title: A liquid chromatography-mass spectrometry-based approach to characterize the substrate specificity of mammalian heparanase.
abstract: Extracellular heparanase activity releases growth factors and angiogenic factors from heparan sulfate (HS) storage sites and alters the integrity of the extracellular matrix. These activities lead to a loss of normal cell matrix adherent junctions and correlate with invasive cellular phenotypes. Elevated expression of heparanase is associated with several human cancers and with vascular remodeling. Heparanase cleaves only a limited fraction of glucuronidic linkages in HS. There have been few investigations of the functional consequences of heparanase activity, largely due to the heterogeneity and complexity of HS. Here, we report a liquid chromatography-mass spectrometry (LC-MS)-based approach to profile the terminal structures created by heparanase digestion and reconstruct the heparanase cleavage sites from the products. Using this method, we demonstrate that heparanase cleaves at the non-reducing side of highly sulfated HS domains, exposing cryptic growth factor binding sites. This cleavage pattern is observed in HS from several tissue sources, regardless of overall sulfation degree, indicating a common recognition pattern. We further demonstrate that heparanase cleavage of HS chains leads to increased ability to support FGF2-dependent cell proliferation. These results suggest a new mechanism to explain how heparanase might potentiate the uncontrolled cell proliferation associated with cancer through its ability to activate nascent growth factor-promoting domains within HS.
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and.
authors.forename: Yang
authors.initials: Y
authors.lastname: Mao
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and.
authors.forename: Yu
authors.initials: Y
authors.lastname: Huang
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and.
authors.forename: Jo Ann
authors.initials: JA
authors.lastname: Buczek-Thomas
authors.affiliation: R&D Systems, Minneapolis, Minnesota 55413.
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Ethen
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Nugent
authors.affiliation: R&D Systems, Minneapolis, Minnesota 55413.
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
authors.affiliation: From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and jzaia@bu.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005109:Extracellular Matrix / Q000737:chemistry* / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D053671:Syndecan-4 / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D000099175:Heparanase
chemical_list: D016222:Fibroblast Growth Factor 2; D005966:Glucuronidase; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D053671:Syndecan-4; D000099175:Heparanase; C508304:SDC4 protein, human
keywords: Glycosaminoglycan; Glycosylation; Heparan Sulfate; Heparanase; Heparin; Heparin-binding Protein