availability of soluble secretory component, simplicity of the method and relative rapidity of the techniques.

Cellular processing limits the heterologous expression of secretory component in mammalian cells.

Recombinant vaccinia-virus-based expression systems are very popular for the overproduction of proteins in mammalian cell lines. Both the double virus T7/vaccinia hybrid system and the single recombinant strategy based on the p11 K late promoter were evaluated for their ability to govern expression and secretion of recombinant human secretory component (SC), a glycoprotein associated with IgA in mucosal secretions. We report here that, while the T7 promoter is transcriptionally 3.4-fold more active than the p11 K promoter, no difference in levels of secreted recombinant human SC is observed using either vaccinia system to infect CV-1 cells. High transcription, and thus translation levels, lead to saturation of early processing steps involved in protein export. Both systems exhibit transient accumulation of comparable amount of recombinant human SC in the endoplasmic reticulum and/or the cis Golgi network, as demonstrated by immunofluorescence and endoglycosidase H (EndoH) sensitivities. Exposure of infected cells to tunicamycin results in similar inhibition of recombinant human SC export, further arguing that N-linked glycosylation is necessary for proper folding and subsequent secretion. Moreover, pulse-chase experiments indicate that newly synthesized recombinant human SC is not completely processed in a mature glycoprotein and that a portion of overexpressed SC might be degraded before it can be secreted. Recombinant human SC behaves identically to native SC in terms of kinetics of secretion and IgA-binding capacity. Our results indicate that optimization of expression systems should not only rely on the design of effective vectors, but also on the identification and clearance of the cellular bottlenecks associated with maturation of the secreted proteins.

Characterization of a novel monoclonal antibody which identifies chicken secretory component.

A mouse monoclonal antibody (MAb) produced against chicken biliary IgA (bIgA) was characterized. This MAb, designated A63, reacted with purified chicken biliary IgA (bIgA) but not with serum IgA in ELISA. In Western blot analysis, it recognized an 80-kDa protein associated with bIgA. MAb A63, but not a chicken IgA-specific MAb, reacted strongly with chicken embryo allantoic fluid, a rich source of SC, in immunoblots. In immunohistostained sections of chicken intestines A63 stained intracytoplasmic vacuoles in the enterocytes consistent with goblet cells, whereas the IgA-specific MAb predominantly stained plasma cells in the lamina propria. Whereas the IgA-specific MAb only reacted with the homologous IgA, MAb A63 reacted with bile samples of chicken, turkey, and quail but not of duck or pheasant. These data collectively indicated that MAb A63 recognized an avian secretory component (sc).

Secretory component, the receptor for polymeric immunoglobulin, has nothing to do with beta-galactosyltransferase in human milk.

Secretory component (SC) in external secretions is a soluble form of the polymeric immunoglobulin-receptor that is expressed on the cell membrane of mucosal epithelial cells. beta-(1-4)galactosyl transferase (beta-GT) is an enzyme that transfers galactose to non-reducing N-acetylglucosamine residues on various glycoproteins and is present in a soluble form in secretions as well as in a membrane-bound form. beta-GT is considered to have affinity for glycoproteins, including IgA in secretion. It has been claimed that these two proteins are related to or identical with each other. In the present study, we defined that the SC and the beta-GT are each independent molecules by the following facts; (1) both molecules are separable either by antibody-affinity chromatography, conventional ion-exchange or molecular exclusion chromatography, (2) conventionally purified SC from human milk contained neither enzymatic activity or antigenic determinants of the beta-GT, (3) recombinant beta-GT does not show reactivity with antibodies to SC, and (4) the SC showed no reactivity with antibody to beta-GT.

Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli.

The non-immunoglobulin component of human milk responsible for the inhibition of Escherichia coli cell adhesion (haemagglutination) mediated by colonisation factor antigen 1 (CFA1) was determined by chromatographic fractionation of human whey proteins with Sephadex G-200, DEAE cellulose and heparin-sepharose. Pure free secretory component (fSC) and pure lactoferrin (Lf) were isolated and both compounds inhibited the haemagglutination induced by E. coli CFA1+. The lowest concentrations of purified fSC and Lf able to inhibit the haemagglutination induced by E. coli strain TR50/3 CFA1+ were 0.06 mg/ml and 0.1 mg/ml respectively. Commercially available lactoferrin from human milk and transferrin from human serum, which has a great structural analogy to lactoferrin, also inhibited the haemagglutination. The lowest concentrations of the commercial lactoferrin and transferrin able to inhibit the haemagglutination induced by E. coli TR50/3 CFA1+ were 0.03 mg/ml and 0.4 mg/ml, respectively. These results indicate that fSC and Lf may be important non-specific defence factors against enterotoxigenic E. coli infections.

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells.

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.

Expression and purification of biologically active domain I of the human polymeric immunoglobulin receptor.

Previous studies using proteolytic fragments and synthetic peptides have indicated that domain I of human polymeric immunoglobulin receptor (PIgR) is necessary for ligand binding. The expression in E. coli, and subsequent IgM-affinity purification of domain I of human PIgR is described. The recombinant domain I protein (rDI) was similar in structure to native SC domain I in that it bound specifically to MAb 6G11, an antibody which recognizes a critical portion of the PIg binding site in domain I. The biological activity of rDI was indicated by high affinity binding to PIgA (Kd = 1.6 x 10(-7) M) and IgM (Kd = 5.1 x 10(-7) M). Domain I of human SC is therefore sufficient for binding to PIg.

Immunoassays of secretory IgA and secretory component.

1. A sandwich-type enzyme-linked immunosorbent assay (ELISA) is described for quantitation of secretory IgA (sIgA) in human serum, as well as an ELISA and a radioimmunoassay (RIA) for measurement of secretory component (SC) in human serum. Samples were reduced and alkylated prior to the measurement of SC. 2. Healthy individuals (N = 53) presented low levels of SC (median, 0.9 mg/l). The protein levels were significantly elevated when compared with the controls, in sera of women during the second (N = 31; median, 1.5 mg/l) and third (N = 35; median, 4.2 mg/l) trimesters of pregnancy and in sera of patients with alcoholic cirrhosis (N = 38; median, 2.4 mg/l) and acute viral hepatitis (N = 25; median, 2.4 mg/l). SC levels of women in the first trimester of pregnancy (N = 24; median, 0.5 mg/l) did not differ from the controls. 3. sIgA levels were also significantly elevated when sera of women in the third trimester of pregnancy (N = 41; median, 25.4 mg/l) and sera of patients with alcoholic cirrhosis (N = 32; median, 75.0 mg/l) or acute viral hepatitis (N = 38; median, 28.5 mg/l) were compared with controls (N = 49; median, 9.0 mg/l). Women in the first (N = 25; median, 7.7 mg/l) and second (N = 29; median, 10.2 mg/l) trimester of pregnancy did not present levels statistically different from the controls. 4. The results obtained for SC by RIA and ELISA were positively correlated (rs = 0.88; P less than 0.001). sIgA levels determined by ELISA were also positively correlated with the results of RIA-SC (rs = 0.77; P less than 0.001) or ELISA-SC (rs = 0.79; P less than 0.001). 5. The assays described are specific, relatively simple to perform, and can be useful for the study of the secretory immune system.

Immunoassays of secretory IgA and secretory component

1. A sandwich-type enzyme-linked immunosorbent (ELISA) is described for quantitation of secretory IgA (sIgA) in human serum, as well as an ELISA and a radioimmunoassay (RIA) for measurement of secretory component (SC) is human serum. Samples were reduced and alkylated prior to the measurement of SC. 2. Healthy individuals (N = 53) presented low levels of SC (median, 0.9 mg/l). The protein levels were significantly elevated when compared with the controls, in sera of women during the second (N = 31; median, 1.5 mg/l) and third (N = 35; median, 2.4 mg/l) and acute viral hepatitis (N = 25; median 2.4 mg/l). SC levels of women in the first trimester of pregnancy (N = 24; median, 0.5 mg/l) did not differ from the controls. 3. sIgA levels were also significantly elevated when sera of women in the third trimester of pregnancy (N = 41; median, 25.4 mg/l) and sera of patients with alcoholic cirrhosis (N = 32; median, 75.0 mg/l) or acute viral hepatitis (N = 38; median, 28.5 mg/l) were compared with controls (N = 49; median, 9.0 mg/l). women in the first (N = 25; median, 7.7 mg/l) and second (N = 29; median, 10.2 mg/l trimester of pregnancy did not present levels statistically different from the controls. 4. The results obtained for SC by RIA and ELISA were positively correlated (rs = 0.88; P < 0.001). sIgA levels determined by ELISA were also positively correlated with the results of RIA-SC(rs = 0.77;P<0.001) or ELISA-SC (rs = 0.79; P < 0.001). 5. The assays described are specific, relatively simple to perform, and can be useful for the study of the secretory system

Molecular characteristics of IgA in infant saliva.

Saliva was collected from 57 infants aged 6 weeks to 2.5 years and the molecular form of IgA was studied by centrifugation on sucrose density gradients. Two distinct populations were identified. Seventy-two per cent of the children had secretory IgA in their salivary secretions, while 28% had a molecular form corresponding to monomeric IgA. No samples with concurrent monomeric and secretory forms were detected. Monomeric IgA was not detected in any infant over 12 months of age. Secretory component was detected in all samples but was not associated with monomeric IgA. Forty-seven per cent of the samples contained IgA fragments of approximately 40,600 molecular weight. The presence of fragments dominated in the group of children with monomeric IgA. The presence of monomeric IgA in infant saliva did not result from degradation due to storage or proteolysis. The study demonstrated an apparent maturation sequence in the molecular form of IgA present in the salivary secretions of infants.