Alpha 1,3 galactosyltransferase: new sequences and characterization of conserved cysteine residues.
Nucleotide sequences were determined for alpha1,3 galactosyltransferases (alpha1,3 GalTs) from several species (bat, mink, dog, sheep, and dolphin) and compared with those previously determined for this enzyme and members of the alpha1,3 galactosyl/N-acetylgalactosyltransferase (alpha1,3 Gal(NAc)Ts) family of enzymes. Sequence comparison of the newly characterized alpha1,3 GalT nucleotide and predicted amino acid sequences with those previously characterized for other alpha1,3GalT enzymes demonstrated a remarkable level of sequence identity at the nucleotide and amino acid level. The identity of each sequence as an alpha1,3 GalT was confirmed by expressing the encoded protein and characterizing the resulting enzyme. The alpha1,3 GalTs have a significant degree of sequence homology with A and B transferases, the alpha1,3 GalNAcT that catalyzes the synthesis of Forssman antigen, and the recently cloned iso-globotriaosylceramide synthase. Among the conserved residues, there are two Cys residues. To determine if these conserved residues are free or involved in the formation of a disulfide bond, bovine alpha1,3 GalT was characterized by chemical modification and mass spectrometry. Each peptide containing a Cys residue was chemically labeled with an alkylating reagent demonstrating that these enzymes do not contain disulfide bonds. Similar results have recently been reported for A and B transferases (Yen et al., 2000, J. Mass. Spectrom., 35, 990-1002). Thus, the highly conserved Cys residues found in these members of the alpha1,3 Gal(NAc)Ts family of enzymes are likely involved in other important aspects of enzyme structure/function within this enzyme family.
Shetterly
S
S
ORSP, Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA.
Tom
I
I
Yen
T Y
TY
Joshi
R
R
Lee
L
L
Wang
P G
PG
Macher
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002685:Chiroptera; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics*; D004285:Dogs; D004289:Dolphins; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008907:Mink; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016415:Sequence Alignment; D012756:Sheep; D013552:Swine
D005700:Galactosyltransferases; D003545:Cysteine