Delineation of the minimal catalytic domain of human Gal beta 1-3GalNAc alpha 2,3-sialyltransferase (hST3Gal I)

The CMP-Neu5Ac:Gal beta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2 .4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Gal beta1-3GalNAc disaccharide structur es found on O-glycans and glycolipids. In order to gain further insight int o the structure/function of this sialyltransferase, we studied protein expr ession, N-glycan processing and enzymatic activity upon transient expressio n in the COS-7 cell line of various constructs deleted in the N-terminal po rtion of the protein sequence. The expressed soluble polypeptides were dete cted within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amin o acids 26-340 (hST3-Delta 25) and 57-340 (hST3-Delta 56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta 76 and hST3-Delta 105 gave also rise to various poly peptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted f orms were determined and shown to be in close agreement with those of the r ecombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Ch em. 269 (1994)). In addition, the present study demonstrates that the recom binant hST3Gal I polypeptides transiently expressed in COS-7 cells are glyc osylated with complex and high mannose type glycans on each of the five pot ential N-glycosylation sites. (C) 2001 Elsevier Science B.V. All rights res erved.