Structure of apolipoprotein C-IIToronto, a nonfunctional human apolipoprotein.

Abstract

We previously reported a family with apolipoprotein C-II (apoC-II) deficiency characterized by the presence of a mutant apoC-il, apoC-llT0r~0t. We now report the purification and primary structure of apoC-IITr..t. The sequence of apoC-llTrOft. is identical to that of normal apoC-il from residues 1-68. It differs from residue 69, where AspGln7O-Val7l-Leu72_Ser73-Val74-Leu75-Lys76-Gly77-Glu78 Glu79 is replaced by Thr69-Lys70-phe7l-Phe72-Leu73-Cys74. This is consistent with the deletion of a nucleotide in the codon for either Thr'8 (9) to allow the isolation of chylomicrons and very low density lipoproteins from 700 ml of plasma in a single run. The Ti 15 rotor was brought to 3000 rpm and loaded sequentially with 700 ml of 0.15 M NaCl (1.006 g/ml), 700 ml of plasma adjusted with KBr to 1.063 g/ml, and 300 ml of 0.15 M NaCl adjusted with KBr to 1.25 g/ml (solution A). The chylomicrons were floated by centrifugation at 20,000 rpm for 1 hr and, after slowing the rotor to 3000 rpm, they were displaced with 60 ml of solution A. The plasma remaining in the rotor was spun at 30,000 rpm for 18 hr, the rotor was slowed to 3000 rpm, and the very low density lipoproteins were displaced with 50 ml of solution A. Chylomicrons and very low density lipoproteins adhering to the rotor core were isolated separately after the rotor had come to a complete stop. Each fraction was dispersed in 0.15 M NaCl/0.01% Na2EDTA/0.02% NaN3 and reisolated by centrifugation in an 80 Ti rotor for 16 hr at 45,000 rpm.The C apolipoproteins were isolated from these fractions by treatment with acetone (10, 11). The lyophilized protein was delipidated with ethanol/diethylether (11). The analysis of this acetone-soluble product by analytical polyacrylamide isoelectric focusing using a pH 4-6.5 gradient showed it to contain apoC-IIT and the isomorphs of apolipoprotein C-IIIi.e., C-III-0, C-III-1, and C-III-2 (8).ApoC-IIT was purified to homogeneity by a single preparative flat-bed isoelectric focusing step using Ultradex granulated gel and pH 4-6 ampholines (LKB). Approximately 50 mg of acetone-soluble protein was dissolved in 3 ml of 1% decyl sodium sulfate (NaDecSO4)/0.01 M TrisHCl, pH 8.2/10 mM dithiothreitol, applied at the cathodic end of the gel and electrophoresed at 8 W for 20 hr at 12°C in a Hoeffer Isobox apparatus (12). The gel containing apoC-IIT was removed and eluted first with 1 ml and then with 2.5 ml of 0.5% NaDecSO4/0.01 M Tris HCl, pH 8.2. The combined eluates were mixed with ice-cold acetone (9 vol of acetone to 1 vol of eluate) and allowed to stand at 4°C for 10 min. The precipitated protein was pelleted by centrifugation at 2000 rpm for 20 min. The precipitate was washed twice with 10 ml of ice-cold acetone. The viscous oil-like residue present was extracted with 5 ml of chloroform/methanol (2:1) at room temperature to produce a white powder. ApoC-IIT was identified by analytical isoelectric focusing and shown to be homogeneous (8). Normal apoC-II was isolated from a control subject following the identical protocol.Amino iT...

A Novel Apolipoprotein C-II Mimetic Peptide That Activates Lipoprotein Lipase and Decreases Serum Triglycerides in Apolipoprotein E–Knockout Mice

“…It contains a relatively high hydrophobic moment of 9.68 mH/n and is known to have high affinity for lipids and lipoproteins, and thus was used to anchor the peptides to the lipoproteins. The second helix for both peptides is based on the third and last helix of apoC-II, which is known to activate LPL (Musliner et al, 1979;Connelly et al, 1987). This helix has a relatively low hydrophobic moment of 4.68 mH/n ( Fig.…”

Role of ApoCs in Lipoprotein Metabolism

“…42,43 In addition, a variety of single-amino acid substitutions in the APOC2 gene has been described ( Table 2) that either resulted in the inability to initiate apoC2 synthesis 44 or in the production of nonfunctional apoC2. [45][46][47] For 2 APOC2 variants (APOC2 SanFrancisco and the APOC2 Lys 19 3 Thr mutation), a direct relationship between this mutant form of apoC2 and lipoprotein abnormalities could not be established. 48 -51 Several lines of evidence have implicated apoC3 as possibly contributing to the development of hypertriglyceridemia.…”

Interaction between variant apolipoproteins C-II and E that affects plasma lipoprotein concentrations.

“…17 The molecular basis for CII-T is a DNA frameshift, resulting from a single DNA base deletion 18 that causes a change in the amino acid sequence from residue 69 onward. 19 CII-T homozygotes have frank hyperchylomicronemia. 17 Among CII-T heterozygotes, plasma levels of triglycerides and VLDL cholesterol vary from normal to hypertriglyceridemic (hyperlipoproteinemia type V).…”