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-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-beta-D-glucuronidase initially detected in B16 melanoma cells, has been described as a M(r) 96,000 glycoprotein with pI 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 immunoselect heparanase-expressing clones from a KNRK-cell-specific lambda gt11 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 beta-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 recombinant revealed that it represents a rat homologue of M(r) 94,000 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.
De Vouge
M W
MW
Ottawa Regional Cancer Centre, Ontario, Canada.
Yamazaki
A
A
Bennett
S A
SA
Chen
J H
JH
Shwed
P S
PS
Couture
C
C
Birnboim
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002417:Cattle; D002472:Cell Transformation, Viral / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D011905:Genes, ras / Q000502:physiology; D016678:Genome; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000276:immunology* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins; D007668:Kidney / Q000201:enzymology / Q000502:physiology; D008546:Melanoma, Experimental / Q000201:enzymology / Q000235:genetics; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000235:genetics*; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics; D000099175:Heparanase
D000906:Antibodies; D018076:DNA, Complementary; D005966:Glucuronidase; D006026:Glycoside Hydrolases; D018840:HSP70 Heat-Shock Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D000099175:Heparanase; C053387:endoplasmin; C018551:glucose-regulated proteins