Human B-lymphocytes Express α2-6-Sialylated 6-Sulfo-N-acetyllactosamine Serving as a Preferred Ligand for CD22/Siglec-2

Naoko Kimura,Katsuyuki Ohmori,Keiko Miyazaki,Mineko Izawa,Yuji Matsuzaki,Yosuke Yasuda,Hiromu Takematsu,Yasunori Kozutsumi,Akihiko Moriyama,Reiji Kannagi

doi:10.1074/jbc.m702341200

Abstract

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.

Highlights

CD22/Siglec-2 is an important inhibitory receptor on B-lymphocytes
The 6sulfation-dependent clones were further subjected to a second screening using SW480/HEC-GlcNAc6ST cells treated with sialidase S or sialidase A
Our results indicate that ␣2– 6-sialylated 6-sulfated LacNAc determinants are really expressed in situ on human B-lymphocytes in peripheral blood as well as in lymphoid tissues such as peripheral lymph nodes and can confer significant binding of CD22 at the cellular level

Summary

Introduction

CD22/Siglec-2 (sialic acid-binding immunoglobulin-like lectins) is an important inhibitory receptor on B-lymphocytes. For generation of antibodies specific to the ␣2– 6-sialylated 6-sulfo-LacNAc determinant, SW480 cells were transfected with expression vector pIRESneo containing the gene for human high endothelial cell GlcNAc-6-sulfotransferase (HECGlcNAc6ST), a GlcNAc␤:6-O-sulfotransferase [23], using Lipofectamine 2000 (Invitrogen) and screened in culture medium containing 400 ␮g/ml G418. ␣2– 6-Sialylated 6-Sulfo-LacNAc Determinant as CD22 Ligand nant) and parental SW480 cells (expressing the non-sulfated ␣2– 6-sialylated LacNAc determinant) were cultured in monolayer in 24-well plates.

Results

Conclusion