Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment

Abstract

Heparanase is an endo-␤-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and on the cell surface. Human proheparanase is produced as a latent protein of 543 amino acids whose activation involves excision of an internal linker segment (Ser 110 -Gln 157 ), yielding the active heterodimer composed of 8-and 50-kDa subunits. Applying cathepsin L knock-out tissues and cultured fibroblasts, as well as cathepsin L gene silencing and overexpression strategies, we demonstrate, for the first time, that removal of the linker peptide and conversion of proheparanase into its active 8 ؉ 50-kDa form is brought about predominantly by cathepsin L. Excision of a 10-amino acid peptide located at the C terminus of the linker segment between two functional cathepsin L cleavage sites (Y156Q and Y146Q) was critical for activation of proheparanase. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrates that the entire linker segment is susceptible to multiple endocleavages by cathepsin L, generating small peptides. Mass spectrometry demonstrated further that an active 8-kDa subunit can be generated by several alternative adjacent endocleavages, yielding the precise 8-kDa subunit and/or slightly elongated forms. Altogether, the mode of action presented here demonstrates that processing and activation of proheparanase can be brought about solely by cathepsin L. The critical involvement of cathepsin L in proheparanase processing and activation offers new strategies for inhibiting the prometastatic, proangiogenic, and proinflammatory activities of heparanase.

Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumorigenesis in mice

“…The 50-kDa enzyme represents an N-terminally processed heparanase, which is at least 100-fold more active than the 65-kDa proenzyme (7). CatL is the predominant protease responsible for processing and activation of proheparanase (10). In accordance, increased levels of CatL mRNA ( Figure 1C) and protein (Figure 1, D and G) were detected in the mouse colonic tissue at corresponding time points (discussed below).…”

“…Posttranslational processing represents an additional key regulatory mechanism. Heparanase is produced as a latent 65-kDa proenzyme whose activation involves proteolytic cleavage, brought about predominantly by cathepsin L (CatL), yielding an enzymatically active heterodimer composed of 8- and 50-kDa subunits (10).…”

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

“…Processing and activation of heparanase occur after proteolytic processing by cathepsin L, a characteristic lysosomal cysteine proteinase of the papain superfamily of peptidases that is implicated in multiple physiological and pathological processes (23)(24)(25). To investigate whether PRRSV infection activates heparanase via cathepsin L, cathepsin L activity in Marc-145 cells was initially assessed at various times or MOIs after PRRSV infection using a Magic Red cathepsin L detection kit.…”

NK cell heparanase controls tumor invasion and immune surveillance

“…There is only 1 enzymatically active form of heparanase in mammals that is expressed at very low levels in normal tissues, and heparanase deficiency in mice causes no obvious pathophysiologies (5,6). Heparanase is secreted as an enzymatically inactive pro-heparanase and requires reuptake into the cell and processing by cathepsin-L in lysosomes (7) or α granules (8) in order to give rise to the enzymatically active heparanase protein. In recent years, it has become evident that heparanase is a complex protein with both enzymatic and nonenzymatic activities, depending on its location and cleavage (9)(10)(11)(12)(13).…”