Our ongoing studies on the structure-function relationship of proteins specifically of glycosyltransferases and their modifiers like alpha-lactalbumin, continued this year. We report for the first time that the cDNA sequence of bovine beta (1-4) galactosyltransferase (GT) (constructed from a partial cDNA clone and a genomic fragment) engineered in an Okayama-Berg vector, upon transfection of COS-7 cells, codes for an enzymatically active CT. There is an approximately 12 fold increase in the GT activity upon transfection of COS-7 cells with sense cDNA compared to antisense or pSV(2)Neo or mock transfected cells. Polyclonal and monoclonal antibodies directed against synthetic peptide corresponding to the amino-terminal region of the GT protein, bind the expressed protein and the immunoprecipitates exhibit GT enzymatic activity. The expressed GT activity is modulated by alpha-lactalbumin to change the acceptor specificity to glucose, to synthesize lactose. These results show that the expressed GT protein is fully functional and that the binding sites for UDP-galactose, N-acetylglucosamine, glucose and alpha-lactalbumin are all intact and operative. We have also engineered the cDNA sequences of galactosyltransferase and alpha-lactalbumin, in such a way that the expression of the cDNA sequences have a potential to produce either soluble and secreted forms or membrane anchored forms of the protein.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CB008389-03
Application #
3813372
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Division of Cancer Biology and Diagnosis
Department
Type
DUNS #
City
State
Country
United States
Zip Code