Molecular cloning and expression of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from mouse brain.
DNA clones encoding beta-galactoside alpha 2,3-sialyltransferase have been isolated from mouse brain cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open reading frame coding for 337 amino acids, and the deduced amino acid sequence showed 80% identity with that of porcine submaxillary gland Gal beta 1,3GalNAc alpha 2,3-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the NH2-terminal part including the cytoplasmic tail, signal-anchor domain and stem region was replaced with an immuno-globulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. This enzyme exhibited the transferase activity toward only the disaccharide moiety of Gal beta 1,3GalNAc of glycoproteins and glycolipids, no significant activity being detected for the other substrates tested.
Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Japan.
Y C
YC
Lee
N
N
Kurosawa
T
T
Hamamoto
T
T
Nakaoka
S
S
Tsuji
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004247:DNA; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D004247:DNA; D012799:Sialyltransferases; C029156:N-acetyllactosaminide alpha-2,3-sialyltransferase