Human heparanase. Purification, characterization, cloning, and expression.
Heparan sulfate and heparan sulfate proteoglycans are present in the extracellular matrix as well as on the external cell surface. They bind various molecules such as growth factors and cytokines and modulate the biological functions of binding proteins. Heparan sulfate proteoglycans are also important structural components of the basement membrane. Heparanase is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this study, we report the purification of a human heparanase from an SV40-transformed embryonic fibroblast cell line WI38/VA13 by four sequential column chromatographies. The activity was measured by high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. The enzyme was purified to homogeneity, yielding a peptide with an apparent molecular mass of 50 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Using the amino acid sequences of the N-terminal and internal heparanase peptides, a cDNA coding for human heparanase was cloned. NIH3T3 and COS-7 cells stably transfected with pBK-CMV expression vectors containing the heparanase cDNA showed high heparanase activities. The homology search revealed that no homologous protein had been reported.
Toyoshima
M
M
Discovery Research, Takarazuka Research Institute, Novartis Pharma K. K., 10-66 Miyuki-cho, Takarazuka 665-8666, Japan. motowa.nakajima@pharma.novartis.com
Nakajima
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D000099175:Heparanase
D016650:Fluorescein-5-isothiocyanate; D005966:Glucuronidase; D006026:Glycoside Hydrolases; D000099175:Heparanase