title: Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells.
abstract: The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.
authors.affiliation: Oxford GlycoSciences (OGS), 10 The Quadrant, Abingdon Science Park, Abingdon, Oxon OX14 3YS, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: McKenzie
authors.forename: Kathryn
authors.initials: K
authors.lastname: Young
authors.forename: Margaret
authors.initials: M
authors.lastname: Hircock
authors.forename: James
authors.initials: J
authors.lastname: Bennett
authors.forename: Maina
authors.initials: M
authors.lastname: Bhaman
authors.forename: Robert
authors.initials: R
authors.lastname: Felix
authors.forename: Paul
authors.initials: P
authors.lastname: Turner
authors.forename: Alasdair
authors.initials: A
authors.lastname: Stamps
authors.forename: David
authors.initials: D
authors.lastname: McMillan
authors.forename: Giles
authors.initials: G
authors.lastname: Saville
authors.forename: Stanley
authors.initials: S
authors.lastname: Ng
authors.forename: Sean
authors.initials: S
authors.lastname: Mason
authors.forename: Daniel
authors.initials: D
authors.lastname: Snell
authors.forename: Darren
authors.initials: D
authors.lastname: Schofield
authors.forename: Haiping
authors.initials: H
authors.lastname: Gong
authors.forename: Reid
authors.initials: R
authors.lastname: Townsend
authors.forename: John
authors.initials: J
authors.lastname: Gallagher
authors.forename: Martin
authors.initials: M
authors.lastname: Page
authors.forename: Raj
authors.initials: R
authors.lastname: Parekh
authors.forename: Colin
authors.initials: C
authors.lastname: Stubberfield
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001792:Blood Platelets / Q000201:enzymology*; D002846:Chromatography, Affinity; D019281:Dimerization; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins; D017384:Sequence Deletion; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology*; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D000099175:Heparanase
chemical_list: D000581:Amidohydrolases; D005343:Fibrinolytic Agents; D005966:Glucuronidase; D006493:Heparin; D006497:Heparan Sulfate; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins; D000099175:Heparanase