CARBOXYL-TERMINAL DOMAIN TRUNCATION ALTERS APOLIPOPROTEIN-A-I IN-VIVOCATABOLISM

Apolipoprotein A-I (apoA-I), the major protein of high density lipopro teins, facilitates reverse cholesterol transport from peripheral tissu e to liver. To determine the structural motifs important for modulatin g the in vivo catabolism of human apoA-I (h-apoA-I), we generated carb oxyl-terminal truncation mutants at residues 201 (apoA-I-201), 217 (ap oA-I-217), and 226 (apoA-I-226) by site directed mutagenesis. ApoA-I w as expressed in Escherichia coil 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 for ms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/da y (+/-S.D.) for apoA-I-201, 6.34 +/- 0.81/day for apoA-I-217, and 4.42 +/- 0.51/day for apoA-I-226 were much faster than the FCR of recombin ant 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 ver y high density lipoproteins, whereas the intact recombinant apoA-I (r- apoA-I) and h-apoA-I associated with HDL(2) and HDL(3), Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA -I-201 associated with a phospholipid-rich rabbit apoA-I containing pa rticle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alph a position as did r-apoA-I. These results indicate that the carboxyl-t erminal region (residue 227-243) of apoA-I is critical in modulating t he association of apoA-I with lipoproteins and in vivo metabolism of a poA-I.