Molecular cloning and expression of chick embryo Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Comparison with the mammalian enzyme.
DNA clones encoding beta-galactoside alpha 2,6-sialyltransferase have been isolated from chick embryonic 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 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region and a large C-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Gal beta 1,4GlcNAc in glycoproteins.
Kurosawa
N
N
Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Japan.
Kawasaki
M
M
Hamamoto
T
T
Nakaoka
T
T
Lee
Y C
YC
Arita
M
M
Tsuji
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008322:Mammals; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase