A soluble form of alpha1,3-galactosyltransferase functions within cells to galactosylate glycoproteins.
It has been assumed that membrane-bound glycosyltransferases function within the Golgi apparatus to glycosylate glycoproteins. We now report, however, that a truncated, soluble recombinant form of the murine alpha1,3-galactosyltransferase expressed in human 293 cells is highly efficient and comparable to the full-length enzyme in alpha-galactosylating both newly synthesized membrane-associated and secreted glycoproteins. Although the soluble enzyme was secreted by cells as expected, we also found that the full-length, membrane-associated form was secreted. Unexpectedly, both secreted forms are cleaved identically at two primary sites within the stem region by endogenous protease(s) at the indicated positions in the sequence 73KDWW (downward arrow) FPS (downward arrow) WFKNG. These results demonstrate that the soluble alpha1,3-galactosyltransferase is functional within the cell and that specific proteolysis occurs in the stem region. The widespread occurrence of different soluble glycosyltransferases secreted by cells suggests that normal glycoconjugate biosynthesis may involve cooperation between membrane-bound and soluble enzymes.
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma Center for Molecular Medicine, Oklahoma City, Oklahoma 73190, USA.
Cummings
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts; D003470:Culture Media; D005690:Galactose / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D003470:Culture Media; D006023:Glycoproteins; D037102:Lectins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C037959:beta-D-galactosyl-N-acetylglucosaminylglycopeptide alpha-1,3-galactosyltransferase; D005690:Galactose