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The structure of apoA‐II on HDL reveals novel insights into its regulation of lipoprotein composition and function
Author(s) -
Melchior John T,
Street Scott E,
Jerome W. Jay,
Hart Rachel C,
Heinecke Jay W,
He Yi,
Davidson W. Sean
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.238.2
Subject(s) - chemistry , dimer , intermolecular force , apolipoprotein b , intramolecular force , lipoprotein , molecule , function (biology) , negative stain , biophysics , biochemistry , crystallography , cholesterol , stereochemistry , biology , electron microscope , genetics , organic chemistry , physics , optics
High‐density lipoproteins (HDL) are compositionally and functionally diverse. HDL can host at least 95 different proteins and studies suggest many of these interactions are governed by specific conformations of HDL's primary scaffold protein, apolipoprotein (apo)A‐I. Previously, we showed that the presence of HDL's second most abundant scaffold protein, apoA‐II, impacts particle composition and function; presumably through interactions with apoA‐I. The goal of this study was to determine the structural conformation of apoA‐II on HDL to understand how its presence impacts HDL metabolism. We used chemical cross‐linking and mass spectrometry to derive spatial information on homogenous populations of recombinant HDL (rHDL) generated in vitro . The rHDL was created using a mixture of wild‐type and isotopically‐labeled apoA‐I to unambiguously identify the molecular span of the cross‐linked peptides; i.e. within a molecule of apoA‐I (intramolecular) or between two molecules of apoA‐I (intermolecular). Two rHDL populations were studied: (i) rHDL that contained only apoA‐I (LpA‐I) and (ii) rHDL containing both apoA‐I and apoA‐II (LpA‐I/A‐II). Cross‐linking and SDS‐PAGE showed both populations contained two molecules of apoA‐I while the LpA‐I/A‐II rHDL contained a single dimer of apoA‐II. Analysis by negative stain electron microscopy revealed both populations were discoidal in shape with no differences in size. The intermolecular cross‐linking pattern between apoA‐I molecules were consistent with the “double‐belt” model of apoA‐I on HDL and, surprisingly, the presence of apoA‐II didn't appear to perturb the pattern. However, we observed strong cross‐links between apoA‐II and the C‐terminus of apoA‐I. In a parallel approach, native LpA‐I and LpA‐I/A‐II HDL was isolated from human plasma using immunoaffinity and size exclusion chromatography. Shotgun proteomics and cross‐linking revealed accessory proteins were mostly associated with LpA‐I HDL and interactions were primarily localized to the N‐ and C‐termini of apoA‐I. As with the rHDL, we found apoA‐II specifically interacts with the C‐terminus of apoA‐I in the LpA‐I/A‐II particles. Taken together, this suggests that apoA‐II's compositional and functional effects on HDL are exerted via its interaction with the C‐terminus of apoA‐I possibly limiting its availability to interact with the accessory proteins. We have gone on to generate preliminary all‐atoms models of apoA‐II on HDL to illustrate this structural mechanism. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .