Studies are proposed to investigate colon adenocarcinoma- associated alpha (1-2) fucosyltransferase which is specific for glycolipid acceptors and is expressed in colon carcinoma and gastric carcinoma cell lines but not in normal colon mucosa. This tumor-associated activity results in the biosynthesis of difucosylated structures such as Leb, Y and H-2 in which the terminal fucosyl residue is joined by an alpha (1-2) linkage. The Leb and Y structures have been detected with monoclonal antibodies using immunopathological techniques, intact cell analysis and chemical analysis. Our preliminary daa indicate that the tumor-associated alpha (1-2) fucosyltransferase clearly differs from any of the known forms of this enzyme. It remains unknown whether alpha (1-2) fucosyltransferase activity in tumor cells involves more than one enzyme, an enzyme with multiple specificities or a modifier of this enzyme activity. This enzyme(s) will be purified from human colon and gastric adenocarcinoma cell lines. Its detailed substrate specificity for different glycolipid precursors, synthetic and chemically defined oligosaccharides and deoxydisaccharides will be established. Also, kinetic properties and requirements for divalent cations, pH, temperature and detergents will be determined. These parameters will be compared with those determined in analogous studies for the alpha (1-2) fucosyltransferase isolated from human sera and milk of """"""""nonsecretor"""""""" and """"""""secretor"""""""" individuals, respectively. Human x rodent cell hybrids will be produced and used to assign the chromosomal location of the alpha (1 greater than 2) fucosyltransferase gene. A monoclonal antibody against purified alpha (1-2) fucosyltransferase will be produced and used for: affinity chromatography purification of large quantities of the enzyme from human tumors, immunodetection of the enzyme molecule in somatic cell hybrids for chromosomal assignment, and for immunopathological studies of early stages of colon carcinoma development. Affinity-purified protein will be subjected to partial sequencing and a synthetic oligonucleotide probe will be prepared. The oligomer in combination with antibody probes will be used to screen colon carcinoma cDNA libraries for alpha 1 greater than 2 fucosyltransferase cDNA. The cDNA will be used to confirm the chromosomal assignment and to fine map the alpha (1-2) fucosyltransferase gene on the chromosome, to investigate homologous structural genes, and to examine the differential expression of this gene in tumor progression and familial polyposis coli.
