title: In-vitro characterization of the six clustered variants of NPC1L1 observed in cholesterol low absorbers.
abstract: OBJECTIVE
  Niemann-Pick C1-like 1 (NPC1L1) has been shown to be involved in cholesterol transport. Among nonsynonymous variants found from cholesterol low absorbers, six variants were located within only 39 amino acids in the predicted extracellular loop of NPC1L1 protein, suggesting the importance of the region with regard to the function of NPC1L1. In this study, we performed in-vitro analysis to determine the protein expression, cellular localization, and intrinsic activity of these variants. As alpha-tocopherol is also transported by NPC1L1, we compared the transport activity of NPC1L1 variants between cholesterol and alpha-tocopherol.
  
  
  RESULTS
  Expression vectors for the variants or wild type of NPC1L1 were constructed and transiently transfected into Caco-2 cells, which revealed that four kinds of variants (D398G, T413M, R417W, and G434R) are associated with the reduced expression level and altered subcellular localization of NPC1L1 protein. As four variants (A395V, G402S, R417W, and G434R) are expressed to some extent on the apical membrane, we constructed Caco-2 cells stably overexpressing these variants. All of these variants showed significantly lower transport activity of cholesterol and alpha-tocopherol than the wild-type NPC1L1, although the transport was ezetimibe-sensitive.
  
  
  CONCLUSIONS
  These results account for the reduced intestinal cholesterol absorption in subjects with these six kinds of variants and suggest the possibility of reduced alpha-tocopherol absorption in carriers of the six variants, due to their decreased expression level, altered subcellular localization or lower intrinsic activity compared with wild-type NPC1L1.
authors.affiliation: Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Yamanashi
authors.forename: Tappei
authors.initials: T
authors.lastname: Takada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D016000:Cluster Analysis; D005786:Gene Expression Regulation; D006801:Humans; D007408:Intestinal Absorption; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D024502:alpha-Tocopherol / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C410800:NPC1L1 protein, human; D012333:RNA, Messenger; D002784:Cholesterol; D024502:alpha-Tocopherol