GLYCOPROTEINS IN DIFFERENTIATION

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The overall objective of this proposed study is to define the basic biochemical alterations occurring in natural killer (NK) T200 glycoprotein during differentiation, maturation, lymphokine (IL-2) induced proliferation and cytolytic function. The relative role of developmental stage, tissue specificity and alternate exon usage in determining glycosylation of T200 glycoprotein isoforms will be studied. Considerable evidence exists to support an important function for T200 in NK-target binding and lysis. T200 is a molecularly heterogenous family of transmembrane glycoproteins ubiquitously expressed on the surface of all mammalian lymphopoietic cells. T200 heterogeneity is due in part to three variable N-terminal peptide sequences resulting from alternate use of combinations of three exons. Eight possible T200 isoforms are possible (with 0 to 3 exons), with cells of different origins expressing different phenotypes. Exon usage will be analyzed in NK cells of different differentiative and functional stages using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR primers, which flank the area of alternate exon usage, will be used to amplify CDNA prepared from the cells to be analyzed. The exons present will be determined from the amplified sequence. Basic glycosylation of unique T200 isoforms will be determined by metabolic radiolabeling, gel and affinity purification, pronase digestion and serial lectin analysis. The particular focus of this study is on oligosaccharides containing poly-N-acetyllactosamine structures. The unique glycosylation pattern apparent on different isoforms of T200 will be characterized to delineate the relationship between exon usage and glycosylation. Functional studies of T200 glycoprotein will be performed using homogenous IL-2-activated NK cells and functionally relevant, epitopespecific T200 monoclonal antibodies. These studies will provide new insights into the basic mechanisms by which cytotoxic effector lymphocytes interact with susceptible tumor cells and may lead to the development of new approaches for the immunotherapy of cancer.