N-glycosylation site occupancy of rat alpha-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity

All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized h ave potential N-glycosylation sites, but the role of these sites in enzymat ic activity or localization has not been investigated. When one member of t his family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosyl ation sites of rFuc-TIV were mutated to determine site occupancy and the ef fect of site occupancy on enzyme activity and targeting of this enzyme. Res ults obtained with singly mutated forms of rFuc-TIV indicate that both site s are occupied in mammalian cells. Lack of glycosylation at sites 117-119, 218-220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme. These r esults show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218-220 playing the major role. However, N-glyco sylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of stai ning for the unglycosylated mutants as is seen for the wild-type rFuc-TIV i n transfected cells. (C) 2000 Elsevier Science B.V. All rights reserved.