The glycan units of mammalian glycoconjugates serve many functions. Alterations in their synthesis result in disease and are correlated with cancer. Therefore it is important to understand how they are synthesized and what factors regulate their expression. Glycosylation mutants with a specific alteration in glycan synthesis provide direct access to glycosylation pathways and have been used to identify new steps in glycan synthesis, to expression clone glycosyltransferase genes, and to identify recognition determinants for lectins, including selectins. Under the auspices of this grant, 30 Chinese hamster ovary (CHO) glycosylation mutants have been characterized. Most recently, two gain-of-function CHO mutants, LEC 14 and LEC 18, were found to possess novel N-glycan core structures, never observed previously. The overall aim of this proposal is to exploit these and other CHO glycosylation mutants to clone new glycosylation genes, to identify the glycosylation defect in new CHO mutants, to define specific inactivating glycosylation mutations, and to develop reagents for studies of glycan function.
The specific aims of this proposal are: 1. To clone genes expressed by novel gain-of-function CHO glycosylation mutants. Expression cloning will he used to obtain a cDNA that encodes each new transferase. Expression patterns in normal and cancerous tissues will be determined. 2. To identify the glycosylation defect expressed by the gain of-function CHO mutants Lec-19, Lec-22, Lec-24 and Lec-25. Carbohydrate changes will be determined by compositional analysis, oligosaceharide mapping, 1H-NMR spectroscopy, mass spectrometry and methylation linkage analysis of glycopeptides unique to each mutant. Biochemical assays will be developed to identify the activity missing in each mutant. 3. To identify the affected gene in Lec-32 and other loss-of-function glycosylation mutants. Expression cloning will be used to obtain a complementary cDNA and the corresponding mutant gene will be characterized. 4. To use CHO glycosylation mutants to develop approaches for identifying glycan functions. Monoclonal antibodies specific for N-glycans with the different core structures of LEClO, LEC14, LEC18 and LEC33 cells will be developed for studies of glycan function.
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