In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) to apolipoprotein AI in rabbits.
In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) from E. coli to apolipoprotein (apo) AI was investigated. rh-Met-proapo AI was labeled with 125I, and then administered intravenously to rabbits. Blood was sampled periodically for 6 days. The plasma decay curves of radioiodinated rt-Met-proapo AI were similar to those of human mature apo AI (fractional catabolic rate (FCR); 1.018 +/- 0.090/day vs. 0.976 1 0.031/day, respectively). In vivo conversion of rh-Met-proapo AI to mature apo AI was examined by autoradiography of the isoelectric focusing (IEF) slab gel, i.e., the HDL fraction from each sampling point was semiquantitatively applied to IEF. It was found that the radioactivity of rh-Met-proapo AI migrated to more acidic isoproteins, the conversion was complete within 24 h, and the FCR of rh-Met-proapo AI was 9.20 +/- 1.34/day. Although the plasma decay curves of both human pro (rh-Met-proapo AI) and mature apo AI were significantly steeper than those of rabbit mature apo AI4 and apo AI5 (FCR; 0.703 +/- 0.027/day and 0.795 +/- 0.031/day, respectively), the conversion rate of human rt-Met-proapo AI to mature apo AI in rabbit was assumed to be 1:1. In vitro incubation of rh-Met-proapo AI with rabbit serum produced mature apo AI isoproteins, as determined by the apo AI immunoblotting method. Prediction of the amino acid sequence at the NH2 terminus of rabbit proapo AI showed that the prosegment consisted of an alpha helix with a high probability of a beta turn at Pro9, which is close to that in humans. Thus, (1) the proteolytic cleavage of proapo AI is an extracellular event, (2) the converting enzyme in rabbits can also process human proapo AI, (3) this converting enzyme does not specifically and directly attack the Gln6-Asp7 bond which links the carboxyl-terminal residue of the hexapeptide to the amino-terminal residue of human mature apo AI. The conformation of proapo AI at the NH2 terminus (alpha helix of the prosegment and a beta turn at Pro9) may have a key role in this cleavage, and (4) the examination of rh-Met-proapo AI in rabbits helps to explain the early events of HDL biogenesis.
Saku
K
K
Department of Internal Medicine, Fukuoka University School of Medicine, Japan.
Liu
R
R
Ohkubo
K
K
Bai
H
H
Hirata
K
K
Yamamoto
K
K
Morimoto
Y
Y
Yamada
K
K
Arakawa
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000302:isolation & purification / Q000378:metabolism*; D001054:Apolipoproteins A / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D011498:Protein Precursors; D011994:Recombinant Proteins