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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Media type:
E-Article
Title:
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
Description:
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.