Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II.
Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine-sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine-glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.
Rhomberg
A J
AJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Shriver
Z
Z
Biemann
K
K
Sasisekharan
R
R
D001665:Binding Sites; D001704:Biopolymers; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry*; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001704:Biopolymers; D006025:Glycosaminoglycans; D006493:Heparin; D011133:Polysaccharide-Lyases; C114663:heparinase II