Novel Anti-carbohydrate Antibodies Reveal the Cooperative Function of Sulfated N- and O-Glycans in Lymphocyte Homing (original) (raw)

2010, Journal of Biological Chemistry

Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defucosylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and L-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N-and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N-and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N-and O-glycans in lymphocyte homing and immune surveillance. Complex carbohydrates play roles in a variety of biological processes, including differentiation, development, immunity, tumor metastasis, and protein quality control (1). To clarify the functions of complex carbohydrates in vivo, it is essential to determine when, where, and which complex carbohydrate chains are expressed in a particular biological setting of interest. For this purpose, anti-carbohydrate mAbs are particularly useful. However, it is difficult to establish anti-carbohydrate mAbs, largely because a wide variety of complex carbohydrates are intrinsically expressed in mice and rats, which are widely used to generate mAbs. Some of the most well studied functions of complex carbohydrates in immunity are in immune cell trafficking. In particular, lymphocyte homing and recruitment to the peripheral lymph nodes (PLNs) 2 through a specialized endothelium called the high endothelial venule (HEV) have been extensively examined (2, 3). The homing receptor L-selectin, which is expressed on the surface of lymphocytes, specifically interacts with a unique complex carbohydrate structure known as 6-sulfo sialyl Lewis X (sialic acid␣2-3Gal␤1-4[Fuc␣1-3(sulfo-6)]GlcNAc␤1-R) (4, 5), which is present at the nonreducing terminus of various O-glycans and N-glycans (Fig. 1A). The essential role of this unique carbohydrate structure for lymphocyte homing was revealed mainly through studies using glycosyltransferase-or sulfotransferase-deficient mice. Studies using double-null mice deficient in fucosyltransferase (FucT)-IV and-VII revealed that the fucosylation of HEV ligands is critical for their interaction with L-selectin (6, 7). Studies with mice deficient in core 1 ␤1,3-N-acetylglucosaminyltransferase (C1␤3GnT) and core 2 ␤1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), which are involved in the biosynthesis of the O-glycan core structures required for the modification with 6-sulfo sialyl Lewis X (supplemental Fig. S1), showed that both N-and O-glycans are important in lym