Carboxyl-terminal domain truncation alters apolipoprotein A-I in vivo catabolism.
Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3. Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.
Schmidt
H H
HH
Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Remaley
A T
AT
Stonik
J A
JA
Ronan
R
R
Wellmann
A
A
Thomas
F
F
Zech
L A
LA
Brewer
H B
HB
Hoeg
J M
JM
D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion
D016632:Apolipoprotein A-I; D017931:DNA Primers; D011994:Recombinant Proteins