The carbohydrates attached to mammalian glycoconjugates markedly affect molecular properties and can function as recognition molecules in various biological phenomena. However much remains to be learned about the specific biological functions of carbohydrates and the many enzymes that regulate their biosynthesis. To address these questions we have isolated glycosylation-defective Chinese hamster ovary (CHO) cell mutants and used them to define the pathways of carbohydrate biosynthesis and as unique cell lines with which to study both intra- and intermolecular functions of mammalian in such studies is in direct proportion to the extent to which they are understood at the biochemical level. It is the major aim of this grant to characterize new CHO mutants that are genetically novel and phenotypic properties typical of glycosylation mutants. Initial studies will focus on the investigation of novel, dominant CHO mutants. Each mutant synthesizes developmentally-regulated carbohydrates due to the expression of a specific glycosyltransferase not expressed in parental CHO cells. LEC29, LEC30, LEC31 and LEC32 transfer alpha(1,3)fucose residues to lactosamine units and LEC33 transfers the bisecting G1cNAc residue to N-linked carbohydrates. The alpha(1,3)fucosyltransferases expressed by the former four mutants are of particular interest as they synthesize ligands recognized by LEC-CAM molecules.
The second aims i s to identify the biochemical defect expressed by five new, recessive mutants (Lec19, Lec20, Lec22, Lec24, Lec25) as reflected by change(s) in the N-linked carbohydrates of vesicular stomatitis virus (VSV) grown in mutant cells. Once a structural defect has been identified, it will be determined whether the lesion is confined to the N-linked biosynthetic pathway and assays to identify the specific glycosylation reaction affected in each mutant will be developed.
The third aim of this proposal is to identify the precise nucleotide change(s) that gave rise to five glycosylation mutations: lec1 and lec1A (defective G1cNAc-TI), lec4 and lec4A (defective G1cNAc-TV) and lec23 (defective alpha-glucosidase I). The results should provide insight into how two G1cNAc-transferases (lec1, lec4) and an alpha-glucosidase (lec23) may be inactivated, or acquire an altered Km (lec1A) or become mislocalized intracellularly (lec4A).
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