Molecular cloning and functional expression of a fifth-type alpha 2,3-sialyltransferase (mST3Gal V: GM3 synthase).
The cDNA encoding a new type of alpha 2,3-sialyltransferase (mST3Gal V) was cloned from mouse brain cDNA library by PCR-based cloning approach using a pair of degenerate primers deduced from the nucleotide sequence information of mouse ST3Gal III and IV. The predicted amino acid sequence of mST3Gal V showed 27.3% and 26.4% identity to mST3Gal III and IV, respectively. The recombinant soluble mST3Gal V fused with protein-A, which expressed in the culture media of COS-7 cells, showed activity toward lactosylceramide (LacCer), and synthesized GM3. The apparent Km value for LacCer was 9.3 microM. mST3Gal V did not exhibit any activity toward other substrates we tested in this study, including glycolipids, glycoproteins and disaccharides. The mST3Gal V cDNA transfected F28-7 cells, which express large amount of lactosylceramide and very small amount of GM3 at native stage, expressed a large amount of GM3. The ST3Gal V gene was strongly expressed in mouse brain and liver, which contained a large amount of ganglioside. The gene expression seemed to be coincident with ganglioside expression in mouse. Thus, we conclude that mST3Gal V is the fifth-type alpha 2,3-sialyltransferase carrying GM3 synthetic activity.
Kono
M
M
Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Takashima
S
S
Liu
H
H
Inoue
M
M
Kojima
N
N
Lee
Y C
YC
Hamamoto
T
T
Tsuji
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004789:Enzyme Activation / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D017422:Sequence Analysis, DNA; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D012799:Sialyltransferases; C021343:haematoside synthetase