Dataset Information

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

ABSTRACT: Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPB(KKK-) [where KKK- refers to the substitutions K3A(Lys3-->Ala)/K4A/K5A] and CyPB(DeltaYFD) (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.

SUBMITTER: Vanpouille C

PROVIDER: S-EPMC1133832 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

Vanpouille Christophe C Denys Agnès A Carpentier Mathieu M Pakula Rachel R Mazurier Joël J Allain Fabrice F

The Biochemical journal 20040901 Pt 2

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requi ...[more]

PMID: 15109301

Similar Datasets

Project description:In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAbeta1-4GlcNSO(3)alpha1-](n)), or of O-desulphated heparin (predominantly [4IdoAalpha1-4GlcNSO(3)alpha1-](n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of (3)H(2)O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of (3)H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5-(3)H-labelled polysaccharide products showed that the (3)H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmström (1996) Biochem. J. 313, 589-596].

Dataset Information

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

Publications

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets