Site-directed Mutagenesis, Proteolytic Cleavage, and Activation of Human Proheparanase

Abstract

Heparanase is an endo-␤-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and cell surfaces. Human proheparanase is produced as a latent 65-kDa polypeptide undergoing processing at two potential proteolytic cleavage sites, located at Glu 109 -Ser 110 (site 1) and Gln 157 -Lys 158 (site 2). Cleavage of proheparanase yields 8-and 50-kDa subunits that heterodimerize to form the active enzyme. The fate of the linker segment (Ser 110 -Gln 157 ) residing between the two subunits, the mode of processing, and the protease(s) engaged in proheparanase processing are currently unknown. We applied multiple site-directed mutagenesis and deletions to study the nature of the potential cleavage sites and amino acids essential for processing of proheparanase in transfected human choriocarcinoma cells devoid of endogenous heparanase but possessing the enzymatic machinery for proper processing and activation of the proenzyme. Although mutagenesis at site 1 and its flanking sequences failed to identify critical residues for proteolytic cleavage, processing at site 2 required a bulky hydrophobic amino acid at position 156 (i.e. P2 of the cleavage site). Substitution of Tyr 156 by Ala or Glu, but not Val, resulted in cleavage at an upstream site in the linker segment, yielding an improperly processed inactive enzyme. Processing of the latent 65-kDa proheparanase in transfected Jar cells was inhibited by a cell-permeable inhibitor of cathepsin L. Moreover, recombinant 65-kDa proheparanase was processed and activated by cathepsin L in a cell-free system. Altogether, these results suggest that proheparanase processing at site 2 is brought about by cathepsin L-like proteases. The involvement of other members of the cathepsin family with specificity to bulky hydrophobic residues cannot be excluded. Our results and a threedimensional model of the enzyme are expected to accelerate the design of inhibitory molecules capable of suppressing heparanase-mediated enhancement of tumor angiogenesis and metastasis.

Two Heparanase Splicing Variants with Distinct Properties Are Necessary in Early Xenopus Development

“…The latent 65-kDa form of heparanase, which does not show significant enzymatic activity, is cleaved to generate an active 50-kDa enzyme (45). By Western blot, we observed no cleavage of either short or long heparanase when overexpressed in a mammalian system.…”

Adaptive evolution of heparanase in hypoxia-tolerantSpalax: Gene cloning and identification of a unique splice variant

“…Human heparanase is synthesized as a latent 65-kDa precursor whose activation involves proteolytic cleavage at two potential sites located at the N-terminal region of the molecule (Glu-109-Ser-110 and Gln-157-Lys-158), resulting in the formation of two subunits that heterodimerize to form the active heparanase enzyme (13)(14)(15). Homologous cleavage sites were identified in the Spalax heparanase at Glu-140-Pro-141 and Gln-188-Lys-189.…”

“…Despite earlier reports on the existence of several distinct mammalian HS-degrading endoglycosidases (heparanases), the cloning of the same single gene by several groups (6,7,11,12) suggests that mammalian cells express primarily a single dominant functional heparanase enzyme. Human heparanase is synthesized as a latent 65-kDa precursor whose processing involves proteolytic cleavage and formation of an active enzyme composed of 50-and 8-kDa subunits (13)(14)(15). Because heparanase promotes angiogenesis and cancer progression, we decided to investigate the evolution of this unique enzyme in a wild mammal that was exposed to underground hypoxic stress throughout the family Spalacidae evolutionary history (16).…”

Reactive oxygen species mediate high glucose-induced heparanase-1 production and heparan sulphate proteoglycan degradation in human and rat endothelial cells: a potential role in the pathogenesis of atherosclerosis

“…Western blot HPR1 in conditioned media and cell lysates was enriched by sepharose-heparin beads (Amersham Biosciences, Piscataway, NJ, USA) as previously published [31]. HPR1 protein in the supernatant fractions, presenting mainly as the 65 kDa proenzyme, was detected by Western blot with a rabbit polyclonal antibody (Santa Cruz) that recognises the 65 kDa form of HPR1.…”