Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases

Abstract

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/ N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O-and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4 ؉ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.Initially identified as cyclosporin A-binding proteins, cyclophilins are peptidyl-prolyl cis-trans isomerases involved in various biological processes, including protein folding, mitochondrial functions, apoptosis, and regulation of trafficking and signaling (1, 2). Besides the repertoire of intracellular functions in which they have been implicated, secreted cyclophilins A and B (CyPB) 2 were reported to be mediators of inflammation and innate immunity. They trigger chemotaxis of neutrophils, T lymphocytes, and monocytes/macrophages by way of interactions with CD147 and cell surface heparan sulfate (HS) (3-7). CyPB also induces integrin-mediated adhesion of CD4 ϩ T lymphocytes and monocytes/macrophages to fibronectin, by a mechanism that requires interaction with the HS moieties of syndecan-1 and association of CD147 with CD98, the latter being an activator of ␤1 integrins (4,8,9).HS consists of alternating N-acetyl/N-sulfate glucosamine (GlcNAc/GlcNS) and GlcUA/IdoUA residues clustered in a series of domains of relatively high IdoUA content and sulfate density (NS domains), bound by short transition zones with intermediate sulfation patterns and separated by N-a...

Heparan sulfate 3-O-sulfation: A rare modification in search of a function

“…In the mouse, Hs3st-6 is expressed predominantly in the liver and kidney with lower expression in the heart, brain, lung and testis (Xu et al, 2005). At least one Hs3st gene is expressed in nearly every cell line examined thus far and many express multiple Hs3st genes simultaneously (Deligny et al, 2010; Girardin et al, 2005; Vanpouille et al, 2007). Notable exceptions are Chinese hamster ovary cell line and Engelbreth-Holm-Swarm mouse tumor, which synthesize heparan sulfate devoid of 3- O -sulfation (Pejler et al, 1987a; Zhang et al, 2001b).…”

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Cyclophilin B Attenuates the Expression of TNF-α in Lipopolysaccharide-Stimulated Macrophages through the Induction of B Cell Lymphoma-3

“…The transcript of GAPDH was used as a control to normalize for total mRNA input and confirm efficiency of cDNA synthesis. PCR reactions were performed essentially as described in Deligny et al (29). The synthetic primers were designed by using Primer Premier 5.0 (Biosoft International), according to the published cDNA sequences of TNF-a and Bcl-3 (accession numbers: NM_000594.2 and NM_005178.3, respectively).…”

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