Expression of cellular retinoic acid-binding protein (type II) in Escherichia coli. Characterization and comparison to cellular retinoic acid-binding protein (type I).
Cellular retinoic acid-binding protein type II, CRABP(II), has been expressed efficiently in Escherichia coli from the mouse cDNA and compared to E. coli-expressed cellular retinoic acid-binding protein type I, CRABP(I). CRABP(II) had a molecular weight approximately 15,700, a pI of 5.46, an Amax of 350 nm with A350/A280 of 1.8, a fluorescence excitation maximum at 347 nm, and a fluorescence emission maximum at 465 nm (holoprotein). All-trans-retinoic acid and 3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, and 18-hydroxy-retinoic acids bind CRABP(II) and CRABP(I) stoichiometrically (Kd values no greater than approximately 10-20 nM). 9-cis-Retinoic acid exhibited saturation binding to both CRABP(II) and CRABP(I) with similar affinities, Kd = approximately 50-70, as did 13-cis-retinoic acid, Kd = approximately 160-240 nM, demonstrating that CRABP(II) and CRABP(I) have similar orders of selectivity for known retinoids. HoloCRABP(II) transferred approximately 70% of its all-trans-retinoic acid to CRABP(I), however, indicating that CRABP(II) has approximately a 3-fold lower affinity for stoichiometrically binding retinoids than CRABP(I). Stern-Volmer plots of both native and denatured CRABP(II) and CRABP(I) were consistent with comparable loci for the tryptophan residues and similar three-dimensional structures. These results suggest that CRABP(II) and CRABP(I), when expressed in vivo in excess of total retinoids, bind several ligands, and functional dissimilarities between the two proteins would not be related to unique preferences for known endogenous retinoids. Rather, CRABP(I) and CRABP(II) may modulate the steady-state concentrations of retinoids to different set points.
Fiorella
P D
PD
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
Giguère
V
V
Napoli
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004926:Escherichia coli; D007526:Isoelectric Point; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012176:Retinoids / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D008024:Ligands; D018168:Receptors, Retinoic Acid; D012176:Retinoids; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular