Characterization of the high‐affinity oligosaccharide‐binding site of the 205‐kDa porcine large granular lymphocyte lectin, a member of the leukocyte common antigen family

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Abstract
Membrane lectins of mammalian large granular lymphocytes are thought to be important receptors in their non-major-histocompatibility complex-restricted activation. A triantennary desialylated oligosaccharide has been reported as the most effective triggering structure [PospÍS̆il M., Kubrycht J., Bezous̆ka K., Táborský O., Novák M. & Kocourek J. (1986) Immunol. Lett. 12, 83–90] while its cell surface receptor has recently been identified in pig natural killer cells as a 205-kDa membrane lectin resembling the proteins of the leukocyte common antigen family (LCA). In this study we have prepared 4-azidophenyl (photoactivatable) and 4-hydroxyphenyl (radio-iodinatable) derivatives of triantennary oligosaccharides by a new procedure which allows the natural conformation of the N-glycosidic linkage between the oligosaccharide and the respective labeling group to be retained. We used these high-affinity ligands to investigate the oligosaccharide-combining site of the 205-kDa lectin. Photoaffinity labeling of the whole cells and solubilized proteins confirmed that a 205-kDa polypeptide constitutes the major cell-surface cacium-independent receptor for triantennary oligosaccharides in pig lymphocytes. Isolation and manual sequencing of two ligand-labeled and eleven other peptides proved that the 205-kDa lectin represents a member of the LCA family expressing exons 4 and 6 during alternative splicing and that the high-affinity binding site is localized in the N-terminal 70-kDa extracellular domain. Binding studies with radiolabeled oligosaccharides and the above carbohydrate-recognition domain subjected to various chemical and enzymatic treatments indicated that the binding of oligosaccharides might be significantly modulated by sialylated O-glycosidically linked lineage-specific carbohydrate epitopes localized within this domain. Affinity chromatography of LCA isolated by conventional methods on immobilized oligosaccharides revealed that only a fraction of these cell-surface glycoproteins expressed high-affinity binding sites for the oligosaccharide ligands. Thus, N-linked oligosaccharide moieties of cell-surface glycoproteins seem to represent possible ligands of LCA that may be important in intercellular adhesion and oligosaccharide-mediated activation of lymphocytes.