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キーワード:
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要旨:
OSBP (oxysterol-binding protein) homologues, ORPs (OSBPrelated
proteins), constitute a 12-member family in mammals.
We employed an in vitro [3H]25OH (25-hydroxycholesterol)-
binding assay with purified recombinant proteins as well as live
cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH
(photo-cholesterol), to investigate sterol binding by the
mammalian ORPs. ORP1 and ORP2 [a short ORP consisting
of an ORD (OSBP-related ligand-binding domain) only]
were in vitro shown to bind 25OH. GST (glutathione Stransferase)
fusions of the ORP1L [long variant with an Nterminal
extension that carries ankyrin repeats and a PH
domain (pleckstrin homology domain)] and ORP1S (short
variant consisting of an ORD only) variants bound 25OH with
similar affinity (ORP1L, Kd =9.7×10−8 M; ORP1S, Kd =8.4× 10−8 M), while the affinity of GSTORP2 for 25OH was lower
(Kd =3.9×10−6 M). Molecular modelling suggested that ORP2
has a sterol-binding pocket similar to that of Saccharomyces cerevisiae
Osh4p. This was confirmed by site-directed mutagenesis
of residues in proximity of the bound sterol in the structural
model. Substitution of Ile249 by tryptophan or Lys150 by alanine
markedly inhibited 25OH binding by ORP2. In agreement with
the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked
with photo-25OH in live COS7 cells. Furthermore, in experiments
with either truncated cDNAs encoding the OSBP-related
ligand-binding domains of the ORPs or the full-length proteins,
photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6,
ORP7,ORP8, ORP10 and ORP11. In addition, theORP1L variant
and ORP3, ORP5, and ORP8 were cross-linked with photoCH.
The present study identifies ORP1 and ORP2 as OSBPs and suggests
that most of the mammalian ORPs are able to bind sterols.
