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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA070740-04
Application #
6150220
Study Section
Pathology B Study Section (PTHB)
Program Officer
Sathyamoorthy, Neeraja
Project Start
1997-02-15
Project End
2002-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
4
Fiscal Year
2000
Total Cost
$343,143
Indirect Cost
Name
Northwest Hospital and Medical Center
Department
Type
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98133
Shetterly, Susan; Jost, Franziska; Watson, Susan R et al. (2007) Site-specific fucosylation of sialylated polylactosamines by alpha1,3/4-fucosyltransferases-V and -VI Is defined by amino acids near the N terminus of the catalytic domain. J Biol Chem 282:24882-92
Sherwood, Anne L; Upchurch, David A; Stroud, Mark R et al. (2002) A highly conserved His-His motif present in alpha1-->3/4fucosyltransferases is required for optimal activity and functions in acceptor binding. Glycobiology 12:599-606
Sherwood, A L; Stroud, M R; Levery, S B et al. (2001) An amino acid region at the N-terminus of rat hepatoma alpha1-->2 fucosyltransferase modulates enzyme activity and interaction with lipids: strong preference for glycosphingolipids containing terminal Galbeta1-->3GalNAc-structures. Biochemistry 40:5708-19
de Vries, T; Knegtel, R M; Holmes, E H et al. (2001) Fucosyltransferases: structure/function studies. Glycobiology 11:119R-128R
Sherwood, A L; Davis, W C; Ho, S et al. (2000) A GDP-fucose-protected, pyridoxal-5'-Phosphate/NaBH(4)-sensitive lys residue common to human alpha1-->3Fucosyltransferases corresponds to Lys(300) in FucT-IV. Biochem Biophys Res Commun 273:870-6
Holmes, E H; Yen, T Y; Thomas, S et al. (2000) Human alpha 1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites. J Biol Chem 275:24237-45
Sherwood, A L; Holmes, E H (1999) Analysis of the expression and enzymatic properties of alpha1-->3fucosyltransferase from human lung carcinoma NCI-H69 and PC9 cells. Glycobiology 9:637-43
Vo, L; Lee, S; Marcinko, M C et al. (1998) Human alpha1,3/4-fucosyltransferases. II. A single amino acid at the COOH terminus of FucT III and V alters their kinetic properties. J Biol Chem 273:25250-5
Sherwood, A L; Nguyen, A T; Whitaker, J M et al. (1998) Human alpha1,3/4-fucosyltransferases. III. A Lys/Arg residue located within the alpha1,3-FucT motif is required for activity but not substrate binding. J Biol Chem 273:25256-60
Sherwood, A L; Holmes, E H (1998) Cloning and expression of the catalytic domain from rat hepatoma H35 cell GDP-fucose:GM1 alpha 1-->2fucosyltransferase, an enzyme which is activated during early stages of chemical carcinogenesis in rat liver. Arch Biochem Biophys 355:215-21

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