Rabbit Secretory Component Research Articles

A Pathogen-specific Epitope Inserted into Recombinant Secretory Immunoglobulin A Is Immunogenic by the Oral Route

Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F beta strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses.

Cloning and characterization of the bovine polymeric immunoglobulin receptor-encoding cDNA

Trans-epithelial transport of polymeric immunoglobulins (pIg) into mucosal and glandular secretions is carried out by the pIg receptor (pIgR). Therefore, expression of the pIgR gene in epithelial cells of mucosal and glandular tissues is an absolute requirement for achieving mucosal immunity. We report the cloning and characterization of the bovine pIgR cDNA. Three overlapping cDNA clones with a total length of 3608 bp yielded an open reading frame encoding a 757-amino-acid (aa) transmembrane (TM) glycoprotein. Although polymorphism was found in two separate clones, Northern blot analysis showed a single pIgR mRNA (approx. 3.8 kb) to be present in the mammary gland, liver, lung, kidney and intestine of a lactating cow. There was no detectable expression of pIgR in the spleen of the same animal. Comparison of the deduced bovine pIgR aa sequence with those of rat, mouse, man and rabbit shows that this receptor is highly conserved both in aa sequence and structural organization. The degree of conservation in the TM sequence and the C-terminal cytoplasmic tail, which contains the various signals for intracellular trafficking of the receptor, is 65–73%. We also find a high degree of conservation (61–66%) in the ectoplasmic part of the receptor, known as the secretory component (SC), with an exception for that of the rabbit SC, which is much lower (47%). Among the five Ig-like domains in the SC, the N-terminal domain I, where the primary pIg-binding site is located, showed the highest (72–83%) aa sequence conservation.

Generation and assembly of secretory antibodies in plants.

Four transgenic Nicotiana tabacum plants were generated that expressed a murine monoclonal antibody kappa chain, a hybrid immunoglobulin A-G heavy chain, a murine joining chain, and a rabbit secretory component, respectively. Successive sexual crosses between these plants and filial recombinants resulted in plants that expressed all four protein chains simultaneously. These chains were assembled into a functional, high molecular weight secretory immunoglobulin that recognized the native streptococcal antigen I/II cell surface adhesion molecule. In plants, single cells are able to assemble secretory antibodies, whereas two different cell types are required in mammals. Transgenic plants may be suitable for large-scale production of recombinant secretory immunoglobulin A for passive mucosal immunotherapy. Plant cells also possess the requisite mechanisms for assembly and expression of other complex recombinant protein molecules.

Rabbit secretory components of different allotypes vary in their carbohydrate content and their sites of N-linked glycosylation.

The asparagine-linked glycosylation sites in rabbit high and low Mr secretory components (SC) have been determined for the three known allotypes, t61, t62, and t63. Purified SC polypeptides were subjected to mild periodate oxidation of terminal nonreducing sugars followed by selective reduction with [3H]sodium borohydride, SC polypeptides were further proteolytically cleaved, and the 3H-labeled peptides were isolated and characterized. Both high and low Mr SCs of the three allotypes possess a common glycosylation site at the asparagine residue position 400, whereas the second site, in the amino-terminal domain of SC, was found to be variable: the t61 and t63 allotypes contained a glycosylation site at positions 70 and 90, respectively. Moreover, although the t62 allotype was found to contain a triplet acceptor site (N-X-S) at positions 90-92, analyses showed that less than 30% of the t62 allotype peptides encompassing this region were glycosylated at residue 90. Furthermore, the amino acid sequence of the t61 SC in the region of residues 69-90 varies by 8 and 10 amino acid substitutions when compared with the t62 and t63 allotype sequences, respectively. However, neither the variation in amino acid sequence nor the variation in degree or site of glycosylation measurably affected the non-covalent binding of domain 1 to dimeric IgA.

Recent studies of the interaction of rabbit dimeric IgA with its polymeric immunoglobulin receptor

Rabbit secretory components (SC) constitute a highly heteregeneous population of glycoprotein molecules that are present in secretions as free or bound forms to polymeric immunoglobulins (Ig). Two SC families are known, one of high molecular weight (⋍ 80 Kd) composed of five (perhaps six) domains related to Ig variable domains, and one of low molecular weight (⋍ 55 Kd) An account of our most recent experimental data is reviewed in this article. We have shown: 1. 1) that both the high and low Mr SC families possess the same relative avidity for binding to dimeric IgA of the g-sublanguage; 2. 2) that the first NH 2-terminal domain of SC derived from the high and low Mr polypeptides is necessary and sufficient for efficient non-covalent binding to dimeric IgA of the g-sublanguage; 3. 3) that the low Mr SC polypeptide derives from the high Mr SC by the internal deletion of the entire second and third domains, suggesting that these domains are not involved in the binding reaction with polymeric Ig; 4. 4) that the heterogeneity of rabbit secretory components is, in large part, due to the expression of several polymorphic forms (allotypes) susceptible to be recognized by specific alloantisera; the biochemical characterization of the three known SC allotypes (t61, t62 and t63) reveals that t62 and t63 are structurally very similar to each other and markedly divergent from the t61 homologue; 5. 5) that by using non-cross-reactive alloantisera, the major immunodominant allotopes are confined within the COOH-terminal domains 3, 4 and 5 of SC; 6. 6) that the location of the residues involved in the attachment of the carbohydrate unit within domain 1 varies according to the allotype: t61 is N-linked glycosylated at position 70, whereas about 75 % of t62 molecules are devoid of sugars; the remaining 25 % of t62 molecules are glycosylated at residue position 90; these oligosaccharide chain units are linked to asparagine residues in the acceptor site consensus sequence, Asn-X-Thr/Ser; 7. 7) that the presence of the carbohydrate unit in domain 1 is not required for efficient binding of this domain to polymeric Ig: indeed, after enzymatic deglycosylation, domain 1 exhibits a relative binding avidity which is indistinguishable from that of the native glycosylated domain 1.

Structural variability of rabbit secretory components. Allotype-associated differences in the third, fourth, and fifth domains.

We have previously shown (Frutiger, S., Hughes, G. J., Hanly, W. C., Kingzette, M., and Jaton, J.-C. (1986) J. Biol. Chem. 261, 16673-16681) that limited tryptic digestion of the high Mr form of rabbit secretory component of allotypes t61, t62, and t63 generates two major fragments, the NH2-terminal domain and a 40-kDa fragment encompassing domains 3, 4, and 5. Similarly, from the low Mr form of secretory component, (SC) the NH2-terminal domain, together with a 30-kDa fragment containing domains 4 and 5, were released. These fragments were used as inhibitors in a sensitive competitive binding radioimmunoassay with noncross-reactive rabbit alloantisera to study the distribution and localization of the major allotype-specific allotopes within the SC polypeptide. The 40-kDa fragments were shown to inhibit the 125I-labeled intact SC/anti-SC allotype reaction to the extent of 90%, i.e. nearly as well as the intact homologous high Mr SC form. In contrast, the NH2-terminal fragments (domain 1) were not inhibitory. The low Mr SC of each allotype was less inhibitory on a molar basis than the homologous high Mr SC polypeptide, an observation compatible with the deletion of domains 2 and 3 in the smaller polypeptide (Deitcher, D. L., and Mostov, K. E. (1986) Mol. Cell. Biol. 6, 2712-2715; Frutiger, S., Hughes, G. J., Fonck, Ch., and Jaton, J.-C. (1987) J. Biol. Chem. 262, 1712-1715). The structural correlates of the allotypic specificities were evaluated by comparative peptide mapping of the 40-kDa fragments (allotypes t61, t62, and t63). The data suggest that the t61 allotype structure differs significantly from the t62 and t63 structures, the latter two being much more related to each other than to t61. These findings are in full agreement with the serological data. The inhibition results suggest that the major allotype-specific, noncross-reactive allotopes of SC are distributed throughout domains 3, 4, and 5, even though domain 4 appears to be more conserved than domains 3 and 5 between the allotypes t61 and t63. Seven amino acid substitutions between t61 and t63 have been detected within domains 3, 4, and 5.

High and low molecular weight rabbit secretory components. Evidence for the deletion of the second and third domains in the smaller polypeptide.

Rabbit secretory components exist in two forms which differ in apparent mass by about 25 kDa. Each of these two forms were reduced, carboxymethylated, and extensively digested with trypsin. The resulting peptides were purified by reverse-phase high performance liquid chromatography and characterized by NH2- and COOH-terminal sequence determination and/or amino acid analysis. They were aligned with the protein sequence predicted from the cDNA nucleotide sequence encoding the rabbit poly(Ig) receptor (Mostov, K. E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). All peptides belonging to the fourth and fifth domains except one (positions 488-496) were accounted for in both forms. In addition, limited tryptic proteolysis of the native low Mr secretory components produced the intact 18-kDa NH2-terminal domain (positions 1-117) and the 30-kDa fragment encompassing the fourth and fifth domains. These results suggest that the smaller polypeptide derives from the larger secretory component form by the deletion of the second and third domains.

The amino-terminal domain of rabbit secretory component is responsible for noncovalent binding to immunoglobulin A dimers.

Rabbit secretory components (SC) constitute a family of markedly heterogeneous glycoproteins which are released in the secretions as free SC or as SC bound to polymeric immunoglobulins. The aim of this work was to determine the region of the SC polypeptides which is involved in IgA binding. The high and the low Mr forms of free SC (or IgA-dissociated bound SC) and the native secretory IgA complex were subjected to limited tryptic digestion. Chemically characterized peptides ranging in apparent size from 15 to 20 kDa, depending upon the allotype, were shown to be necessary and sufficient for efficient noncovalent binding to IgA dimers (subclass g). These fragments encompass the amino-terminal first domain of SC, i.e. residues 1-126, when aligned with the predicted amino acid sequence from a cDNA clone encoding the rabbit polymeric Ig receptor (Mostov, K.E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). The high and the low Mr forms of SC exhibited the same relative affinity for IgA dimers, suggesting that the postulated internal deletion in the smaller polypeptide (Kühn, L. C., Kocher, H.-P., Hanly, W.C., Cook, L., Jaton, J.-C., and Kraehenbuhl, J.-P. (1983) J. Biol. Chem. 258, 6653-6659) does not impair the IgA dimer recognition function.

Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts.

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.

40] Biosynthesis, processing, and function of secretory component

Publisher Summary This chapter discusses the biosynthesis, processing, and function of secretory component (SC). The chapter focuses on SC from both rabbits and humans. The rabbit system has the advantage that messenger RNA (mRNA) for cell-free translation studies may be easily extracted from tissues that make SC, such as liver and lactating mammary gland. However, rabbit SC is a heterogeneous family of at least four polypeptides, each of which is made as a separate primary translation product. This complicates interpretation of data. The SC from humans, in contrast, apparently consists of one polypeptide that is made as a single primary translation product. Obtaining mRNA from tissues is problematic, because the tissue must be fresh to prevent degradation of the mRNA. There is a well-differentiated colon adenocarcinoma cell line (HT29) that makes SC and transports IgA from the basolateral to the apical surface of the cells. Pulse-chase experiments can be performed with these cells and small amounts of mRNA for in vitro translation studies can be extracted.

Interaction of rabbit secretory component with rabbit IgA dimer.

Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.

On the relationship between human and rabbit secretory components

Human and rabbit secretory components were compared in several different ways. They cross-react weakly with chicken antiserum, share about half their tryptic peptides in common, and differ in regard to size and NH 2-terminal amino acid sequences.

On the relationship between human and rabbit secretory components

On the relationship between human and rabbit secretory components

Studies on the Subunit Structure of Rabbit Secretory IgA

Abstract The nature of the intersubunit bonds in rabbit secretory IgA dimer was investigated by employing gel filtration on 2% and 4% agarose columns equilibrated with 6 M guanidine-HCl. Under these conditions, rabbit secretory IgA behaved as a dimer [(H2L2)2] which established that the bonds linking the monomer subunits were covalent in nature. Rabbit secretory component, however, dissociated from the native molecule in 6 M guanidine-HCl, which indicated that the secretory component was bound to the native dimer by non-covalent bonds. Mild reduction and alkylation with dithiothreitol (DTT) in aqueous buffer resulted in dissociation of the rabbit secretory IgA dimer molecule to monomer subunits and lower molecular weight material. Reduction with varying quantities of DTT showed that rabbit secretory IgA dimer was more resistant to reduction than human secretory IgA dimer which in turn was more resistant to reduction than a human IgA dimer myeloma protein.