Molecular cloning and characterization of beta-1,4-galactosyltransferase expressed in mouse testis

Eur J Biochem. 1997 Mar 15;244(3):706-12. doi: 10.1111/j.1432-1033.1997.00706.x.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Escherichia coli / genetics
  • Female
  • Gene Expression
  • Male
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • N-Acetyllactosamine Synthase / chemistry
  • N-Acetyllactosamine Synthase / genetics*
  • N-Acetyllactosamine Synthase / metabolism
  • Protein Structure, Secondary
  • Rabbits
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Restriction Mapping
  • Sequence Homology, Amino Acid
  • Testis / enzymology*

Substances

  • DNA, Complementary
  • Recombinant Fusion Proteins
  • N-Acetyllactosamine Synthase

Associated data

  • GENBANK/D37790
  • GENBANK/D37791