Differential binding of lectins IL-2 and CSL to Candida albicans and cancer cells

Jean-Pierre Zanetta 2 Roger Bonaly 1 2 Susanna Maschke 0 2 Grard Strecker 2 Jean-Claude Michalski 2 0 Laboratoire de Biochimie Structurale, Faculte de Medecine , Place de Verdun, 59045 Lille Cedex , France 1 Faculte des Sciences Pharmaceutiques et Biologiques, Universite Nancy I , 5 rue A. Lebrun, 54001 Nancy , France 2 Laboratoire de Chimie Biologique , CNRS UMR 111, 59655 Villeneuve d'Ascq Cedex , France 3To whom correspondence should be addressed The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor , IL-2 associates the latter which the CD3/TCR complex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al., 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R by p56lck, the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2-dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous amplifier of activation signals (Zanetta et al., 1995, Biochem. J., 311, 629-636). Introduction In a previous paper (Zanetta et al., 1996), we demonstrated that human interleukin-2 (IL-2) is a calcium-independent carbohydratebinding protein (lectin). This lectin activity, specific for oligomannosidic N-glycans with five and six mannose residues, is still preserved when IL-2 is bound to its receptor (IL-2R). As a bifunctional extracellular molecule, IL-2 associates the IL-2R with the CD3/TCR receptor complex (and induces the subsequent phosphorylation of the intracytoplasmic domain of IL-2R by the p56lck tyrosine kinase, initially associated with CD3/TCR), through a carbohydrate-dependent mechanism. This association can be inhibited by oligomannosidic N-glycans with five and six mannose residues, the high affinity ligands of IL-2 (Zanetta et al., 1996). Another mannose-binding lectin important for the immune system is the calcium-independent mannose-binding lectin, CSL (Zanetta et al., 1987), also involved in the activation process of human lymphocytes (Zanetta et al., 1995). This polyvalent lectin is early expressed upon human lymphocyte stimulation and acts as an amplifier of activation signal. CSL recognized N-linked glycans at the surface of T and B cells including glycosylated forms of CD3 on T cells, CD24 on B cells and a few number of unidentified glycoproteins. The externalization of CSL, and its binding to its surface ligands, induce the shift in tyrosine phosphorylation between p56lck and p59fyn observed in the early stages of lymphocyte stimulation, described as resulting from the clustering of cell surface complexes, especially CD3/TCR complex on T cells. In contrast with IL-2, CSL recognizes only the conformation of the Man6GlcNAc2Asn structure (Marschal et al., 1989), because the Man(a 12) residue of the C branch interacts with the GlcNAc(14)GlcNAc(1-) structure of the N-glycan core (Wyss et al., 1995). We made the hypothesis that oligomannosides bound to pathogenic cells or microorganisms could perturb the IL-2- and CSL-dependent immune response. Consequently, we addressed the problem to know if these two lectins could bind pathogenic cells and microorganisms provoking immunodeficiencies. This study demonstrates the specific binding of IL-2 to the pathogenic yeast Candida albicans and to cancer cells, contrasting with that of CSL, binding only to cancer cells. Results Binding of IL-2 and CSL to yeast cell wall constituents As illustrated in Figure 1, IL-2 bound to Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis cell wall material, the binding being reversed using 105 M of Man5GlcNAc2 but not using 104 M of Man9GlcNAc, indicating a carbohydrate-dependent mechanism with the same lectin specificity as IL-2. This binding was saturable (Figure 2) with a maximum binding of 7.5 ng, 7.5 ng and 15 ng of IL-2/m g mannose equivalents of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis, respectively (Table I). When using the same amount of immobilized cell wall material from Candida albicans (Figure 2), all the IL-2 (up to 2 m g/well) was bound. In fact, the mannoproteins of Candida albicans showed extremely higher binding of IL-2 (0.75 m g IL-2/m g mannose equivalents; i.e., 5 m g IL-2/m g cell wall protein) than the other species. The binding was reversed using 105 M Man5GlcNAc2 and Man6GlcNAc2Asn but not by 103 M Man9GlcNAc, indicating a specific binding due to the lectin activity of IL-2. Consequently, the cell wall material of Candida albicans showed 50100 fold more IL-2 binding sites than the other yeast species. In contrast, the binding of CSL to the cell wall material of Candida albicans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis was very similar (Figure 3, Table I). The fixation of CSL to all yeast species was inhibited by Man6GlcNAc2Asn (105 M), but not by Man5GlcNAc2 and Man9GlcNAc (104 M), indicating a specific binding of CSL. However, the quantity of fixed CSL on Candida albicans cell wall material was by far lower than that of IL-2 (2 orders of magnitude). Due to the specific affinity of CSL for Man6GlcNAc2Asn, the ligands of CSL represented around 0.1% of the cell wall neutral sugars. Binding of IL-2 and CSL to C6 glioblastoma cell glycoproteins C6 glioblastoma cells bound high amounts of IL-2 (Table II). The carbohydrate-dependence of the interaction was demonstrated by the reversibility of the IL-2 binding using Man56 oligomannosides and the absence of effect using Man9GlcNAc. Furthermore, the binding of IL-2 was absent from the material from C6 cells treated with inhibitors of N-glycan processing tunicamycin (which blocks the addition of the first GlcNAc residue to the polyisoprenic intermediate and inhibits the formation of N-glycans), deoxynojirimycin, and castanospermine (which produce immature N-glycans (Glc23Man9GlcNAc2)). In contrast to the yeast cell wall material, C6 glioblastoma cells had a strong capacity to bind CSL (Table II). This binding was reversed using the CSL ligand Man6GlcNAc2Asn, but not using Man5GlcNAc2 and Man9GlcNAc. Furthermore, no binding was observed on material of C6 cells treated with tunicamycin, deoxynojirimycin and castanospermine. In fact, the number of molecules of IL-2 and CSL bound by C6 glioblastoma material were virtually the same: 0.25 m g of IL-2 and 0.5 m g of CSL for 10 m g C6 cell protein (i.e., about 16.6 pmol of both IL-2 and CSL bound to 10 m g C6 cell protein). Because of the carbohy (...truncated)