טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLevy-Adam Flonia
SubjectCharacterization of Protein Domains Critical for
Heparanase Functions
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endo-β-D-glucuronidase involved in cleavage of heparan sulfate (HS) residues and hence participates in extracellular matrix (ECM) degradation and remodeling. The heparanase protein is synthesized as an inactive ~65 kDa protein which undergoes a proteolytic cleavage, yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus and a 6 kDa linker peptide that resides in-between.

In this work, we have shown that heterodimer formation between the 8 kDa and 50 kDa heparanase subunits is essential for heparanase enzymatic activity, and identified protein domains involved in heterodimer formation, which may be potential sites for design of specific heparanase inhibitors In addition, we have identified and characterized two protein domains (Lys158-Asp171 and Gln270-Lys280) that mediate the interaction of heparanase with its HS substrate. A peptide (KKDC), corresponding to Lys158-Asp171 region, binds to heparin and inhibits heparanase enzymatic activity. Deletion of Gln270-Lys280 region (Δ10) from the 65 kDa protein resulted in accumulation of the protein in the cell culture medium, apparently due to the lack of HS-mediated heparanase uptake.

Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions, among them enhanced adhesion of tumor-derived cells and primary T-cells. We provide evidence that the KKDC peptide interacts with cell membrane HS, and results in clustering of Syndecan-1 and Syndecan-4, clusters that remain on the cell surface, in contrast to heparanase-syndecan complex which is subjected to rapid internalization. We demonstrate that heparanase/KKDC peptide efficiently stimulates the adhesion and spreading of primary fibroblasts, CHO, glioma and multiple myeloma cells, which involve activation of the PKC pathway, Src and Rho GTPases. These results support, and further expand the notion that heparanase function is not limited to its enzymatic activity, and highlight heparin binding domains as a valid target for the development of anti-heparanase reagents.   

Heparanase 2 protein has been identified as a heparanase homolog based on sequence homology. Heparanase 2 appears to be expressed by several normal human tissues and cell lines, yet enzymatic, molecular and cellular properties of the protein have not been so far revealed. Our initial characterization of the Hpa 2 protein reveals that the Hpa 2c splice variant shows higher homology to heparanase as compared to Hpa 2b and 2a, however efforts to detect HS degrading ability in different cell lines expressing Hpa 2a/2b/2c have failed, leaving Hpa 1 the only active HS endoglycosidase expressed by mammalian cells.