Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I).
The CMP-Neu5Ac:Galbeta1-3GalNAc 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 Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites.
Vallejo-Ruiz
V
V
Unité de Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Haque
R
R
Mir
A M
AM
Schwientek
T
T
Mandel
U
U
Cacan
R
R
Delannoy
P
P
Harduin-Lepers
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D012799:Sialyltransferases / Q000737:chemistry*; D014162:Transfection; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D007527:Isoenzymes; D011994:Recombinant Proteins; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; C000722087:ST3GAL1 protein, human