Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme.
We have recently identified and cloned the gene for hyaluronan (HA) synthase, hasA, from group A Streptococci [DeAngelis, P.L., Papaconstantinou, J., & Weigel, P.H. (1993) J. Biol. Chem. 268, 19181-19184]. We have now generated two polyclonal monospecific antibodies against synthetic peptides corresponding to portions of the deduced protein. Both antibodies recognize a protein with an apparent molecular weight of 42,000 either from wild-type Streptococcus pyogenes or from Escherichia coli containing the cloned gene on a plasmid. Immobilized affinity-purified antibody depleted HA synthase activity from functional detergent extracts of streptococcal membranes in a specific fashion. The immobilized protein displayed HA synthase activity, and HasA was the major bound polypeptide. The recombinant HA synthase behaves identically to that from Streptococci, with respect to sugar nucleotide specificity and polysaccharide production. Only the authentic sugar nucleotides UDP-glucuronic acid and UDP-N-acetylglucosamine support HA polymerization. The recombinant enzyme elongates HA in a processive manner and rapidly produces polymers on the order of > or = 5 x 10(6) Da at rates of about 10-30 monosaccharides/s at three times the apparent Km of substrates.
DeAngelis
P L
PL
Department of Human Biological Chemistry and Genetics, University of Texas, Medical Branch, Galveston 77555-0647.
Weigel
P H
PH
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014166:Transferases; D029867:Xenopus Proteins
D000906:Antibodies; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases