Molecular cloning and expression of Galbeta1,3GalNAc alpha2, 3-sialyltransferase from human fetal liver.
Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galbeta1, 3GalNAc alpha2,3-sialyltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS-1, Escherichia coli, and Pichia pastoris. The recombinant protein expressed in COS-1 could catalyse the transfer of NeuAc from CMP-NeuAc to asialo-fetuin. No enzyme activity was detected with a 32-kDa protein in E. coli and both 32-kDa and 41-kDa proteins in P. pastoris. These results suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.
Shang
J
J
State Key Laboratory of Microbial Resources, Institute of Microbiology,Academia Sinica, Beijing, China.
Qiu
R
R
Wang
J
J
Liu
J
J
Zhou
R
R
Ding
H
H
Yang
S
S
Zhang
S
S
Jin
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D003001:Cloning, Molecular; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D004926:Escherichia coli; D060748:Fetuins; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006801:Humans; D008099:Liver / Q000196:embryology / Q000201:enzymology*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010843:Pichia; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017385:Sequence Homology; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics*; D000509:alpha-Fetoproteins / Q000378:metabolism; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D001212:Asialoglycoproteins; D060748:Fetuins; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase