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.
Highlights
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]
Our previous studies [6, 26, 27] demonstrated that efficient binding of cyclophilins A and B (CyPB) to heparin and cell surface heparan sulfate (HS) expressed on T cells was dependent on the interaction with a highly sulfated HS sequence, in which a critical modification was the presence of a 3-O-sulfated GlcNH2 residue
GlcNH2 residues have been reported to be preferentially located in NA domains and in transition zones between NA and NS domains, 3-O-sulfated GlcNH2-containing sequences have been characterized within heparin and the highly sulfated NS domain of some HS species [21, 23, 46]
Summary
CyPB, cyclophilin B; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; dp, degree of polymerization; DPBS, Dulbecco’s phosphate-buffered saline; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; GlcNH2, N-unsubstituted D-glucosamine; GlcNS, N-sulfated D-glucosamine; HS, heparan sulfate; MAPK, mitogen-activated protein kinase; NDST, N-deacetylase/N-sulfotransferase; OST, O-sulfotransferase; siRNA, small interfering RNA; VSV, vesicular stomatitis virus; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid. Seven 3-OST isoforms have been recognized in humans, with 3-OST1, 3-OST3 (3A and 3B), and 3-OST5 being the most widely expressed in various tissues and cell types These isoenzymes exhibit fine differences in substrate specificity, suggesting their involvement in making tissue-specific HS with different biological functions. Following the demonstration that N-, 2-O-, and 3-O-sulfations of HS sequences were critically important to support the efficient binding of CyPB, we showed that activated/memory CD4ϩ T lymphocytes, monocytes/macrophages, Jurkat and THP-1 cells, which are target cells of CyPB, expressed high levels of mRNA encoding NDST2 and 3-OST3 isoforms. We found that NDST1 and NDST2 have no redundant activities but rather have complementary activities in making N-sulfated HS motifs with CyPB-binding properties These results suggest a regulatory mechanism in which the expression of some HS sulfotransferases determines the specificity of binding of CyPB to target cells
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