title: Apolipoprotein B is intracellularly associated with an ER-60 protease homologue in HepG2 cells.
abstract: Two ALLN (N-acetyl-leucyl-leucyl-norleucinal)-sensitive endoplasmic reticulum (ER)-localized proteases (ER-60 and ER-72) were recently purified from rat liver. We used an antibody to rat ER-60 to investigate the possible role of this protease in apolipoprotein B (apoB) degradation. First, immunoprecipitation and immunoblotting experiments with the anti-rat ER-60 antibody suggested that HepG2 cells contain a homologue of ER-60 with an approximate molecular mass of 58-60 kDa. The ER-60 homologue was mostly associated with the luminal contents of HepG2 microsomes. Evidence from co-immunoprecipitation and cross-linking experiments appear to suggest that the ER-60 homologue in HepG2 cells is associated with apoB intracellularly. A small pool of apoB was recovered when HepG2 lysates were subjected to immunoprecipitation with anti-rat ER-60 antibody followed by a second immunoprecipitation with anti-apoB antibody. Furthermore, cross-linking of permeabilized cells with dithiobis(succinimidylpropionate) further demonstrated association of apoB with the ER-60 homologue in HepG2 cells. Three polypeptides with molecular masses of 78, 66, and 50 kDa were consistently found to be associated with apoB as well as the 58-kDa ER-60 homologue. The 78-kDa protein associated with both apoB and ER-60 appeared to represent immunoglobulin heavy chain-binding protein (BiP) based on immunoprecipitation with a monoclonal antibody. Cross-linking and immunoblotting experiments suggested the association of the 78-kDa BiP with both the 58-kDa ER-60 homologue as well as the 550-kDa apoB. In summary, the data suggests that HepG2 cells contain a 58-kDa protein which is homologous to the rat liver ER-60 in size, antigenecity, and intracellular localization. The ER-60 homologue in HepG2 cells appears to be closely associated with apoB, as well as other proteins possibly representing ER chaperones such as BiP. We hypothesize that the ER-60 homologue may be involved in the degradation of apoB in the ER lumen of HepG2 cells.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, N9B 3P4 Canada. adeli@uwindsor.ca
authors.forename: K
authors.initials: K
authors.lastname: Adeli
authors.forename: J
authors.initials: J
authors.lastname: Macri
authors.forename: A
authors.initials: A
authors.lastname: Mohammadi
authors.forename: M
authors.initials: M
authors.lastname: Kito
authors.forename: R
authors.initials: R
authors.lastname: Urade
authors.forename: D
authors.initials: D
authors.lastname: Cavallo
mesh_terms: D000818:Animals; D001055:Apolipoproteins B / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats
chemical_list: D001055:Apolipoproteins B; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D003546:Cysteine Endopeptidases; C077733:ER-60 protease