Heparanases produce distinct populations of heparan sulfate glycosaminoglycans in Chinese hamster ovary cells.
Once internalized, cell-associated heparan sulfate proteoglycans are degraded to short glycosaminoglycans by the action of endoglycosidases or heparanases. We have begun to address the question of how many heparanases are responsible for this process by analyzing short heparan sulfate chains produced in vivo by Chinese hamster ovary (CHO) cell heparanases. Short heparan sulfate chains were purified from CHO cells and labeled at the reducing end with [3H]NaBH4. Hydrolysis of the chains to monosaccharides and analysis of the 3H-sugar alcohols indicate that heparanase activities in CHO cells are endo-beta-glucuronidases. The modification state of the heparanase-derived glycosaminoglycans was examined by treating the [3H]heparan sulfate chains with nitrous acid or bacterial heparin lyases, which cut the chain at specific sequences, and analyzing the products by P2 gel filtration chromatography. Two populations of short chains were identified that differ in the extent of modification on the nonreducing side of the heparanase cleavage site. One class of chains is unmodified for at least 9 residues from the reducing end, while the other group has a modified domain within 3-7 residues from the heparanase cleavage site. Our results suggest a model of heparanase action where the enzymes recognize differences in sulfate content between modified and unmodified regions and bind to sites that encompass both domains. The enzymes then cleave the glycosaminoglycan at junctions between the modified and unmodified sequences to produce the different populations of short heparan sulfate chains.
Bame
K J
KJ
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110, USA.
Robson
K
K
D000818:Animals; D002850:Chromatography, Gel; D002854:Chromatography, Paper; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005966:Glucuronidase / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010053:Ovary / Q000378:metabolism*; D000099175:Heparanase
D005966:Glucuronidase; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D000099175:Heparanase