Presentation Title

Oxidative Modification of Apolipoprotein E3 by Myeloperoxidase

Faculty Mentor

Vasanthy Narayanaswami

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

111

Session

poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

High density lipoproteins (HDL) are large spherical anti-atherogenic complexes composed of ~ 70 different proteins and ~ 200 different types of lipids. A major class of proteins on HDL are apolipoproteins (apo) such as apoAI, apoAII, apoE, which are exchangeable proteins with an ability to exist in lipid-free and lipid-associated state. In lipid associated state, these proteins, along with amphipathic lipids such as phospholipids encompass a core of non-polar lipids. HDL plays a critical role in reverse cholesterol transport from macrophages to liver for eventual disposal, a process that earned its popular designation as the “good cholesterol.” In addition, it also bears anti-inflammatory, antithrombotic, and antioxidant properties. However, there are naturally occurring oxidative processes that could impair HDL function; one such process is oxidation mediated by myeloperoxidase (MPO), an enzyme expressed by granulocytes to mediate antimicrobial activity. In the current study we hypothesized that MPO mediated oxidative modification impairs the structure and function of apoE3. The specific objectives are to: (i) prepare purified recombinant apoE3, (ii) modify purified apoE3 by MPO, and (iii) confirm modification by Western blot and mass spectrometry. Recombinant apoE3 bearing a His-tag at the N-terminal end was overexpressed, isolated and purified by affinity chromatography from E. coli. Purified apoE3 was modified by MPO buffered in sodium phosphate solution (pH=7.4) with the addition of pentetic acid (DTPA), sodium chloride, and hydrogen peroxide. SDS-PAGE analysis under reducing conditions revealed the presence of a monomeric and a dimeric band suggesting possible formation of a covalently cross-linked species. In-gel digestion of the monomeric and dimeric band by trypsin was performed for tandem mass spectrometry MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization- Time of Flight) revealed modification of tyrosine 91 in apoE3 monomer and tyrosine 179 in apo E3 dimer to 3-chlorotyrosine. Further studies are required to determine the effect of MPO modification on the secondary structure and tertiary fold of apoE3 by circular dichroism and fluorescence spectroscopy respectively, and to identify potential functional alterations to apoE in terms of lipid binding activity, cholesterol efflux ability, and LDL receptor binding activity. The findings from this study will aid in understanding the role of MPO in rendering HDL dysfunctional and identifying factors that reduce the anti-atherogenic properties of HDL.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Oxidative Modification of Apolipoprotein E3 by Myeloperoxidase

111

High density lipoproteins (HDL) are large spherical anti-atherogenic complexes composed of ~ 70 different proteins and ~ 200 different types of lipids. A major class of proteins on HDL are apolipoproteins (apo) such as apoAI, apoAII, apoE, which are exchangeable proteins with an ability to exist in lipid-free and lipid-associated state. In lipid associated state, these proteins, along with amphipathic lipids such as phospholipids encompass a core of non-polar lipids. HDL plays a critical role in reverse cholesterol transport from macrophages to liver for eventual disposal, a process that earned its popular designation as the “good cholesterol.” In addition, it also bears anti-inflammatory, antithrombotic, and antioxidant properties. However, there are naturally occurring oxidative processes that could impair HDL function; one such process is oxidation mediated by myeloperoxidase (MPO), an enzyme expressed by granulocytes to mediate antimicrobial activity. In the current study we hypothesized that MPO mediated oxidative modification impairs the structure and function of apoE3. The specific objectives are to: (i) prepare purified recombinant apoE3, (ii) modify purified apoE3 by MPO, and (iii) confirm modification by Western blot and mass spectrometry. Recombinant apoE3 bearing a His-tag at the N-terminal end was overexpressed, isolated and purified by affinity chromatography from E. coli. Purified apoE3 was modified by MPO buffered in sodium phosphate solution (pH=7.4) with the addition of pentetic acid (DTPA), sodium chloride, and hydrogen peroxide. SDS-PAGE analysis under reducing conditions revealed the presence of a monomeric and a dimeric band suggesting possible formation of a covalently cross-linked species. In-gel digestion of the monomeric and dimeric band by trypsin was performed for tandem mass spectrometry MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization- Time of Flight) revealed modification of tyrosine 91 in apoE3 monomer and tyrosine 179 in apo E3 dimer to 3-chlorotyrosine. Further studies are required to determine the effect of MPO modification on the secondary structure and tertiary fold of apoE3 by circular dichroism and fluorescence spectroscopy respectively, and to identify potential functional alterations to apoE in terms of lipid binding activity, cholesterol efflux ability, and LDL receptor binding activity. The findings from this study will aid in understanding the role of MPO in rendering HDL dysfunctional and identifying factors that reduce the anti-atherogenic properties of HDL.