Hyaluronan biosynthesis by class I streptococcal hyaluronan synthases occurs at the reducing end.
Previous studies reached different conclusions about whether class I hyaluronan synthases (HASs) elongate hyaluronic acid (HA) by addition to the reducing or the nonreducing end. Here we used two strategies to determine the direction of HA synthesis by purified class I HASs from Streptococcus equisimilis and Streptococcus pyogenes. In the first strategy we used each of the two UDP-sugar substrates separately to pulse label either the beginning or the end of HA chains. We then quantified the relative rates of radioactive HA degradation by treatment with beta-glycosidases that act at the nonreducing end. The results with both purified HASs demonstrated that HA elongation occurred at the reducing end. In the second strategy, we used purified S. equisimilis HAS, UDP-glucuronic acid, and UDP[beta-32P]-Glc-NAc to radiolabel nascent HA chains. Under conditions of limiting substrate, the 32P-labeled products were separated from the substrates by paper chromatography and identified as HA-[32P]UDP saccharides based on their degradation by snake venom phosphodiesterase or hyaluronidase and by their binding to a specific HA-binding protein. The 32P radioactivity was chased (released) by incubation with unlabeled UDP-sugars, showing that the HA-UDP linkages turn over during HA biosynthesis. In contrast, HA-[32P]UDP products made by the purified class II Pasteurella multocida HAS were not released by adding unlabeled UDP-sugars, consistent with growth at the nonreducing end for this enzyme. The results demonstrate that the streptococcal class I HAS enzymes polymerize HA chains at the reducing end.
Tlapak-Simmons
Valarie L
VL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Baron
Christina A
CA
Gotschall
Russell
R
Haque
Dewan
D
Canfield
William M
WM
Weigel
Paul H
PH
D001710:Biotin / Q000737:chemistry; D049109:Cell Proliferation; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D008956:Models, Chemical; D016979:Pasteurella multocida / Q000201:enzymology; D010710:Phosphates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology*; D018502:Streptococcus equi / Q000201:enzymology; D013297:Streptococcus pyogenes / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014530:Uridine Diphosphate / Q000737:chemistry
D010710:Phosphates; D014530:Uridine Diphosphate; D001710:Biotin; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases