Abstract
Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.
Highlights
Mutations in the human gene RLBP1 encoding the cellular retinaldehyde-binding protein (CRALBP)1 cause retinal pathology and have been associated with autosomal recessive retinitis pigmentosa [1], Bothnia dystrophy [2, 3], retinitis punctata albescens [4], fundus albipunctatus [5], and Newfoundland rod-cone dystrophy [6]
Toward a better understanding of the molecular basis of retinal pathology associated with RLBP1 gene defects, we report here characterization of Recombinant CRALBP (rCRALBP) containing the M225K or R233W disease-causing mutations (Fig. 1)
Impaired cellular retinaldehyde binding protein (CRALBP) was first associated with autosomal recessive retinitis pigmentosa in 1997 when the missense mutation R150Q was found in the RLBP1 gene from a family in India [1]
Summary
Ated 11-cis-retinol was produced by reduction of 11-cis-retinal with NaB[3H]4 [15]. Mutagenesis and Production of rCRALBP Mutants M225K and R233W—Mutant rCRALBP cDNA carrying either the R233W or M225K substitutions were created using The QuikChange site-directed mutagenesis method (Stratagene). To substitute a Trp for an Arg at residue 233 (underlined) in the R233W mutant, complimentary oligonucleotides were used: sense, 5Ј-ATTCCTTCCCAGCCTGGTTCAAAGCCATCC-3Ј; antisense, 5Ј-GGATGGCTTTGAACCAGGCTGGGAAGGAAT-3Ј. Each mutagenesis mix was transformed into Escherichia coli strain XL1Blue (Stratagene), mutant clones identified by restriction mapping with NspI for M225K and MspI for R233W, were amplified, cleaved with XbaI and HindIII, and ligated back into expression vector pET19b (Novagen).
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