Identification of Active-Site Residues of the Pro-Metastatic Endoglycosidase Heparanase
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- 作者：Mark D. Hulett ; June R. Hornby ; Stephen J. Ohms ; Johannes Zuegg ; Craig Freeman ; Jill E. Gready ; and Christopher R. Parish
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：December 26, 2000
- 年：2000
- 卷：39
- 期：51
- 页码：15659 - 15667
- 全文大小：256K
- 年卷期：v.39,no.51(December 26, 2000)
- ISSN：1520-4995
文摘
Heparanase is a 
-D-endoglucuronidase that cleaves heparan sulfate (HS) and has beenimplicated in many important physiological and pathological processes, including tumor cell metastasis,angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of humanheparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identifiedbetween heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) fromglycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the criticalactive-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes.Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (
/)8 TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a numberof glycosyl hydrolases from GH-A, Glu225 and Glu343 of human heparanase were identified as the likelyproton donor and nucleophile residues, respectively. The substitution of these residues with alanine andthe subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degradingcapacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues(Glu378 and Glu396), both predicted to be outside the active site, did not affect heparanase activity. Thesedata suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalyticmechanism that involves two conserved acidic residues, a putative proton donor at Glu225 and a nucleophileat Glu343.
-D-endoglucuronidase that cleaves heparan sulfate (HS) and has beenimplicated in many important physiological and pathological processes, including tumor cell metastasis,angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of humanheparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identifiedbetween heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) fromglycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the criticalactive-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes.Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (/)8 TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a numberof glycosyl hydrolases from GH-A, Glu225 and Glu343 of human heparanase were identified as the likelyproton donor and nucleophile residues, respectively. The substitution of these residues with alanine andthe subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degradingcapacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues(Glu378 and Glu396), both predicted to be outside the active site, did not affect heparanase activity. Thesedata suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalyticmechanism that involves two conserved acidic residues, a putative proton donor at Glu225 and a nucleophileat Glu343.