IMMUNOSELECTION OF GRP94 ENDOPLASMIN FROM A KNRK CELL-SPECIFIC LAMBDA-GT11 LIBRARY USING ANTIBODIES DIRECTED AGAINST A PUTATIVE HEPARANASE AMINO-TERMINAL PEPTIDE

Induction of an invasive phenotype by metastatic tumour cells results in part from inappropriate expression of extracellular matrix-degradin g enzymes normally involved in embryonic morphogenesis, tissue remodel ling, angiogenesis and wound healing. Such enzymes include endoglycosi dases that degrade heparan sulfate (HS) in endothelial basement membra ne, as well as better characterized proteases. Heparanase, an endo-bet a-D-glucuronidase initially detected in B16 melanoma cells, has been d escribed as a M(r) 96 000 glycoprotein with pI of 5.2, and has been im munolocalized to the cell surface and cytoplasm. We have utilized a po lyacrylamide-gel-based HS degradation assay to demonstrate that KNRK, a rat kidney fibroblast cell line transformed by v-K-ros, exhibits HS- degrading activity similar to that of B16F10 mouse melanoma cells. To immunoselect heparanase-expressing clones from a KNRK-cell-specific la mbdagt11 cDNA library, we have also prepared a rabbit anti-serum direc ted against a putative amino-terminal peptide of B16F10 cellular hepar anase. Lysogens from one clone expressed a beta-galactosidase fusion p rotein whose staining with peptide anti-serum was inhibited by competi tion with excess peptide. Dideoxy-mediated sequencing of the insert te rmini of this recombinant revealed that it represents a rat homologue Of M(r) 94,000 glucose-regulated protein (GRP94/endoplasmin), a molecu lar chaperone that contains the exact amino-terminal sequence previous ly attributed to heparanase. Our results call into question the specif icity of this peptide sequence, as well as previous immunolocalization studies of heparanase carried out using such anti-sera. (C) 1994 Wile y-Liss, Inc.