Structure-Function Analysis of Human 1,3-Fucosyltransferase

Abstract

A series of molecular biology experiments were car- ried out to identify the catalytic domain of two human ␣1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V), and to identify amino acids that function in acceptor substrate binding. Sixty-one and 75 amino ac- ids could be eliminated from the N terminus of FucTs III and V, respectively, without a significant loss of enzyme activity. In contrast, the truncation of one or more amino acids from the C terminus of FucT V resulted in a dramatic or total loss of enzyme activity. Results from the truncation experiments demonstrate that FucT III62–361 (containing amino acids 62–361) and FucT V76–374 (containing amino acids 76–374) are active, whereas shorter forms of the enzymes were inactive. The shortest, active forms of the enzymes are more than 93% identical at the predicted amino acid level, but have distinct acceptor substrate specificities. Thus, FucT III is an ␣1,4-fucosyltransferase, whereas FucT V is an ␣1,3- fucosyltransferase with disaccharide substrates. All but one of the amino acid sequence differences between the two proteins occur near their N terminus. Results ob- tained from domain swapping experiments demon- strated that the single amino acid sequence difference near the C terminus of these enzymes did not alter the enzyme’s substrate specificity. However, swapping a re- gion near the N terminus of the truncated form of FucT III into an homologous region in FucT V produced a protein with both ␣1,3- and ␣1,4-fucosyltransferase ac- tivity. This region contains 8 of the amino acid sequence differences that occur between the two proteins