Intracellular degradation of apolipoprotein B generates an N-terminal 70 kDa fragment in the endoplasmic reticulum.
Regulated apoB degradation in HepG2 cells occurs in the endoplasmic reticulum (ER), is catalyzed by an N-acetylleucylleucylnorleucinal (ALLN)-sensitive proteinase, and generates a specific 70 kDa fragment (Adeli, K., 1994, J. Biol. Chem. 269, 9166-9175) [corrected]. In the present report, we have characterized the 70 kDa fragment by immunoprecipitation of permeabilized HepG2 cells with a battery of monoclonal antibodies against various sites on the apoB molecule. N-Terminal monoclonal antibodies (1D1 and 2D8) were capable of binding to the 70 kDa fragment suggesting that this polypeptide is an N-terminal fragment of the intact apoB. Subcellular fractionation of permeabilized cells and carbonate extraction resulted in the detection of the 70 kDa fragment in the ER lumen. Endoglycosidase H treatment confirmed that the fragment is N-linked glycosylated. We hypothesize that the ALLN-sensitive proteinase which may be located on the luminal side of the ER membrane, catalyzes an initial cleavage of apoB near the N-terminus generating a 70 kDa fragment, which is then released into the ER lumen.
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
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Sallach
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Adeli
D000818:Animals; D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B / Q000276:immunology / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006596:Hexosaminidases; D010446:Peptide Fragments / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D010446:Peptide Fragments; D006596:Hexosaminidases