Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.
Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.
Gillespie
W
W
UCLA School of Medicine, Department of Biological Chemistry 90024.
Kelm
S
S
Paulson
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013363:Submandibular Gland / Q000201:enzymology; D013552:Swine; D014162:Transfection; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase