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Mechanism of activation of human heparanase investigated by protein engineering


  • Nardella C, Lahm A, Pallaoro M, Brunetti M,
  • Vannini A.,
  • Steinkühler C


The aim of this study was to investigate the mechanism of activation of human heparanase, a key player in heparan sulfate degradation, thought to be involved in normal and pathologic cell migration processes. Active heparanase arises as a product of a series of proteolytic processing events. Upon removal of the signal peptide, the resulting, poorly active 65 kDa species undergoes the excision of an intervening 6 kDa fragment generating an 8 kDa polypeptide and a 50 kDa polypeptide, forming the fully active heterodimer. By engineering of tobacco etch virus protease cleavage sites at the N- and C-terminal junctions of the 6 kDa fragment, we were able to reproduce the proteolytic activation of heparanase in vitro using purified components, showing that cleavage at both sites leads to activation in the absence of additional factors. On the basis of multiple-sequence alignment of the N-terminal fragment, we conclude that the first beta/alpha/beta element of the postulated TIM barrel fold is contributed by the 8 kDa subunit and that the excised 6 kDa fragment connects the second beta-strand and the second alpha-helix of the barrel. Substituting the 6 kDa fragment with the topologically equivalent loop from Hirudinaria manillensis hyaluronidase or connecting the 8 and 50 kDa fragments with a spacer of three glycine-serine pairs resulted in constitutively active, single-chain heparanases which were comparable to the processed, heterodimeric enzyme with regard to specific activity, chromatographic profile of hydrolysis products, complete inhibition at NaCl concentrations above 600 mM, a pH optimum of pH approximately 5, and inhibition by heparin with IC(50)s of 0.9-1.5 ng/microL. We conclude that (1) the heparanase heterodimer (alpha/beta)(8)-TIM barrel fold is contributed by both 8 and 50 kDa subunits with the 6 kDa connecting fragment leading to inhibition of heparanase by possibly obstructing access to the active site, (2) proteolytic excision of the 6 kDa fragment is necessary and sufficient for heparanase activation, and (3) our findings open the way to the production of recombinant, constitutively active single-chain heparanase for structural studies and for the identification of inhibitors.

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