Immunoselection of GRP94/endoplasmin from a KNRK cell‐specific λGTLL library using antibodies directed against a putative heparanase amino‐terminal peptide

Publisher Website
Google Scholar
Abstract
Induction of an invasive phenotype by metastatic tumour cells results in part from inappropriate expression of extracellular matrix‐degrading enzymes normally involved in embryonic morphogenesis, tissue remodelling, angiogenesis and wound healing. Such enzymes include endoglycosidases that degrade heparan sulfate (HS) in endothelial basement membrane, as well as better characterized proteases. Heparanase, an endo‐β‐D‐glucuronidase initially detected in B16 melanoma cells, has been described as a Mr 96 000 glycoprotein with pl of 5.2, and has been immunolocalized to the cell surface and cytoplasm. We have utilized a polyacrylamide‐gel‐based HS degradation assay to demonstrate that KNRK, a rat kidney fibroblast cell line transformed by v‐K‐ras, exhibits HS‐degrading activity similar to that of B16F10 mouse melanoma cells. To immuno‐select heparanase‐expressing clones from a KNRK‐cell‐specific λgtII cDNA library, we have also prepared a rabbit anti‐serum directed against a putative amino‐terminal peptide of B16F10 cellular heparanase. Lysogens from one clone expressed a β‐galactosidase fusion protein whose staining with peptide anti‐serum was inhibited by competition with excess peptide. Dideoxy‐mediated sequencing of the insert termini of this reco.mbinant revealed that it represents a rat homologue of Mr94,000 glucose‐regulated protein (GRP94/endoplasmin), a molecular chaperone that contains the exact amino‐terminal sequence previously attributed to heparanase. Our results call into question the specificity of this peptide sequence, as well as previous immunolocalization studies of heparanase carried out using such anti‐sera.