Unexpected Consequences of Deletion of the First Two Repeats of the Ligand-binding Domain from the Low Density Lipoprotein Receptor

Abstract

Heterozygosity for a 5-kilobase (kb) deletion of the first two ligand-binding repeats (exons 2 and 3) of the low density lipoprotein (LDL) receptor (R) gene (LDL-R ⌬5kb) confers familial hypercholesterolemia (FH).The FH phenotype is unexpected based on previous sitedirected mutagenesis showing that deletion of exons 2 and 3 resulted in little or no defect in LDL-R activity. In the present study, we took unique advantage of the ability to distinguish the LDL-R ⌬5kb from the normal receptor on the basis of size, in order to resolve this apparent discrepancy. Fibroblasts from heterozygotes for the LDL-R ⌬5kb displayed 50% of normal capacity to bind LDL and ␤-VLDL, apparently due to lower receptor number. Cellular mRNA for the ⌬5kb allele was at least as abundant as that for the normal allele. Immunoblotting and cell binding assays with anti-LDL-R antibody IgG-4A4 demonstrated normal synthesis and transport of the ⌬5kb receptor. Ligand blotting demonstrated that the ⌬5kb receptor displayed minimal or no ability to bind LDL or ␤-VLDL. Thus, in contrast to transfected cell lines, in human fibroblasts, the first two cysteinerich repeats of the LDL-R appear functionally necessary. These characteristics of the LDL-R ⌬5kb in human fibroblasts explain the in vivo phenotype of carriers. The low density lipoprotein (LDL)1 receptor (R) binds and catabolizes apolipoprotein E-containing chylomicron and VLDL remnants and LDL. In the liver, LDL-R functions to remove these lipoproteins from plasma for eventual excretion of the cholesterol into the bile. In peripheral cells, it functions to provide the cell with cholesterol needed for membrane synthesis. The LDL-R contains five major structural domains: a seven-repeat, cysteine-rich ligand binding domain encoded by exons 2-6, an epidermal growth factor-precursor homology domain (exons 7-14), a glycosylation domain (exon 15), a membrane-spanning domain (exon 16), and a cytoplasmic tail (exon 17) (1). The LDL-R is one of the few proteins for which knowledge of the structure-function relationship has been generated both from site-directed mutagenesis and from numerous naturally occurring human mutations.Mutations in the LDL-R gene resulting in a dysfunctional receptor cause a codominantly inherited disorder of plasma cholesterol catabolism known as familial hypercholesterolemia (FH). Human LDL-R mutations have been assigned to five classes of defects based on their phenotypic effects on the receptor protein (1). We have previously described a deletion of approximately 5 kb, which removes exons 2 and 3 of the LDL-R gene (LDL-R ⌬5kb) (2). In site-directed mutagenesis experiments, deletion of the first repeat (exon 2) has no effect on the binding or internalization of LDL or ␤-VLDL or recycling of receptors in transfected mammalian cells (3). Simultaneous deletion of exons 2 and 3 has resulted in a receptor which binds LDL 70% as well as the normal receptor and which binds ␤-VLDL equally as well (4). These results have led to the suggestion that the first two repeats of the LDL-R ligandb...

Phenotypic consequences of a deletion of exons 2 and 3 of the LDL receptor gene

“…The two other heterozygous relatives pos-sessing the ⌬2-3 mutation also had relatively high levels of total serum cholesterol. These findings are in agreement with those of Sass et al (36), who found that FH heterozygous French Canadians carrying a 5 kb deletion of exons 2 and 3, had unexpectedly high values of total serum cholesterol. Thus, heterozygotes for this deletion seem to be relatively more severely affected than the homozygotes.…”

“…The data of Russell et al (35) and those of ours are at variance with those of Sass et al (36). Based upon ligand blotting of cell protein extracts using either 125 I-labeled LDL or 125 I-labeled ␤VLDL, they concluded that the mutant receptor caused by a 5 kb deletion of exons 2 and 3 was unable to bind both LDL and VLDL.…”

“…Northern blot analysis of mRNA from fibroblasts from the homozygote cultured in the absence of a statin revealed that mRNA was at least as abundant as that of a normal cell line. A 50% overexpression of the deleted allele was also found in 7 French-Canadian heterozygotes with a 5 kb deletion of exons 2 and 3 (36). Taken together with the clinical findings, the Northern blot analysis could indicate that the transcription might already be at its maximum.…”

Calcium Is Essential for the Structural Integrity of the Cysteine-Rich, Ligand-Binding Repeat of the Low-Density Lipoprotein Receptor

“…Early mutational studies showed that repeats 3 to 7 were necessary for the binding of the LDL particle (i.e., recognition of apoB-100), whereas repeat 5 is critical for recognition of the β-VLDL (i.e., apoE binding) (26). Deletion of the first two repeats in a mutant LDLR has, however, recently been shown to eliminate binding to LDL and β-VLDL (27). Furthermore, it has been demonstrated that the LDLR also binds to the receptor-associated protein (28) and to lipoprotein lipase (29).…”

Three‐dimensional NMR structure of the sixth ligand‐binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities

“…Studies on mutant receptors showed that LB3–7 are required for high affinity binding of apoB100‐containing lipoproteins [10–12]. More recent studies on naturally occurring mutant LDL receptors from familial hypercholesterolaemia patients indicate that LB1 and LB2 are also required for the binding of apoB100‐containing lipoproteins [13,14]. The binding of apoE‐containing lipoproteins to the LDLR, however, is mediated primarily by LB5 [12].…”