Activation of lipoprotein lipase by native and synthetic fragments of human plasma apolipoprotein C-II.

Abstract

Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-I and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II coresponding to residues 1-9 and 10-59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60-78 increased hydrolysis 4fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60-78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55-78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-I. By contrast, the synthetic peptide containing residues 66-78 did not activate. Removal ofthe three COOH-terminal residues, Gly-Glu-Glu, from fragment 60-78 decreased the ability to activate LPL by >95%. These studies suggest that the maximal activation of LPL by apoC-I1 requires a minimal sequence contained within residues 55-78.Chylomicrons and very low density lipoproteins (VLDL) are the vehicles for the transport of plasma triglycerides (1, 2). Transfer of fatty acid from the triglyceride-rich lipoproteins to the tissue requires hydrolysis of the triglyceride by lipoprotein lipase at the capillary walls. Apoliprotein C-II from human VLDL (apoC-II) plays an important role in triglyceride metabolism by serving as an activator of lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3) (3, 4). The amino acid sequence of apoC-I1 (Fig. 1) has recently been determined (5). The protein consists of 78 amino acid residues and is lacking cysteine, cystine, and histidine.We now present results of preliminary studies to define the sequence requirement in apoC-II necessary for the activation of LPL. In addition to testing the three cyanogen bromide (CNBr) fragments corresponding to residues 1-9, 10-59, and 60-78, we have also synthesized and tested the fragments representing residues 66-78, 60-78, and 55-78. On the basis of the results of these studies, we suggest that the sequence determinant in apoC-I1 that is required for maximal activation of LPL is contained between residues 55 and 78. MATERIALS AND METHODSIsolation of apoC-II and CNBr Fragments. apoC-II was isolated as described (6, 7) from VLDL obtained from fasting subjects with type IV or type V hyperlipoproteinemia (8). The isolated apoprotein was homogeneous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate and by amino acid analysis. The three CNBr fragments of apoC-II were prepared by chromatography on Bio-Gel P-30 (5).The peptide corresponding to residues 60-75 was prepared from a tryptic digest of ...

Lipoprotein Lipase: Some Effects of Activator Proteins

“…Recent studies on the structure and properties of apolipoprotein CII have indicated that the N-terminal residues 1 -49 are primarily responsible for its ability to bind to lipids [31]. Nonetheless, this part of the molecule is not required for stimulation of lipoprotein lipase activity [4,5]. This supports our conclusion that the main role of CII is not to enhance binding of the lipase to the substrate droplets.…”

“…Activator proteins, present on the lipoproteins, enhance the enzyme activity [2]. The primary structure of the human activator, apolipoprotein CII, is known [3], and the activity has been localized to its C-terminal part [4,5]. How it promotes hydrolysis is however not known.…”

Isolation of Two Distinct Activator Proteins For Lipoprotein Lipase from Ovine Plasma

“…Values reported for maximum activation by apo C-II vary widely between various groups, but commonly range from 1 jlg/ml (Matsuoka et al1981;Wallinder et al 1982) to more than 40 jlg/ml (Kinnunen et al 1977). The relationship between the apparent Km and the activator proteintriolein ratio was the same for all three activator preparations and was similar to that reported for human LPL with apo C-II (Schrecker and Greten 1979).…”

“…The smaller activator is likely to contain some 30 amino acid residues fewer than the larger activator (and human apo C-II), yet it still functions as effectively. Studies on synthetic fragments or cyanogen bromide fragments 'of human apo C-II have indicated that two distinct functional domains exist in the entire molecule; one for binding to lipid (residues 43-51, Morrisett et al 1977;Catapano et al 1979), another for the catalytic activation of LPL (residues 56-78, Kinnunen et al 1977;Catapano et al 1979); both are required for maximal activation. It must be assumed that each of the ovine activators has these two functional regions since they are both as effective as the human apo C-II preparation.…”

In vitro activation of the enzymic activity of hepatic lipase by apoA‐II

“…The modulation of LPL enzymic activity by apolipo- protein C-II has been actively investigated [4,5,[20][21][22] and the physiological importance of this regulation is apparent from the low LPL activity and hypertriglyceridemia reported in patients with apoC-II deficiency [8]. The regulation of HL activity has received much less attention [3,14] and there has been, to our knowledge, no previous report of specific activators of this enzyme.…”