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Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum.

Strøm TB, Tveten K, Laerdahl JK, Leren TP - FEBS Open Bio (2014)

Bottom Line: Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum.This led to reduced amounts of the mature 160 kDa LDLR at the cell surface.It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

View Article: PubMed Central - PubMed

Affiliation: Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.

ABSTRACT
More than 1700 mutations in the low density lipoprotein receptor (LDLR) gene have been found to cause familial hypercholesterolemia (FH). These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

No MeSH data available.


Related in: MedlinePlus

The LDLR transmembrane domain is devoid of residues with charged side chains. Multiple sequence alignment of the human LDLR (residues 764–839) and 29 vertebrate homologs shows a complete lack of charged residues in the transmembrane alpha helix passing through the membrane lipid bilayer. In the cartoon at the top, non-polar-aliphatic and aromatic residues are indicated by blue circles, the highly conserved Pro795 and Gly805 by black circles, basic residues by red circles, and polar, neutral residues by green circles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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f0005: The LDLR transmembrane domain is devoid of residues with charged side chains. Multiple sequence alignment of the human LDLR (residues 764–839) and 29 vertebrate homologs shows a complete lack of charged residues in the transmembrane alpha helix passing through the membrane lipid bilayer. In the cartoon at the top, non-polar-aliphatic and aromatic residues are indicated by blue circles, the highly conserved Pro795 and Gly805 by black circles, basic residues by red circles, and polar, neutral residues by green circles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mentions: Mutation G805R is a rare mutation in Norway. Only two of a total of 1850 unrelated patients provided with a molecular genetic diagnosis of familial hypercholesterolemia, carry mutation G805R. Segregation analysis among family members of one of the index patients revealed that mutation G805R segregated with hypercholesterolemia through 14 meioses (Supplementary Fig. S1). The probability that this co-segregation occurred by chance is (1/2)14 = 0.006%. Mutation G805R was predicted to be pathogenic by the software programs PolyPhen2 (Score: “Probably Damaging”), SIFT (Score: “Not Tolerated”) and Mutation Taster (Score: “Disease Causing”). A multiple sequence alignment of the human LDLR (residues 764–839) and orthologs from 18 mammals and 8 additional vertebrate species found by searching the UniProt [9] and Ensembl [10] databases, is shown in Fig. 1. This alignment shows Gly805 to be highly conserved and there is a complete lack of residues with charged side chains in the transmembrane domain. Together, these data indicate that mutation G805R is pathogenic. The alignment also shows poor residue conservation in the ectodomain and in the transmembrane domain, but the latter is strongly enriched in hydrophobic residues. Three conserved positively charged residues of the cytoplasmic domain (Lys811, Arg814, and Lys816 in the human LDLR, Fig. 1) appear to be necessary for inserting the receptor with correct topology in the membrane, according to the positive-inside rule [11].


Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum.

Strøm TB, Tveten K, Laerdahl JK, Leren TP - FEBS Open Bio (2014)

The LDLR transmembrane domain is devoid of residues with charged side chains. Multiple sequence alignment of the human LDLR (residues 764–839) and 29 vertebrate homologs shows a complete lack of charged residues in the transmembrane alpha helix passing through the membrane lipid bilayer. In the cartoon at the top, non-polar-aliphatic and aromatic residues are indicated by blue circles, the highly conserved Pro795 and Gly805 by black circles, basic residues by red circles, and polar, neutral residues by green circles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048843&req=5

f0005: The LDLR transmembrane domain is devoid of residues with charged side chains. Multiple sequence alignment of the human LDLR (residues 764–839) and 29 vertebrate homologs shows a complete lack of charged residues in the transmembrane alpha helix passing through the membrane lipid bilayer. In the cartoon at the top, non-polar-aliphatic and aromatic residues are indicated by blue circles, the highly conserved Pro795 and Gly805 by black circles, basic residues by red circles, and polar, neutral residues by green circles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mentions: Mutation G805R is a rare mutation in Norway. Only two of a total of 1850 unrelated patients provided with a molecular genetic diagnosis of familial hypercholesterolemia, carry mutation G805R. Segregation analysis among family members of one of the index patients revealed that mutation G805R segregated with hypercholesterolemia through 14 meioses (Supplementary Fig. S1). The probability that this co-segregation occurred by chance is (1/2)14 = 0.006%. Mutation G805R was predicted to be pathogenic by the software programs PolyPhen2 (Score: “Probably Damaging”), SIFT (Score: “Not Tolerated”) and Mutation Taster (Score: “Disease Causing”). A multiple sequence alignment of the human LDLR (residues 764–839) and orthologs from 18 mammals and 8 additional vertebrate species found by searching the UniProt [9] and Ensembl [10] databases, is shown in Fig. 1. This alignment shows Gly805 to be highly conserved and there is a complete lack of residues with charged side chains in the transmembrane domain. Together, these data indicate that mutation G805R is pathogenic. The alignment also shows poor residue conservation in the ectodomain and in the transmembrane domain, but the latter is strongly enriched in hydrophobic residues. Three conserved positively charged residues of the cytoplasmic domain (Lys811, Arg814, and Lys816 in the human LDLR, Fig. 1) appear to be necessary for inserting the receptor with correct topology in the membrane, according to the positive-inside rule [11].

Bottom Line: Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum.This led to reduced amounts of the mature 160 kDa LDLR at the cell surface.It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

View Article: PubMed Central - PubMed

Affiliation: Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.

ABSTRACT
More than 1700 mutations in the low density lipoprotein receptor (LDLR) gene have been found to cause familial hypercholesterolemia (FH). These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

No MeSH data available.


Related in: MedlinePlus