Walcheck Bruce, Leppanen Anne, Cummings Richard D, Knibbs Randall N, Stoolman Lloyd M, Alexander Shelia R, Mattila Polly E, McEver Rodger P
Department of Veterinary PathoBiology, University Minnesota Academic Health Center, University of Minnesota, St Paul 55108, USA.
Blood. 2002 Jun 1;99(11):4063-9. doi: 10.1182/blood-2001-12-0265.
Core 2 O-glycans terminated with sialyl-Lewis x (sLe(X)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLe(X) extended from a core 2 branch (C2-O-sLe(X)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLe(X) was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(X) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(X)-related glycans. Unlike anti-sLe(X) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both.
以唾液酸化路易斯x（sLe(X)）为末端的核心2 O-聚糖是具有重要功能的寡糖，它能赋予特定大分子与选择素家族高亲和力的聚糖配体。迄今为止，尚未有识别白细胞上这些结构的抗体被报道。我们在此鉴定了一种这样的单克隆抗体（mAb）（CHO-131）。通过含有精确O-聚糖结构的合成糖肽直接检测了CHO-131的结合特异性。CHO-131与从核心2分支延伸出的sLe(X)（C2-O-sLe(X)）结合，但如果这种寡糖缺乏岩藻糖或sLe(X)从核心1分支延伸，则CHO-131无反应性。利用转染细胞系，我们发现CHO-131结合需要糖基转移酶α2,3-唾液酸转移酶、α1,3-岩藻糖转移酶-VII和核心2β1,6 N-乙酰葡糖胺转移酶（C2GnT）的功能活性。C2-O-sLe(X)基序主要出现在唾液粘蛋白上，并且已直接表明它有助于P-选择素与高亲和力P-选择素糖蛋白配体-1结合。事实上，用O-糖蛋白酶去除唾液粘蛋白后，中性粒细胞的CHO-131染色减弱，并且它与转染造血细胞系的反应性与P-选择素配体的表达相关。CHO-131还对一小部分淋巴细胞进行了染色，这些淋巴细胞主要是CD3(+)、CD4(+)和CD45RO(+)，并且代表了皮肤淋巴细胞相关抗原（CLA）T细胞的一个亚群（37.8%±18.3%），可通过检测与sLe(X)相关聚糖的单克隆抗体HECA-452来区分。与抗sLe(X)单克隆抗体不同，CHO-131的结合还表明了C2GnT活性，并证明CLA T细胞基于它们合成的聚糖结构是异质性的。这些发现支持了以下证据，即不同的C2GnT活性导致表达E-选择素、P-选择素或两者配体的T细胞亚群。
