Isolation and biochemical characterization of the iC3b receptor of Candida albicans

Abstract

In an effort to identify the protein structure on Candida albicans, pseudohyphal forms which had been shown earlier to bind human iC3b, a protein of about 42 kDa (p42), were obtained from lysates of pseudohyphal forms by absorption with C3(H20)-Sepharose. An antiserum raised in rabbits against this protein effectively inhibited adherence of sheep erythrocytes carrying iC3b (EAC3bi) to pseudohyphal forms. p42 cross-reacted with OKM-1, a monoclonal antibody directed against the human complement receptor type 3 (CR3, CD11b). This protein, p42, was designated p42-CR3. The antiserum against p42-CR3 was used for further purification of lysates by affinity chromatography. Three proteins of 66, 55, and 42 kDa were isolated. All were recognized by OKM-1 in immunoblots (p66-, p55-, and p42-CR3). The different proteins were separated and treated with neuraminidase and endoglycosidase F. Almost complete deglycosylation of the p66-CR3 protein was obtained after treatment with neuraminidase, indicating a high degree of glycosylation. Neuraminidase also had an effect on p55-CR3, but not on p42-CR3. Endoglycosidase F did not alter any of the three proteins. In ligand blots, p42-CR3 bound C3(H20), C3b, and iC3b but not C3d; p55-CR3 clearly reacted with C3(H20) and weakly reacted with C3b and iC3b. p66-CR3 never showed reactivity. It is suggested that p55 and p66 represent glycosylated forms of p42-CR3. Although C. albicans CR3 and human CR3 cross-react and bind identical ligands, the two receptors differ in structure. Candida albicans pseudohyphal forms, in contrast to yeast forms, bind sheep erythrocytes carrying iC3b or C3d (EAC3bi, EAC3d). This observation, initially described by Heidenreich and Dierich (10), was confirmed later by several other investigators (3, 4, 6). Eigentler et al. (5) have shown that the expression of the iC3b receptor on C. albicans is dependent on the growth temperature. C. albicans grown at 30°C bound EAC3bi strongly, whereas those cells grown at 38.5°C were completely devoid of that feature. The C3d-binding proteins of C. albicans were purified by ion-exchange and affinity chromatography. Two proteins with molecular masses of 62 and 70 kDa were identified by silver stain, by Western blots (immunoblots) with CA-A, a monoclonal antibody against C. albicans that inhibited interaction of C. albicans with EAC3d, and by size exclusion high-pressure liquid chromatography (2, 3, 11). Ollert et al. (12) reported data on a human serum which inhibited the iC3b-mediated adherence almost completely; this serum reacted with 71-, 68-, 55-, and 50-kDa molecules in immunoblots of extracts C. albicans pseudohyphae. Here we report on the isolation of iC3b binding molecules from C. albicans. MATERIALS AND METHODS Reagents. OKM-1, a mouse monoclonal antibody of the immunoglobulin G2b (IgG2b) subtype directed against the alpha chain of human complement receptor type 3 (CR3, CD11b), was purchased from Ortho Diagnostic Systems, Raritan, N.J. C3, C3(H20), and the C3 fragments C3b, iC3b,

Dangerous liaisons: complement, coagulation, and kallikrein/kinin cross‐talk act as a linchpin in the events leading to thromboinflammation

“…150 In the case of platelet-leukocyte complexes formation, the integrin CD11b/CD18 (also known as CR3 or Mac-1) is involved. 151 154 and a CR3 (CD11b/CD18)-like molecule from Candida albicans, 155 and we have confirmed that the platelet-bound C3(H 2 O)/iC3(H 2 O) binds to soluble CR1 (CD35). 40 The mechanism by which this activation takes place is not known, but a very similar form of C3 is also found on neutral liposomes exposed to serum/plasma.…”

The influence of subinhibitory concentrations of conventional and experimental antifungal drugs on the expression of the iC3b binding protein in Candida albicans strains during filamentation

“…However, the di¡erences in the presence compared to in the absence of drug appear too minor to support this hypothesis. As the same two major bands, with apparent M r of approximately 42 kDa, which was also found in a previous study [34], and 32 kDa, were observed after treatment with antifungal agents, a major drug-induced cleavage of this protein appears unlikely.…”

Adherence ofCandida albicansto host cells

“…Ligand blots demonstrated binding of C3, C3b, and iC3b to p42-CR3, whereas p55-CR3 reacted with CR3 but only weakly with C3b and iC3b. The 66-kDa component did not show reactivity to any of these proteins [31].…”

“…Antibody to CR3 (OKM-1) identified a 165 + 15kDa protein by Western analysis [29] and was able to immunoprecipitate a 130-kDa species [30]. Most recently, Alaei et al [31] have used C3b affinity chromatography to isolate a 42-kDa glycoprotein which reacts with OKM-1 (termed p42-CR3). An antibody raised to this protein species was able to inhibit adherence of iC3b-coated erythrocytes to pseudohyphae and reacted with a human cell line expressing CR3.…”