RRC ID 77540
Author Sato M, Amemiya K, Hayakawa S, Munakata H.
Title Subcellular localization of human heparanase and its alternative splice variant in COS-7 cells.
Journal Cell Biochem Funct
Abstract Heparanase, the enzyme that degrades heparan sulfate, has been implicated to play important and characteristic roles in organogenesis, tissue organization, cell migration, and tumor metastasis. Clarification of its expression, its intracellular sorting, and its secretion is, therefore, of much importance to understand its role in cell biology. In addition to the 1.7 Kb transcript previously reported, we detected a 1.5 Kb transcript of human heparanase by RT-PCR. The smaller transcript was shown to be an alternatively spliced variant lacking exon 5, which contains the essential glutamic acid residue required for enzyme activity. When expressed in COS-7 cells this variant did not show any heparanase activity. Full-length heparanase and the exon 5-deleted splice variant were expressed in COS-7 cells and examined by confocal laser scanning microscopy. Both proteins co-localized with calnexin, a marker protein for the endoplasmic reticulum, and they co-immunoprecipitated with calnexin. Both proteins were postulated to be precursors based upon the results of SDS-PAGE analyses. Treatment with endoglycosidases revealed that all potential N-glycosylation sites in the proteins were glycosylated. Tunicamycin treatment of transfected COS-7 cells inhibited N-glycosylation but did not change the subcellular localization. These results indicate that overexpressed heparanase and its splice variant localize to the endoplasmic reticulum independent of glycosylation in COS-7 cells.
Volume 26(6)
Pages 676-83
Published 2008-8-1
DOI 10.1002/cbf.1492
PMID 18646256
MeSH Alternative Splicing* Amino Acid Sequence Animals Base Sequence COS Cells Calnexin / metabolism Catalysis / drug effects Chlorocebus aethiops Cloning, Molecular Endoplasmic Reticulum / enzymology* Glucuronidase / biosynthesis Glucuronidase / genetics Glucuronidase / metabolism* Glycoside Hydrolases / metabolism Glycosylation / drug effects Humans Immunoprecipitation Isoenzymes / biosynthesis Isoenzymes / genetics Isoenzymes / metabolism Microscopy, Confocal Molecular Sequence Data Recombinant Proteins / biosynthesis Recombinant Proteins / metabolism Transfection Tunicamycin / pharmacology
IF 2.632
Human and Animal Cells COS-7(RCB0539)