Abstract
Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.
Highlights
Lipoproteins play important physiologic roles in cellular function and regulation of metabolic pathways
low-density lipoprotein (LDL) surface is surrounded by a single copy of Apolipoprotein B-100 (ApoB100), with some regions rich in β -type structures embedded in the lipid domain of the particle[15,16], while residues involved in low-density lipoprotein receptor (LDLR) binding are exposed to the medium[16]
Infrared (IR) spectroscopy complements the information obtained by other methodologies, providing information related to size and density of LDL and, about the secondary structure content of ApoB10024,25
Summary
Lipoproteins play important physiologic roles in cellular function and regulation of metabolic pathways. Other pathogenic alterations have been described, p.(Arg3507Trp), p.(Arg3558Cys), p.(Trp4396Tyr), and recently our group described and characterize two novel mutations, p.(Arg1164Thr) and p.(Gln4494del), located in exon 22 and 29, respectively[8] None of these alterations reside in the consensus region of the ApoB100/LDLR binding or in the region postulated by Krisko and Etchebest[13]. Small angle neutron scattering of lipid-free ApoB100 which describes the modular nature of the protein, with ordered domains connected by flexible linkers[21]; small-angle X-ray scattering which models the LDL core at low-resolution[22], and cryomicroscopy for single particle reconstruction[23]. In the current study we have analysed the particle size of LDL carrying wt ApoB100, and from heterozygote patients carrying p.(Arg3527Gln) (c.10580G> A), p.(Arg1164Thr) (c.3491G>C) and p.(Gln4494del) (c.13480_13482delCAG) variants by dynamic light scattering (DLS) and electron microscopy (EM) and their secondary structure by IR spectroscopy. We have found differences both in particle size and in the ApoB100 secondary structure between the mutant particles compared with wt ApoB100 particles that may underlie the defective binding and uptake of LDL carrying these ApoB100 variants leading to FH
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