EFFECT OF APOLIPOPROTEIN A-II ON THE STRUCTURE OF HIGH-DENSITY LIPOPROTEINS: RELATIONSHIP TO THE ACTIVITY OF LECITHIN: CHOLESTEROL ACYL TRANSFERASE IN VITRO

The crystallization and structure determination of recombinant human apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, is described. The fragment crystallized, residues 44–243 of native apo A-I [apo Δ(1–43)A-I], is very similar to intact native apo A-I in its ability to bind lipid, to be incorporated into high-density lipoproteins and to activate lecithin–cholesterol acyl transferase. Apo Δ(1–43)A-I crystallizes from 1.0–1.4 M sodium citrate pH 6.5–7.5 in space group P212121, with unit-cell parameters a = 97.47, b = 113.87, c = 196.19 Å (crystal form I). The crystals exhibit unusual diffraction intensity spikes and axial extinctions that are discussed in the context of the 4 Å crystal structure. When flash-cooled to 100 K, the crystals diffract synchrotron radiation to 3 Å resolution. Radiation sensitivity and crystal-to-crystal variation have hindered the assembly of a complete 3 Å data set.

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ApoE and apoC-III-defined HDL subtypes: A descriptive study of their lecithin cholesterol acyl transferase and cholesteryl ester transfer protein content and activity

10.21203/rs.2.20333/v3 ◽

2020 ◽

Author(s):

Mateo Amaya-Montoya ◽

Jairo A Pinzón-Cortés ◽

Lina S Silva-Bermúdez ◽

Daniel Ruiz-Manco ◽

Maria C Perez-Matos ◽

...

Keyword(s):

Size Distribution ◽

Plasma Cholesterol ◽

Inverse Correlation ◽

High Density Lipoproteins ◽

Acyl Transferase ◽

Lcat Activity ◽

Cholesterol Acyl Transferase ◽

Lecithin Cholesterol Acyl Transferase ◽

Transfer Protein ◽

Plasma Lcat

Abstract Background The functionality of high-density lipoproteins (HDL) is a better cardiovascular risk predictor than HDL concentrations. One of the key elements of HDL functionality is its apolipoprotein composition. Lecithin-cholesterol acyl transferase (LCAT) and cholesterol-ester transfer protein (CETP) are enzymes involved in HDL-mediated reverse cholesterol transport. This study assessed the concentration and activity of LCAT and CETP in HDL subspecies defined by their content of apolipoproteins E (apoE) and C-III (apoC-III) in humans. Methods Eighteen adults (ten women and eight men, mean age 55.6, BMI 26.9 Kg/m 2 , HbA1c 5.4%) were studied. HDL from each participant were isolated and divided into four subspecies containing respectively: No apoE and no apoC-III (E-C-), apoE but not apoC-III (E+C-), apoC-III but no apoE (E-C+) and both apoE and apoC-III (E+C+). The concentration and enzymatic activity of LCAT and CETP were measured within each HDL subspecies using immunoenzymatic and fluorometric methods. Additionally, the size distribution of HDL in each apolipoprotein-defined fraction was determined using non-denaturing electrophoresis and anti-apoA-I western blotting. Results HDL without apoE or apoC-III was the predominant HDL subtype. The size distribution of HDL was very similar in all the four apolipoprotein-defined subtypes. LCAT was most abundant in E-C- HDL (3.58 mg/mL, 59.6 % of plasma LCAT mass), while HDL with apoE or apoC-III had much less LCAT (19.8%, 12.2% and 8.37% of plasma LCAT respectively for E+C-, E-C+ and E+C+). LCAT mass was lower in E+C- HDL relative to E-C- HDL, but LCAT activity was similar in both fractions, signaling a greater activity-to-mass ratio associated with the presence of apoE. Both CETP mass and CETP activity showed only slight variations across HDL subspecies. There was an inverse correlation between plasma LCAT activity and concentrations of both E-C+ pre-beta HDL (r=-0.55, P =0.017) and E-C- alpha 1 HDL (r=-0.49, P =0.041). Conversely, there was a direct correlation between plasma CETP activity and concentrations of E-C+ alpha 1 HDL (r=0.52, P =0.025). Conclusions The presence of apoE in small HDL is correlated with increased LCAT activity and esterification of plasma cholesterol. These results favor an interpretation that LCAT and apoE interact to enhance anti-atherogenic pathways of HDL.

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Abstract 541: The Molecular Interaction of Apolipoprotein A-I Containing High Density Lipoproteins with Lecithin: Cholesterol Acyl Transferase

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.541 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Allison Cooke ◽

John T Melchior ◽

Jamie C Morris ◽

Rong Huang ◽

W. Gray Jerome ◽

...

Keyword(s):

Protein Interactions ◽

Density Lipoprotein ◽

Negative Control ◽

High Density ◽

High Density Lipoproteins ◽

Acyl Transferase ◽

Lcat Activity ◽

Wild Type ◽

Cholesterol Acyl Transferase ◽

Hdl Function

The structure of apolipoprotein (apo)A-I on a high density lipoprotein (HDL) particle can elucidate the protein interactions and cardioprotective functions of HDL. This includes critical interactions with HDL modifying proteins like lecithin:cholesterol acyl transferase (LCAT) which performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring structure that encapsulates lipid in reconstituted (r)HDL particles. Cross-linking analysis of rHDL particles imply two possible registries: one with the fifth helical repeat of an apoA-I molecule adjacent to the fifth helical repeat of its antiparallel partner (5/5), and the other with the fifth helical repeat adjacent to the second helical repeat of its antiparallel partner (5/2). We hypothesized that apoA-I registry on rHDL can modulate LCAT activity. Site-directed cysteine mutagenesis was used to lock two apoA-I molecules into each of the aforementioned helical registries, and an intermediate registry created as a negative control. rHDL particles were generated using these dimerized mutants, and their ability to activate LCAT was determined. The 5/5 mutant demonstrated higher LCAT activity than wild-type rHDL particles while the 5/2 and intermediate mutants showed dramatically lower LCAT activity (p<0.001). To determine where LCAT interacts with apoA-I, rHDL containing wild-type apoA-I was cross-linked to LCAT. The majority of the cross-links were concentrated between S240 of LCAT and a region encompassing helices 4-6 of apoA-I. We propose that LCAT binds to a discontinuous epitope comprised of two apoA-I molecules in rHDL and that changes in this registry can alter LCAT function. These studies provide a basis for understanding how apoA-I structure may modulate the association and activity of a multitude of different HDL protein partners. This implicates apoA-I conformation as a target for altering HDL function in order to enhance the cardioprotective properties of HDL.

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Neutrophil association and degradation of normal and acute-phase high-density lipoprotein 3

Biochemical Journal ◽

10.1042/bj2480919 ◽

1987 ◽

Vol 248 (3) ◽

pp. 919-926 ◽

Cited By ~ 23

Author(s):

E G Shephard ◽

F C de Beer ◽

M C de Beer ◽

M S Jeenah ◽

G A Coetzee ◽

...

Keyword(s):

Acute Phase ◽

Density Lipoprotein ◽

High Density ◽

High Density Lipoproteins ◽

Human Neutrophils ◽

Amyloid A ◽

Apolipoprotein A ◽

Serum Amyloid A Protein ◽

Alpha 1 Antitrypsin

The interaction of normal and acute-phase high-density lipoproteins of the subclass 3 (N-HDL3 and AP-HDL3) with human neutrophils and the accompanying degradation of HDL3 apolipoproteins have been studied in vitro. The chemical composition of normal and acute-phase HDL3 was similar except that serum amyloid A protein (apo-SAA) was a major apolipoprotein in AP-HDL3 (approx. 30% of total apolipoproteins). 125I-labelled AP-HDL3 was degraded 5-10 times faster than 125I-labelled N-HDL3 during incubation with neutrophils or neutrophil-conditioned medium. Apo-SAA, like apolipoprotein A-II (apo-A-II), was more susceptible than apolipoprotein A-I (apo-A-I) to the action of proteases released from the cells. The amounts of cell-associated AP-HDL3 apolipoproteins at saturation were up to 2.8 times greater than N-HDL3 apolipoproteins; while apo-A-I was the major cell-associated apolipoprotein when N-HDL3 was bound, apo-SAA constituted 80% of the apolipoproteins bound in the case of AP-HDL3. The associated intact apo-SAA was mostly surface-bound as it was accessible to the action of exogenous trypsin. alpha 1-Antitrypsin-resistant (alpha 1-AT-resistant) cellular degradation of AP-HDL3 apolipoproteins also occurred; experiments in which pulse-chase labelling was performed or lysosomotropic agents were used indicated that insignificant intracellular degradation occurred which points to the involvement of cell-surface proteases in this degradation.

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Crystallization of truncated human apolipoprotein A-I in a novel conformation

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999008914 ◽

1999 ◽

Vol 55 (9) ◽

pp. 1578-1583 ◽

Cited By ~ 20

Author(s):

David W. Borhani ◽

Jeffrey A. Engler ◽

Christie G. Brouillette

Keyword(s):

Diffraction Limit ◽

Unit Cell ◽

High Density ◽

High Density Lipoproteins ◽

Original Form ◽

Major Protein ◽

Cholesterol Acyl Transferase ◽

Space Group ◽

Human Apolipoprotein ◽

Apolipoprotein A

The crystallization of recombinant human apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, in a new crystal form is described. The fragment crystallized, residues 44–243 of native apo A-I [apo Δ(1–43)A-I], is very similar to intact native apo A-I in its ability to bind lipid, to be incorporated into high-density lipoproteins and to activate lecithin–cholesterol acyl transferase. Apo Δ(1–43)A-I crystallizes, in the presence of β-D-octylglucopyranoside, in space group I222 or I212121, with unit-cell parameters a = 37.11, b = 123.62, c = 164.65 Å and a diffraction limit of 3.2 Å. These form II crystals grow under conditions of significantly lower ionic strength than the original form I crystals (space group P212121, a = 97.47, b = 113.87, c = 196.19 Å, diffraction limit 3.0 Å). Packing arguments show that the unusual open conformation of apo Δ(1–43)A-I found in the form I crystals cannot be packed into the smaller oddly proportioned form II unit cell. Monomeric apo Δ(1–43)A-I, as either a four-helix bundle (∼75 × 30 × 30 Å) or an extended helical rod (∼150 × 20 × 20 Å), can be packed into the form II unit cell. It is concluded, therefore, that apo Δ(1–43)A-I may have crystallized in one of these distinct conformations in the form II crystals.

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