A novel lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) was recently identified in bovine aortic endothelial cells. It is strongly suggested to have a potential role in the initiation and development of atherosclerosis. In this study, we have isolated cDNA clones encoding the rabbit homologue of LOX-1 by screening a rabbit placenta cDNA library. In amino acid sequence and domain structure organization, the rabbit LOX-1 is highly conserved with the human counterpart. Transfection of rabbit LOX-1 cDNA to HEK-293 cells conferred the activity to bind and internalize oxidized low density lipoprotein. Rabbit LOX-1 was identified as a 45-kDa protein by Western blot analysis with a specific monoclonal antibody. Notably, analyses by reverse trascription-polymerase chain reaction and Western blot revealed that LOX-1 was accumulated in 8-week-old Watanabe heritable hyperlipidemic rabbit aortas compared with normal rabbit aortas. Immunostaining confirmed that the augmented expression of LOX-1 was primarily localized within the intima at the earliest stages of atherogenesis. The most prominent staining was in the endothelial cells of lesions. Furthermore, the distinctive staining of LOX-1 was identified in the endothelium of nonlesion areas of Watanabe heritable hyperlipidemic rabbit aortas. Taken together, these findings support the possibility that LOX-1 might be involved in the initiation of atherosclerosis.

抄録全体を表示

In Japan, as consanguineous marriages are relatively frequent, it is possible to observe comparatively frequently the true homozygotes of FH. These patients inherit two identical LDL receptor gene mutations from their parents. By analyzing the LDL receptor mutations in 14 families with true homozygotes, we identified 9 different mutations. The true homozygotes are useful for characterizing the phenotype of each mutation due to the uniformity of the gene mutations. By analyzing these gene mutations together with their phenotypes in the fibroblasts of the patients, we could characterize several unique phenotypes of the LDL receptor such as internalization-defective, recycling-impaired, processing-impaired, truncated, degradation-enhanced and protein synthesis-impaired forms. From these studies we could determine the structure/function relationship of the LDL receptor protein.
It is known that in some selected populations, limited numbers of mutations of LDL receptor gene predominate. In such areas, diagnosis of FH is easier. To determine if some common mutations exist in Japan, we examined the frequency of each mutation identified in our FH homozygotes. By analyzing 120 unrelated FH heterozygotes, we found that the five mutations, 1) 1845+2T→C, 2) K790X, 3) C317S, 4) P664L, and 5) L547V, are relatively frequent. These mutations comprised about 30% of the mutant alleles of Japanese FH. Although the frequency of each mutation is not as high as those of common mutations found in populations such as French Canadians or Afrikaaners, these frequencies reflect a sort of “founder effect” as Japanese people are mostly uniracial and Japan is geographically isolated.
The natures of the LDL receptor gene mutations contributed to the clinical manifestation of FH in patients. Individuals bearing the receptor-defective type L547V mutation manifested lower plasma levels of LDL-cholesterol and had less aggressive coronary atherosclerosis than those bearing receptor-negative type mutations.

抄録全体を表示

Objective: Animal cells must regulate their cholesterol biosynthesis and uptake to supply sufficent amounts of cholesterol without risking overproduction. This coordination is achieved by a family of membrane-bound transcription factors called sterol regulatory element binding proteins (SREBPs). To enhance transcrtiption of genes encoding enzymes of cholesterol and fatty acid biosynthesis, the active NH2 terminal domain of SREBP is released from membranes of endoplasmic reticulum (ER) by two sequential cleavages. The first, regulated by sterols, is catalyzed Site-1 protease (S1P) that cleaves luminal loop of SREBPs. This reaction is mediated by a polytopic membrane protein called SREBP cleavage activating protein (SCAP) that complexes with SREBPs. SCAP appears to be a regulatory protein and serves as a sterol sensor. The second, not regulated by sterols, catalyzed by a hydrophobic zinc metallprotease, cleaves SREBPs within the first trans-membrane domain. Excess cholesterol block S1P activity thereby inhibits SREBP processing.
A cruial component of this regulatory pathway is the Site-1 protease (S1P) that initiates sterol-regulated release from the ER by making the first cut in the SREBPs. S1P is the target of the feedback regulation, but nothing is known about the structure and properties of S1P. To identify S1P, we carried out expression cloning.
Results: To clone S1P, we newly developed pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol deprived cells, cleavage by both proteases leads to PLAP secretion, but PLAP was not secreted in S1P deficient cells (SRD12B cells). We transfected pCMV-PLAP-BP2 plus pools of CHO cDNAs into SRD12B cells and monitored the PLAP secretion. We succeeded in identifying a cDNA that restores Site-1 cleavage. The cDNA encodes S1P; an intraluminal 1052-amino acid membrane bound subtilisin-like protease. We concluded that S1P is the sterol-regulated luminal protease that cleaves SREBPs and controls lipid metabolism in animal cells.

抄録全体を表示