Purification and Characterization of Human Serum and Secretory IgA1 and IgA2 Using Jacalin.

Human immunoglobulin A (IgA) is found in serum and secretions in several different forms (1), Serum IgA is produced by the bone marrow and is pre-dominately monomeric (M ( r )= 160 kDa). Most of the IgA in secretions (sIgA) is polymeric, predominantly dimeric, comprising two monomer subunits linked together by intersubunit disulfide bonds and a cysteine-rich polypeptide termed J-chain (M ( r )= 16 kDa). Polymeric IgA in secretions, but not in serum, contains an additional heavily glycosylated protein called secretory component (SC) (M ( r )= 70 kDa).

Novel polymer-grafted starch microparticles for mucosal delivery of vaccines.

Recent studies have demonstrated that systemic and mucosal administration of soluble antigens in biodegradable microparticles can potentiate antigen-specific humoral and cellular immune responses. However, current microparticle formulations are not adequate for all vaccine antigens, necessitating the further development of microparticle carrier systems. In this study, we developed a novel microparticle fabrication technique in which human serum albumin (HSA) was entrapped in starch microparticles grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS), a biocompatible silicone polymer. The immunogenicity of HSA was preserved during the microparticle fabrication process. Following intraperitoneal immunization of mice, TS-PDMS-grafted microparticles (MP) dramatically enhanced serum IgG responses compared with ungrafted MP and soluble HSA alone (P < 0.001). When delivered orally, both TS-PDMS-grafted and ungrafted microparticles elicited HSA-specific IgA responses in gut secretions, in contrast to orally administered soluble antigen. Indeed, TS-PDMS-grafted microparticles stimulated significantly stronger serum IgG (P < 0.005) and IgA (P < 0.001) responses compared with those elicited by ungrafted microparticles. These findings indicate that TS-PDMS-grafted starch microparticles have potential as systemic and mucosal vaccine delivery vehicles.

The cleavage of immunoglobulin G in vitro and in vivo by a proteinase secreted by the urinary tract pathogen Proteus mirabilis.

Eighteen different strains of Proteus mirabilis were all shown to produce an EDTA-sensitive proteinase of c. 50 kDa that cleaved the heavy chain, but not the light chain, of IgG. Digestion of pure IgG with small amounts of pure P. mirabilis proteinase generated Fabc'2 and Fab'2 fragments; greater amounts generated Fab and Fc fragments that were comparable in size to those generated by pepsin and papain, respectively. Incubation of neutrophils with IgG digested with P. mirabilis proteinase or papain resulted in a marked decrease in the respiratory burst activity of the neutrophils that coincided with cleavage of the IgG into Fab and Fc fragments. Analysis of urine from patients with P. mirabilis urinary tract infection revealed in many the presence of Fab and Fc fragments of IgG indistinguishable in size from those generated by P. mirabilis proteinase. These results indicate that, in P. mirabilis urinary tract infections, the proteinase is secreted and cleaves IgG to fragments that have defective immune effector functions, thereby limiting the effectiveness of the immune response.

Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients.

The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The other Proteus proteinases had similar patterns but slightly different mobilities. In each case all proteinase activity in culture supernatants was demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be associated with only the triple-band complex; all three bands were proteolytically active. The P. mirabilis proteinase was resistant to inhibitors of both serine and thiol proteinases but strongly inhibited by metal chelators, although it was not affected by phosphoramidon, an inhibitor of the thermolysin group of bacterial metalloproteinases. Active proteinase was detected in urine samples from P. mirabilis-infected patients; this is consistent with our detection of immunoglobulin A fragments of a size suggestive of P. mirabilis proteinase activity.

The production and activity in vivo of Proteus mirabilis IgA protease in infections of the urinary tract.

Immunoblotting of urine from 21 patients of both sexes and of wide age range who had a Proteus mirabilis urinary tract infection (UTI) showed that 14 (64%) specimens contained immunoglobulin A (IgA). In nine (64%) of these the IgA heavy chain had been degraded to fragments of a size identical to those formed when purified IgA was degraded by pure P. mirabilis protease. Urine from patients with clinical evidence of upper UTI contained fragmented IgA and in some of these urine samples P. mirabilis protease activity was detectable. Urine infected with a non-proteolytic strain contained only intact IgA. It is concluded that P. mirabilis IgA protease is produced and is active during infections of the urinary tract.

Purification and characterization of human immunoglobulin IgA1 and IgA2 isotypes from serum.

A method is described for the simultaneous purification of IgA1 and IgA2 from human serum. Ammonium sulphate precipitation, gel filtration and ion-exchange chromatography on DEAE-Sephacel yielded a partially purified IgA preparation which was separated quantitatively into IgA1 and IgA2 by affinity chromatography on jacalin-Sepharose. The IgA1 which bound to the jacalin was eluted with 0.8 M D-galactose. The IgA1 preparation was apparently homogeneous by SDS-PAGE but contained a trace of C1-inhibitor and a second protein detected by immunoelectrophoresis. The IgA2 which did not bind to the jacalin was purified to apparent homogeneity by chromatography on columns of Protein G-Sepharose, Fastflow-S Sepharose and Superose 6. Typical yields were 95% and 58% for IgA1 and IgA2 respectively or 253 mg and 24 mg per 100 ml serum. The IgA1 and IgA2 were characterised by their reactivity with isotype specific monoclonal antibodies and sensitivity to bacterial proteinases. The IgA2 preparation apparently contained both allotypes, IgA2m(1) and IgA2m(2).

A proteolytic enzyme secreted by Proteus mirabilis degrades immunoglobulins of the immunoglobulin A1 (IgA1), IgA2, and IgG isotypes.

Proteus mirabilis strains associated with human urinary tract infections have previously been shown to secrete an extracellular metalloproteinase which cleaves serum immunoglobulin A (IgA). The enzyme has now been purified to apparent homogeneity from culture supernatants of P. mirabilis 64676. The protease activity is associated with a 50-kilodalton (kDa) protein. Unlike that of the classic IgA1 proteases, the substrate specificity of the P. mirabilis protease has been found to extend to both sublcasses of IgA, IgG, and the nonimmunoglobulin substrates, secretory component and casein. Cleavage of IgA1 and IgA2 by the P. mirabilis protease yielded fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis whose sizes were consistent with a cleavage site outside the hinge region. Both secretory IgA1 and IgA2 were also cleaved by P. mirabilis protease, although the secretory IgA2 molecule was less readily cleaved than secretory IgA1. Free and IgA-bound secretory components were degraded to some extent by P. mirabilis protease. Cleavage of IgG, however, occurred at the hinge region as a two-stage process. The first stage was pepsinlike and generated an F(ab')2 fragment of 120 kDa and a small pFc fragment detected on nonreduced polyacrylamide gels. In the second stage, the F(ab')2 product was cleaved to yield papainlike Fab and Fc fragments, visualized as a diffuse band of 40 to 50 kDa.

Evidence for occurrence of passively adsorbed I antigen activity on a cultured strain of Mycoplasma pneumoniae.

The aim of this study was to investigate whether I antigen occurs in association with Mycoplasma pneumoniae in a form that may be immunogenic during natural infection or experimental immunization. I antigen activity was detected by radioimmunoassay in suspensions of M. pneumoniae MY11965 and in the soluble phase of mycoplasma lysates prepared with Triton X-100. There was evidence for the occurrence of I antigen in at least two macromolecular forms. The first form partitioned in the lipid phase following chloroform-methanol extraction and chromatographed on thin-layer chromatograms as a ceramide decasaccharide. The second form was associated with the residue after lipid extraction and was solubilized by treatment with sodium dodecyl sulfate or pepsin; this component was tentatively designated a glycoprotein or polysaccharide and was not investigated further. In a lipid extract from mycoplasmas that had been surface labeled by the galactose oxidase-NaB3H4 method, two 3H-labeled glycolipids were detected as minor components which chromatographed on thin-layer chromatograms in the region of an authentic I-active ceramide decasaccharide. However, no significant radioactivity was incorporated into glycolipids after metabolic labeling with [3H]glucosamine. These observations suggested that the mycoplasmas contained surface-associated glycolipids with I antigen activity that were of exogenous origin. This was supported by the observations that horse, rabbit, and fetal calf sera contained I antigen activity and that the I antigen activity in M. pneumoniae cultures reflected the levels found in the sera included in the culture media. From rabbit serum, which expressed the highest antigen activity, an I-active glycolipid was isolated that chromatographed as a ceramide decasaccharide. I-active substances passively adsorbed onto M. pneumoniae are potentially immunogenic. However, we consider these unlikely to be the main stimulus for autoantibody production in natural infection, since the autoantibodies elicited are restricted to the I carbohydrate antigen and there is a lack of antibodies to other glycolipids that may be adsorbed from serous and cellular components of the host tissues. In our view, the more likely stimulus is the specific complex formed between the mycoplasma and the sialo-oligosaccharide receptors of the Ii antigen type, as suggested previously.

Interaction of Mycoplasma pneumoniae with erythrocyte glycolipids of I and i antigen types.

The role of sialoglycolipids (gangliosides) as receptors for the human pathogen Mycoplasma pneumoniae was investigated by using purified gangliosides of known carbohydrate structures as inhibitors of the binding of 51Cr-labeled erythrocytes to sheet cultures of M. pneumoniae. We found that sialoglycolipids with long carbohydrate backbones of the poly-N-acetyllactosamine type were more potent inhibitors of M. pneumoniae binding than those with short carbohydrate chains. This is in accord with earlier inhibition data for glycoproteins and oligosaccharides. Thus, the inhibitory activity of a fraction of bovine erythrocyte gangliosides containing long backbone structures of I antigen type was approximately 200 times greater than that of the short chain gangliosides GM3 and GT1b. The binding of M. pneumoniae to erythrocytes of I and i antigen types was found to be comparable, indicating that M. pneumoniae in its adhesive specificity may not distinguish between the branched carbohydrate backbones of I type and the linear structures of i type. Thus, the production of autoantibodies to the backbone structures of I type rather than i type after infection with this agent may simply reflect a greater abundance of branched carbohydrate receptors of I type on the surface of host cells with which the mycoplasma forms immunogenic complexes.

Cryptic I antigen activity and Mycoplasma pneumoniae-receptor activity associated with sialoglycoprotein GP-2 of bovine erythrocyte membranes.

The 250-kDa sialoglycoprotein of bovine erythrocyte membranes, GP-2, has been found to be an exceptionally rich source of branched sialo-oligosaccharides of poly-N-acetyllactosamine (I antigen) type with receptor activity for the human pathogen Mycoplasma pneumoniae. Desialylated GP-2 is the most potent I-active substance thus far tested. Since this glycoprotein is hydrophobic and can be readily re-incorporated into cell membranes, it should be useful in future studies of the mechanism of production of autoantibodies to the I antigen which commonly arise following human infection with M. pneumoniae.

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins.

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.

Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type.

Among the pathological effects in man following infection with Mycoplasma pneumoniae is a transient autoimmune disorder characterized by the presence of high-titre erythrocyte autoantibodies (cold agglutinins). These autoantibodies are usually directed against the carbohydrate antigen termed I (ref. 3) which consists of a branched oligosaccharide. The mechanism by which the anti-I antibodies are elicited is unknown. However, sialic acid-containing receptors have been implicated in the adherence of M. pneumoniae to erythrocytes and other cell types, and both I and the related antigen i occur on erythrocytes in sialylated form: i is the predominant antigen on fetal erythrocytes and I is predominant in adults. Anti-I antibodies might arise in M. pneumoniae infection in response to a modification of the 'self' antigen-I as a result of its interaction with this agent. Here we report our study of the specificity of the interaction of M. pneumoniae with human erythrocytes. We found that this interaction is mediated by long chain oligosaccharides of sialic acid joined by alpha 2-3 linkage to the terminal galactose residues of poly-N-acetyllactosamine sequences of Ii antigen type.