epidermal-growth-factor and Hyperlipoproteinemia-Type-II

The low-density lipoprotein receptor normally carries lipoprotein particles into cells, and releases them upon delivery to the low pH milieu of the endosome. Recent structural and functional studies of the receptor, combined with the plethora of prior knowledge about normal receptor function and the effects of disease-associated mutations that cause familial hypercholesterolemia, reveal a detailed molecular model for how the acidic environment of the endosome triggers the release of bound lipoprotein particles. Remarkably, the receptor dynamically interconverts between open (ligand-active) and closed (ligand-inactive) conformations in response to pH, relying on a specific arrangement of fixed and flexible interdomain connections to facilitate efficient binding and release of its lipoprotein ligands.

Topics: Binding Sites; Endosomes; Epidermal Growth Factor; Humans; Hydrogen-Ion Concentration; Hyperlipoproteinemia Type II; Mutation; Protein Structure, Tertiary; Receptors, LDL; Structure-Activity Relationship

Topics: Animals; Arteries; Arteriosclerosis; Blood Platelets; Cell Communication; Child; Diet, Atherogenic; Disease Models, Animal; Endothelium; Epidermal Growth Factor; Humans; Hyperlipoproteinemia Type II; Macrophages; Middle Aged; Monocytes; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; RNA, Messenger

The primary genetic cause of familial hypercholesterolemia (FH) is related to mutations in the LDLR gene encoding the Low-density Lipoprotein Receptor. LDLR structure is organized in 5 different domains, including an EGF-precursor homology domain that plays a pivotal role in lipoprotein release and receptor recycling. Mutations in this domain constitute 51.7% of the total missense variants described in LDLR. The aim of the present work was to analyse how clinically significant variants in the EGF-precursor homology domain impact LDLR. The activity of sixteen LDLR variants was functionally characterized by determining LDLR expression by Western blot and LDLR expression, LDL binding capacity and uptake, and LDLR recycling activity by flow cytometry in transfected CHO-ldlA7 cells. Of the analysed variants, we found six non-pathogenic LDLR variants and ten pathogenic variants distributed as follow: three class 3 variants; four class 2 variants; and three class 5 variants. These results can be incorporated into clinical management of patients by helping guide the appropriate level of treatment intensity depending on the extent of loss of LDLR activity. This data can also contribute to cascade-screening for pathogenic FH variants.

Topics: Animals; CHO Cells; Cricetulus; Epidermal Growth Factor; Humans; Hyperlipoproteinemia Type II; Lipoproteins, LDL; Mutation, Missense; Phenotype; Polymorphism, Genetic; Protein Domains; Receptors, LDL

Topics: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Epidermal Growth Factor; Humans; Hyperlipoproteinemia Type II; Ligands; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Receptors, LDL; Structure-Activity Relationship

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.

Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Computer Graphics; Conserved Sequence; Crystallography, X-Ray; Epidermal Growth Factor; Humans; Hydrogen Bonding; Hyperlipoproteinemia Type II; Models, Molecular; Molecular Sequence Data; Point Mutation; Protein Structure, Tertiary; Receptors, LDL; Repetitive Sequences, Amino Acid; Sequence Alignment

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear 1H-15N NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta-strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. 15N longitudinal and transverse relaxation rates, and [1H]-15N heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85+/-0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides.

Topics: Amino Acid Sequence; Binding Sites; Calcium; Disulfides; Epidermal Growth Factor; Humans; Hyperlipoproteinemia Type II; Ligands; Lipoproteins, LDL; Models, Molecular; Molecular Sequence Data; Mutation, Missense; Nuclear Magnetic Resonance, Biomolecular; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, LDL; Sequence Alignment

We describe the characterization of a novel mutation in the low density lipoprotein receptor (LDL-R) gene in a patient with true homozygous familial hypercholesterolemia (FH). The combined use of denaturing gradient gel electrophoresis (DGGE) and sequencing of genomic DNA revealed a guanine to adenine base substitution at nucleotide position 1013 of the LDL-R cDNA. This point mutation results in a change from cysteine to tyrosine at amino acid residue 317 of repeat A of the epidermal growth factor (EGF) precursor homology domain. Binding, uptake and degradation of iodinated LDL in skin fibroblasts from the homozygous patient were less than 10% of normal. In contrast, binding, uptake and degradation of iodinated VLDL was reduced by only 60, 30, and 38%, respectively. Incubation of the patient's fibroblasts in the presence of cholesterol diminished the residual binding of VLDL by 50%, suggesting that the loss of the highly conserved cysteine at position 317 results in a LDL-R that fails to bind LDL, but retains some ability to bind VLDL by interacting with the apolipoprotein E. Both parents were heterozygous for the C317Y mutation. Interestingly, however, the father presented with markedly elevated levels of triglycerides and VLDL cholesterol, whereas his LDL cholesterol was unexpectedly low. The mother of the index patient had only slightly elevated LDL cholesterol. These observations testify to the biological complexity of genotype-environment interactions in individuals carrying mutations at the LDL-R locus and indicate that genetic analysis importantly complements the clinical and biochemical diagnosis of patients with hyperlipidemia.

Topics: Adolescent; Adult; Alleles; Base Sequence; Cells, Cultured; Child; Epidermal Growth Factor; Female; Fibroblasts; Genotype; Haplotypes; Homozygote; Humans; Hyperlipoproteinemia Type II; Lipoproteins; Male; Middle Aged; Mutation, Missense; Pedigree; Receptors, LDL; Repetitive Sequences, Nucleic Acid; Skin

Mutation analysis of the low density lipoprotein receptor (LDLR) gene revealed a novel 8-bp duplication after nucleotide 681 in a Costa Rican patient with familial hypercholesterolaemia. The frameshift caused by this mutation results in a premature termination codon in the EGF precursor homology domain of the mature LDLR, whereby a truncated protein of the first 206 residues with an additional 39 abnormal residues would be created. The insertion overlaps with previously described duplications of 18 bp and 21 bp, thus revealing an insertional hotspot in exon 4 of the LDLR gene. We propose that the structural features of this region of the LDLR gene contribute significantly to genetic instability and the subsequent DNA duplication via an endogenous sequence-directed mechanism of mutagenesis.

Topics: Amino Acid Sequence; Base Sequence; Cholesterol; Cholesterol, HDL; Codon; DNA Mutational Analysis; DNA Primers; DNA Transposable Elements; Epidermal Growth Factor; Exons; Female; Frameshift Mutation; Humans; Hyperlipoproteinemia Type II; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, LDL; Terminator Regions, Genetic; Triglycerides

Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Analysis of single-strand conformation polymorphisms of exons 10 and 11 of the LDL receptor gene from familial hypercholesterolemia heterozygotes indicated the presence of two mutations, which were characterized by DNA sequencing. One mutation (delta N466) was a 3-bp deletion in exon 10 that deletes Asn in codon 466. The other (intron 11 +1, G-->T) was a splice donor mutation at position +1 of intron 11.

Topics: Amino Acid Sequence; Base Sequence; Epidermal Growth Factor; Humans; Hyperlipoproteinemia Type II; Molecular Sequence Data; Norway; Point Mutation; Protein Precursors; Receptors, LDL; Sequence Analysis, DNA; Sequence Homology

A novel mutation of low density lipoprotein (LDL)-receptor gene was found in an Italian family hypercholesterolemia (FH) patient during a screening of 300 FH patients. The proband as well as her daughter were found to be heterozygotes for the mutation. Binding, internalization, and degradation of 125I-labeled LDL by the proband's fibroblasts were reduced to approximately 50% compared to values found in control cells. DNA analysis by Southern blotting showed that the mutant allele was characterized by an insertion of about 10 kb, which resulted from a duplication of exons 9-14 of the LDL-receptor gene. In addition, Northern blot analysis of the proband's RNA showed, besides the normal sized LDL-receptor mRNA (5.3 kb), an additional mRNA of about 6.2 kb. The junction between exon 14 and the duplicated exon 9 was amplified by polymerase chain reaction (PCR) from the cDNA. The sequence of the amplified fragment showed that exon 14 joined the duplicated exon 9 without changing the reading frame. The derived amino acid sequence indicated that the mutated receptor protein had a partial duplication of the EGF precursor homology domain. Ligand and immunoblotting revealed that proband's fibroblasts contained one-half of the normal amount of LDL-receptor protein (molecular mass 130 kDa) and an abnormally large receptor of approximately 160 kDa. The amount of this abnormal receptor as detected by two monoclonal antibodies (10A2 and 4B3) was found to be approximately 30% that of the normal LDL-receptor present in the same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Base Sequence; DNA Transposable Elements; Epidermal Growth Factor; Female; Fibroblasts; Genetic Testing; Humans; Hyperlipoproteinemia Type II; Italy; Lipoproteins, LDL; Middle Aged; Molecular Sequence Data; Point Mutation; Receptors, LDL; RNA, Messenger; Sequence Homology

The multifunctional nature of coated pit receptors predicts that these proteins will contain multiple domains. To establish the genetic basis for these domains (LDL) receptor. This gene is more than 45 kilobases in length and contains 18 exons, most of which correlate with functional domains previously defined at the protein level. Thirteen of the 18 exons encode protein sequences that are homologous to sequences in other proteins: five of these exons encode a sequence similar to one in the C9 component of complement; three exons encode a sequence similar to a repeat sequence in the precursor for epidermal growth factor (EGF) and in three proteins of the blood clotting system (factor IX, factor X, and protein C); and five other exons encode nonrepeated sequences that are shared only with the EGF precursor. The LDL receptor appears to be a mosaic protein built up of exons shared with different proteins, and it therefore belongs to several supergene families.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Complement C9; DNA; Endonucleases; Epidermal Growth Factor; Factor IX; Factor X; Genes; Glycoproteins; Humans; Hyperlipoproteinemia Type II; Molecular Weight; Protein C; Protein Precursors; Protein Processing, Post-Translational; Receptors, LDL; Repetitive Sequences, Nucleic Acid; Single-Strand Specific DNA and RNA Endonucleases; Transcription, Genetic