Prevention of phospholipase‐C induced aggregation of low density lipoprotein by amphipathic apolipoproteins

Liu, Hu; Scraba, Douglas G.; Ryan, Robert O.

doi:10.1016/0014-5793(93)81730-n

Cited by 82 publications

(86 citation statements)

References 27 publications

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“…4 interactions among LDL subclasses and suggests that they can fuse with each other. Taken together with a similar nonadditive behavior of human HDL subclasses during heat denaturation ( 46 ) and with the demonstrated ability of HDL or its major protein apoA-I, to inhibit LDL aggregation and fusion ( 29,(47)(48)(49), this result indicates that different lipoprotein classes and subclasses interact with each other during fusion.…”

Section: Effects Of Ldl Concentration Particle Size and Ph On The Rmentioning

confidence: 63%

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Gantz

Herscovitz

et al. 2012

Journal of Lipid Research

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of LDL cholesterol and, particularly, apoB are the strongest predictors of atherosclerosis and its causative agents ( 3 ). In atherosclerosis, LDL lipids are deposited in the arterial intima; according to the response-to-retention paradigm ( 4, 5 ), LDL retention by the arterial matrix proteoglycans triggers a cascade of pro-atherogenic events culminating in formation of atherosclerotic plaque ( 6-8 ). These events include biochemical modifi cations of LDL, such as oxidation and/or hydrolysis by the resident proteases and lipases, e.g., phospholipase A 2 and sphingomyelinase, which can reduce LDL affi nity for the LDL receptor, increase LDL affi nity for the proteoglycans, and promote LDL fusion (7)(8)(9)(10)(11)(12)(13)(14). In addition, ionic interactions with proteoglycans reduce LDL stability and promote their fusion and rupture (i.e., release of core lipids) ( 15 ). Fusion of lipoproteins prevents their exit from the arterial intima and thereby augments their further modifi cations, enhances LDL uptake by the arterial macrophages, and initiates the formation of atherosclerotic lesions ( 9, 16 ). Therefore, the pro-atherogenic potential of LDL is thought to be linked to their ability to fuse ( 9,10,17 ). Dissecting the pathogenic pathway of LDL fusion and identifying key factors that promote or inhibit this pathway can help obtain new therapeutic targets for atherosclerosis.Structural analysis of intact and modifi ed LDL has been limited to low resolution ( у 16 Å) by the large size and hydrophobicity of apoB and by LDL heterogeneity ( 1,(18)(19)(20)(21). Human plasma LDL consist of subclasses differing Abstract Fusion of modifi ed LDL in the arterial wall promotes atherogenesis. Earlier we showed that thermal denaturation mimics LDL remodeling and fusion, and revealed kinetic origin of LDL stability. Here we report the fi rst quantitative analysis of LDL thermal stability. Turbidity data show sigmoidal kinetics of LDL heat denaturation, which is unique among lipoproteins, suggesting that fusion is preceded by other structural changes. High activation energy of denaturation, E a = 100 ± 8 kcal/mol, indicates disruption of extensive packing interactions in LDL. Size-exclusion chromatography, nondenaturing gel electrophoresis, and negativestain electron microscopy suggest that LDL dimerization is an early step in thermally induced fusion. Monoclonal antibody binding suggests possible involvement of apoB N-terminal domain in early stages of LDL fusion. LDL fusion accelerates at pH < 7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the fi rst kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase t...

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Section: Effects Of Ldl Concentration Particle Size and Ph On The Rmentioning

confidence: 63%

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Gantz

Herscovitz

et al. 2012

Journal of Lipid Research

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“…The first assay involved incubation of human low density lipoprotein (LDL) with phospholipase-C (PL-C) in the presence or absence of apolipoproteins (Liu et al, 1993). LDL was isolated from fresh human plasma by sequential density ultracentrifugation (Schumaker and Puppione, 1986).…”

Section: Methodsmentioning

confidence: 99%

“…Aggregation results in sample turbidity development that can be monitored at 340 nm as a function of time. Exchangeable apolipoproteins are able to prevent this aggregation by associating with the lipolyzed surface (Liu et al, 1993;Singh et al, 1994). The ability of oxidized N40C/L90C apoLp-III to interact with the lipoprotein surface was evaluated in comparison with the reduced form and WT apoLp-III (Fig.…”

Section: Effect Of Disulfide Bond Formation On Lipid Bindingmentioning

confidence: 99%

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Narayanaswami¹,

Wang²,

Kay

et al. 1996

Journal of Biological Chemistry

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Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an exchangeable, amphipathic apolipoprotein that alternately exists in water-soluble and lipid-bound forms. It is organized as a five-helix bundle in solution, which has been postulated to open at putative hinge domains to expose the hydrophobic interior, thereby facilitating interaction with the lipoprotein surface (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608). To test this hypothesis, we engineered two cysteine residues in apoLp-III, which otherwise lacks cysteine, by site-directed mutagenesis at Asn-40 and Leu-90. Under oxidizing conditions the two cysteines spontaneously form a disulfide bond, which should tether the helix bundle and thereby prevent opening and concomitant lipid interaction. N40C/L90C apoLp-III was overexpressed in Escherichia coli and characterized for disulfide bond formation, secondary structure content, and stability, under both oxidizing and reducing conditions. Functional characterization was carried out by comparing the abilities of the oxidized and reduced protein to associate with modified lipoproteins in vitro. While the reduced form behaved like wild type apoLp-III, the oxidized form was unable to associate with lipoproteins. These results suggest that opening of the helix bundle is required for interaction with lipoproteins and provide a molecular basis for the dual existence of water-soluble and lipid-bound forms of apoLp-III. However, in phospholipid bilayer association assays, wild type, reduced, and oxidized N40C/L90C apoLp-III exhibited similar abilities to transform dimyristoylphosphatidylcholine multilamellar vesicles to disclike complexes, as judged by electron microscopy. These data emphasize that underlying differences exist in initiating or maintaining a stable interaction of apoLp-III with phospholipid disc complexes versus spherical lipoprotein surfaces.Exchangeable apolipoproteins belong to a class of amphipathic ␣-helical proteins that regulate the metabolism and dynamics of lipoprotein interconversions. These proteins reversibly associate with the surface of lipoprotein particles in response to hydrophobic surface availability or their intrinsic ability to displace pre-existing apolipoproteins. This functional property implies an ability to exist in both lipid-free and lipidbound forms in plasma, and it has been proposed that a dramatic conformational change is required for initiation and maintenance of interaction with lipid surfaces (Breiter et al., 1991;Weisgraber, 1994). The sole exchangeable apolipoprotein found in insect hemolymph, apolipophorin III (apoLp-III), 1 provides an excellent model to study lipid association-induced conformational changes of amphipathic exchangeable apolipoproteins. ApoLp-III is well characterized in terms of physicochemical and functional properties (see Van der Horst, 1990;Blacklock and Ryan, 1994; Soulages and Wells, 1994, for reviews). Structural informa...

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“…Indeed, the lipid droplets isolated from the arterial intima have features suggesting that they may have been derived from plasma LDL by extensive modification (reviewed by Öörni et al 11 ). Modification of LDL in vitro by proteolytic enzymes, [12][13][14] by oxidative compounds, 15 or by lipolytic enzymes such as sphingomyelinase, 13,[15][16][17] phospholipase C, 18,19 or phospholipase A 2 (PLA 2 ) 17,20 has been shown to induce aggregation, fusion, or both aggregation and fusion of the particles.…”

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confidence: 99%

Lipolysis of LDL by Human Secretory Phospholipase A ₂ Induces Particle Fusion and Enhances the Retention of LDL to Human Aortic Proteoglycans

Hakala

Öörni

Pentikäinen

et al. 2001

ATVB

102

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Abstract-The first morphological sign of atherogenesis is the accumulation of extracellular lipid droplets in the proteoglycan-rich subendothelial layer of the arterial intima. Secretory nonpancreatic phospholipase A 2 (snpPLA 2 ), an enzyme capable of lipolyzing LDL particles, is found in the arterial extracellular matrix and in contact with the extracellular lipid droplets. We have recently shown that in the presence of heparin, lipolysis of LDL with bee venom PLA 2 induces aggregation and fusion of the particles. Here, we studied the effect of human snpPLA 2 on the integrity of LDL particles and on their interaction with human aortic proteoglycans. In addition, the capacity of the proteoglycans to retain PLA 2 -lipolyzed LDL particles was tested in a microtiter well assay. We found that lipolysis of LDL induced fusion of proteoglycan-bound LDL particles, which increased their binding strength to the proteoglycans. Moreover, lipolysis of LDL with snpPLA 2 under physiological salt and albumin concentrations induced a 3-fold increase in the amount of LDL bound to proteoglycans. The results imply a role for PLA 2 in the retention and accumulation of LDL to the proteoglycan matrix in atherosclerosis. Key Words: phospholipases Ⅲ LDL Ⅲ fusion Ⅲ retention Ⅲ proteoglycans I nitiation of atherosclerosis in both humans 1 and experimental animals 2-4 is characterized by the appearance of extracellular lipid droplets in the proteoglycan (PG)-rich subendothelial layer. Experimental models have shown that similar droplets can be formed directly from LDL particles both in situ 2,5 and in vivo. 2,6 Moreover, both chemical analyses 7,8 and measurements of the size 9,10 of the extracellular lipid droplets in human arterial lesions suggest that the majority of the droplets originate from LDL particles. Because native LDL particles do not fuse into such lipid droplets, the LDL particles must undergo modification in the arterial intima. Indeed, the lipid droplets isolated from the arterial intima have features suggesting that they may have been derived from plasma LDL by extensive modification (reviewed by Öörni et al 11 ). Modification of LDL in vitro by proteolytic enzymes, 12-14 by oxidative compounds, 15 or by lipolytic enzymes such as sphingomyelinase, 13,15-17 phospholipase C, 18,19 or phospholipase A 2 (PLA 2 ) 17,20 has been shown to induce aggregation, fusion, or both aggregation and fusion of the particles. See p 884Phospholipase A 2 s are enzymes that catalyze the hydrolysis of the sn-2 fatty acyl ester bond in phospholipids, yielding a free fatty acid and a lysophospholipid. Recently, type II secretory nonpancreatic phospholipase A 2 (snpPLA 2 ), which is capable of lipolyzing LDL, 21 has been shown to be located in both atherosclerotic and nonatherosclerotic arterial intima [22][23][24][25][26] and to be associated with extracellular matrix structures and lipid droplets. 24 Interestingly, lipolysis of LDL by bee venom PLA 2 reduces the size of the LDL particles in the absence of glycosaminoglycans (GAGs) 17,20,27 but ...

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Prevention of phospholipase‐C induced aggregation of low density lipoprotein by amphipathic apolipoproteins

Cited by 82 publications

References 27 publications

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Lipolysis of LDL by Human Secretory Phospholipase A ₂ Induces Particle Fusion and Enhances the Retention of LDL to Human Aortic Proteoglycans

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Prevention of phospholipase‐C induced aggregation of low density lipoprotein by amphipathic apolipoproteins

Cited by 82 publications

References 27 publications

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Lipolysis of LDL by Human Secretory Phospholipase A 2 Induces Particle Fusion and Enhances the Retention of LDL to Human Aortic Proteoglycans

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Lipolysis of LDL by Human Secretory Phospholipase A ₂ Induces Particle Fusion and Enhances the Retention of LDL to Human Aortic Proteoglycans