Molecular behavior of mutant Lewis enzymes in vivo

The expression of type-1 Lewis antigens on erythrocytes and in digestive or gans is determined by a Lewis type alpha(1,3/1,4)-fucosyltransferase (Lewis enzyme) encoded by the Fuc-TIII gene (FUT3 gene; Lewis gene). We have clas sified the Lewis alleles in the Japanese population into four types, the wi ldtype allele (Le) and three mutated alleles, i.e., le1, which has missense mutations T59G and G508A, le2, which has T59G and T1067A, and le3, which h as only T59G. Here we carried out an extensive study on the biological prop erties of the three mutant Lewis enzymes, the le1, le2, and le3 enzymes, us ing native tissues and obtained the following results. (1) In in vivo and i n vitro experiments, the le1 and le2 enzymes were found to be susceptible t o protease digestion probably because the one missense mutation in the cata lytic domains, i.e., Gly170 to Ser in the le1 enzyme and Ile356 to Lys in t he le2 enzyme, makes the three-dimensional structures of the enzymes unstab le, while the le3 and wild-type Lewis enzymes were resistant to protease di gestion. (2) The le1 and le2 enzymes cannot synthesize type 1 Lewis antigen s on either glycolipids or mucins. The le3 enzyme cannot synthesize Lewis-a ctive glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes, while it can synthesize Lewis antigens on mucins in normal an d cancerous colon tissues. The missense mutation, Leu20 to Arg, in the tran smembrane domain reduces retention of the le3 enzyme in the Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the la ck of Lewis antigen synthesis. (3) The Lewis gene dosage actually has effec ts in vivo on the amount of the Lewis enzyme, its activity, and finally the amounts of Lewis carbohydrate antigens. This is the first article that cle arly demonstrates the gene dosage effects on the amount of the glycosyltran sferase protein, its activity, and the amounts of carbohydrate products in vivo.