Subcellular localization of human heparanase and its alternative splice variant in COS-7 cells.
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.
Sato
Mayumi
M
Department of Biochemistry, Kinki University School of Medicine, Osaka-Sayama, Japan.
Amemiya
Kana
K
Hayakawa
Sumio
S
Munakata
Hiroshi
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D037281:Calnexin / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D000099175:Heparanase
D037281:Calnexin; D005966:Glucuronidase; D006026:Glycoside Hydrolases; D007527:Isoenzymes; D011994:Recombinant Proteins; D014415:Tunicamycin; D000099175:Heparanase