Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.
Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.
Denys
A
A
Laboratoire de Chimie Biologique, Unité Mixte de Recherche du Centre National de Recherche Scientifique no. 111, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France.
Allain
F
F
Carpentier
M
M
Spik
G
G
D001665:Binding Sites; D002846:Chromatography, Affinity; D021983:Cyclophilins; D004705:Endocytosis; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D020104:Immunophilins / Q000378:metabolism*; D066298:In Vitro Techniques; D008024:Ligands; D019696:Peptidylprolyl Isomerase; D011994:Recombinant Proteins / Q000378:metabolism; D013499:Surface Properties; D013601:T-Lymphocytes / Q000378:metabolism*
D006025:Glycosaminoglycans; D008024:Ligands; D011994:Recombinant Proteins; C081786:cyclophilin B; D021983:Cyclophilins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase