lsoproteins of human apolipoprotein A 4 : isolation and characterization

In human serum, polymorphism of apoA-I1 predominantly in HDLs could be demonstrated. HDLS-apoA-I1 was composed of four isoproteins, each with a molecular weight of 8600 (reduced form) and identical immunological properties. The isoproteins are designated apoA-11-1 (PI 5.16), apoA-11-2 (PI 4.89) corresponding to the already known apoAI1 monomer band, apoA-11-3 (PI 4.58), and apoA-11-4 (PI 4.31). The amino acid compositions of the A-I1 isoproteins were virtually identical with the published data for apoA-11. Treatment with acid phosphatase, alkaline phosphatase, or neuraminidase before electrophoresis did not alter the apoAI1 pattern. The apoA-I1 isoprotein pattern was studied in ten male and ten female normolipidemic volunteers, in two patients with Tangier disease, and in three patients with abetalipoproteinemia. The isoelectric focusing patterns of apoA-I1 appeared virtually identical in all subjects. However, in Tangier disease, due to the low apoA-I1 concentration, only apoA11-1 and apoA-11-2 were detectable, and in abetalipoproteinemia a different relative distribution pattern of the individual isoforms was found as compared to normal HDLS . ! A Our studies indicate that apoA-11, similar to apoA-I, exists in several isoforms. The relationship of these isoforms to each other is at present unclear. They may originate from relatively basic isoproteins that are modified in charge by post-translational processes such as proteolytic cleavage, sequential deamidation, or other mechanisms-G. Schmitz, K. Ilsemann, B. Melnik, and C. Assmann. Isoproteins of human apolipoprotein A-11: isolation and characterization. J. Lipid Res. 1983. 24: 10211029. Supplementary key words HDLs isoelectric focusing crossed immunoelectrophoresis gel blotting abetalipoproteinemia Apolipoprotein A-I1 (apoA-11) is one of the major protein components of human serum high density lipoproteins (HDL) (1, 2). It consists of two identical polypeptide chains, each containing 77 amino acid residues of known sequence (3-5). The two polypeptide chains are crosslinked by a single disulfide bond at cysteine-6 (3). The minimum molecular weight of the dimer is 17,380. The amino-terminal residue of each chain is pyrrolidone carboxylic acid, and the carboxy-terminal residue is glutamine. The protein contains no carbohydrates and has been shown to lack histidine, arginine, and tryptophan (3, 6, 7). ApoA-I1 readily recombines with phospholipids (phosphatidylcholine, sphingomyelin) to form protein-phospholipid complexes (812), which leads to an increase of a-helicity from 25 to 48%. In addition, apoA-I1 associates with other apoproteins (e.g., apoA-I) by protein-protein interaction (2, 11) or forms apoprotein complexes (e.g., with apoE or apoAI Milano) as a mixed disulfide (13, 14). An interrelationship between apoA-I and apoA-I1 in the interaction with HDL (15, 16) or phospholipid vesicles (17) has been reported and it could be demonstrated that 2 mol of apoA-I1 can displace 1 mol of apoA-I from the HDL surface. It has been recently demonstrated that apoA-I, the major apoprotein of HDL, is composed of at least six isoproteins that differ in charge but not in molecular weight (1 8, 19). It appears from those studies that the basic isoproteins of apoA-I are synthesized and modified to more acidic forms that are the major apoA-I components in plasma. These findings suggest a complicated pattern of processing, involving proteolytic cleavage and charge modification, before the major isoprotein form of apoA-I present in plasma is attained. However, similar isoforms of apoprotein A-I1 have not yet been described. In our recent studies on the HDLJHDL2 interconversion and the formation of HDLl (20, 21), some unknown apoprotein bands were detected in isoelectric focusing gels of HDL subfractions that might be related to apoprotein A-I1 isoforms. We have therefore investigated whether apoprotein A-I1 isoforms exist in human lipoprotein fractions. MATERIALS AND METHODS