Effect of phospholipase C and apolipophorin III on the structure and stability of lipophorin subspecies.

The structural basis for the lipid binding capability of Locusta migratoria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecule (approximately 20 kDa) was deglycosylated with endoglycosidase F (molecular mass of deglycosylated species approximately 18 kDa) and cleaved with endoproteinase Arg-C to yield two fragments with molecular masses of approximately 9 kDa each. The two fragments were purified by reversed-phase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestion pattern obtained compared to that expected from the deduced amino sequence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both exist in buffer in a random coil state. However, addition of trifluoroethanol, a helix-inducing agent, resulted in the formation of an alpha-helix, reflecting an innate propensity of the peptides to adopt a helical conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicative of interaction with the phospholipid. In the presence of phospholipids, a 22 nm blue shift in Trp fluorescence emission was observed in the case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced ability to transform DMPC vesicles to disklike complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultsmentioning

confidence: 99%

Spectroscopic and Lipid Binding Studies on the Amino and Carboxyl Terminal Fragments of Locusta migratoria Apolipophorin III

Narayanaswami¹,

Weers²,

Bogerd³

et al. 1995

“…In experiments designed to verify the hypothesis that LDLp specific resonances (peaks 1 and 4) represent DG molecules in the surface monolayer, we conducted 13C NMR measurements of the HDLp-Wl before and after conversion of its phospholipid moiety into DG by treatment with PL-C (Figure 3). We have previously shown that PL-C catalyzed conversion of lipoprotein phospholipid into DG results in particle instability, aggregation, and fusion, leading to sample turbidity (Liu et al, 1993;Singh et al, 1994). Importantly, however, it has also been demonstrated that inclusion of amphipathic exchangeable apolipoproteins, such as apoLp-, prevents PL-C induced lipoprotein aggregation through formation of a stable binding interaction with lipolyzed particles (Liu et al, 1993;Singh et al, 1994).…”

Section: Resultsmentioning

confidence: 99%

“…We have previously shown that PL-C catalyzed conversion of lipoprotein phospholipid into DG results in particle instability, aggregation, and fusion, leading to sample turbidity (Liu et al, 1993;Singh et al, 1994). Importantly, however, it has also been demonstrated that inclusion of amphipathic exchangeable apolipoproteins, such as apoLp-, prevents PL-C induced lipoprotein aggregation through formation of a stable binding interaction with lipolyzed particles (Liu et al, 1993;Singh et al, 1994). Since the phospholipid molecules are localized on the particle surface, PL-C activity should result in surface associated 1,2-DG.…”

Section: Resultsmentioning

confidence: 99%

“…the basis of 31P NMR and compositional data, it has been hypothesized that partitioning of DG to the surface of LDLp influences apoLp-III binding (Wang et al, 1992). The recent observation that phospholipase C (PL-C) induced conversion of lipoprotein-associated phospholipid into DG induces a stable association of apoLp-III supports this concept (Liu et al, 1993;Singh et al, 1994). The present study was undertaken in an effort to obtain direct spectroscopic evidence for the existence of surface DG in apoLp-IIIcontaining lipophorin particles.…”

mentioning

confidence: 94%

See 1 more Smart Citation

Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR

Wang¹,

Liu²,

Sykes³

et al. 1995

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

“…In lipoprotein binding assays designed in our laboratory using PL-C, it was shown that apoLp-III prevents onset of aggregation of human LDL (Liu et al, 1993) or insect lipoproteins (Singh et al, 1994) by binding to the exposed hydrophobic surfaces created by hydrolysis of phospholipid headgroups. In LDLp it was rationalized (Wang et al, 1992) that apoLp-III intercalates between phospholipid molecules and interacts with diacylglycerol at the particle surface, following opening of the helix bundle and exposure of its hydrophobic core.…”

Section: Discussionmentioning

confidence: 99%

Structural and Binding Characteristics of the Carboxyl Terminal Fragment of Apolipophorin III from Manduca sexta

Narayanaswami¹,

Kay²,

Oikawa³

et al. 1994

The molecular basis of the interaction of apolipophorin III (apoLp-III), an exchangeable apolipoprotein from hemolymph of the sphinx moth. Manduca sexta, with lipoprotein surfaces and phospholipids was studied by investigating the structural and binding properties of the C-terminal fragment of the native protein. A 4K peptide, corresponding to the terminal helical segment of the native protein, was generated by cyanogen bromide treatment, purified by gel filtration and reverse-phase HPLC, and characterized by N-terminal sequencing and amino acid and mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the peptide in buffer indicated a predominantly unstructured state while addition of trifluoroethanol (TFE), a helix-inducing agent, resulted in an alpha-helical structure. Sedimentation equilibrium studies revealed that the 4K peptide was monomeric in buffer. The 4K peptide assumed an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS) and lysolecithin, but was unstructured in the presence of dimyristoylphosphatidylcholine, either when added to preformed vesicles or upon cosonication, indicating an ability to bind to detergent micelles but not to phospholipid bilayers. Unlike native apoLp-III, the 4K peptide did not confer protection against turbidity development to human low density lipoprotein upon incubation with phospholipase C, indicating an inability to interact with the surface of lipoproteins. Upon interaction with SDS micelles, both the 4K peptide and apoLp-III were resistant to urea-induced denaturation when compared to free apoLp-III, as evaluated by CD spectroscopy. The structural stability conferred upon interaction with detergents was similar for both the peptide and the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)