Human proapolipoprotein A-II is cleaved following secretion from Hep G2 cells by a thiol protease.
The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.
D000595:Amino Acid Sequence; D001054:Apolipoproteins A / Q000302:isolation & purification / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002460:Cell Line; D003470:Culture Media; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008113:Liver Neoplasms / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*
D001054:Apolipoproteins A; D003470:Culture Media; D011480:Protease Inhibitors; D011498:Protein Precursors; C039768:preproapolipoprotein A-II; D010450:Endopeptidases; D003546:Cysteine Endopeptidases