Cathepsin L is responsible for processing and activation of proheparanase through multiple cleavages of a linker segment.
Heparanase is an endo-beta-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.
Abboud-Jarrous
Ghada
G
Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Atzmon
Ruth
R
Peretz
Tamar
T
Palermo
Carmela
C
Gadea
Bedrick B
BB
Joyce
Johanna A
JA
Vlodavsky
Israel
I
D000595:Amino Acid Sequence; D001665:Binding Sites; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000099175:Heparanase
D056668:Cathepsin L; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D005966:Glucuronidase; D034741:RNA, Small Interfering; D000099175:Heparanase; C534281:CTSL protein, human