Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase.
cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. 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 the construction of a recombinant sialyltransferase in which the NH2-terminal part (232 amino acid residues) was replaced with the immunoglobulin 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. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.
Kurosawa
N
N
Glyco Molecular Biology, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Saitamo, Japan.
Hamamoto
T
T
Lee
Y C
YC
Nakaoka
T
T
Kojima
N
N
Tsuji
S
S
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002642:Chick Embryo; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics*; D012995:Solubility; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012799:Sialyltransferases; D000116:Acetylgalactosamine