HUMAN FUCOSYLTRANSFERASE STRUCTURE/FUNCTION ANALYSIS

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DESCRIPTION: (adapted from the investigator's abstract) Expression of carbohydrate structures at the cell surface has been found to play an important role in many cellular processes, eg. cell-cell recognition, growth control, and receptor binding. Carbohydrates containing an a1A3-linked fucose on lacto- and neolacto-series core chains are found to be tumor-associated markers in many human cancers. Enzymes capable of catalyzing transfer of fucose into these acceptors have been identified and studied. Presently, at least six distinct a1A3 fucosyltransferases have been identified from human sources. Five of these enzymes have been cloned and their DNA and amino acid sequences determined. These enzymes are characterized by differences in enzymatic properties including acceptor specificity, kinetic properties, and distributions. This family of enzymes is an ideal system for studies of structure function relationships of glycosyltransferases. The research proposed in this application will take advantage of the multiplicity of enzyme forms and properties to identify specific residues and portions of the enzyme involved in substrate binding and catalysis. The initial focus will be on identifying by chemical means specific residues which have previously been determined to be essential for activity based upon results from site-specific chemical reagents. Examples include the GDP-frucose protected, N-ethylmaleamide-sensitive Cys residue present in eg. FT-III and -V and the GDP-fucose protected pyridoxal-P modifiable Lys residue present in each enzyme form. Other studies will use photoaffinity reagents to probe the binding sites for acceptor carbohydrates and donor GDP-fucose and analysis of acceptor binding properties through domain swapped variants. Site-directed mutagenesis and enzyme kinetic studies will be used to analyze the function of these sites in substrate binding and catalysis. These studies will provide information leading to a better understanding of enzyme function and, perhaps, a basis for differences in acceptor specificity. Another aim will focus on cloning and genetic characterization of a novel fucosyltransferase enzyme from NCI-H69 small cell lung carcinoma cells. This enzyme has properties distinct from other known forms. The research proposed in this application will lead to a greater understanding of enzyme complexity, structure-function relationships, and the nature of active sites in this family of enzymes having important function in disease processes.