Molecular cloning and characterization of beta-1,4-galactosyltransferase expressed in mouse testis.
Using an affinity-purified antibody against beta-1,4-galactosyltransferase purified from F9 embryonal carcinoma cells, we screened a lambda gt11 expression library constructed from the same cell line. The cDNA clone obtained encoded a 46-kDa protein. Among adult mouse organs, the testis was found to express large amounts of the corresponding mRNA. An antibody against the 46-kDa protein was raised in a rabbit by immunization with a maltose-binding-protein fusion protein produced in Escherichia coli. The affinity-purified antibody against the cloned 46-kDa protein reacted with a 59-kDa protein in sperm extract on western blotting. Among the proteins in the F9 beta-1,4-galactosyltransferase preparation, which were mostly 68-kDa and 59-kDa species, 59-kDa and 50-kDa bands reacted with the antibody against the cloned 46-kDa protein. The cloned 46-kDa protein had type-II transmembrane topology and some blocks of sequence similarity with the known beta-1,4-galactosyltransferase. Furthermore, predicted secondary structures were similar over large portions of the two proteins. The histidine-tagged 46-kDa protein produced in E. coli was partially adsorbed onto a N-acetylglucosamine-Affi-Gel column and eluted by 20 mM N-acetylglucosamine. The histidine-tagged 46-kDa protein, which was purified to homogeneity, had a small, but significant amount of beta-1,4-galactosyltransferase activity. These results strongly suggest that the 46-kDa protein is a beta-1,4-galactosyltransferase.
Uehara
K
K
Department of Biochemistry, Nagoya University School of Medicine, Showa-ku, Japan.
Muramatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000201:enzymology*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D009237:N-Acetyllactosamine Synthase