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Cellular retinaldehyde binding protein (CRALBP) is a 36-kD water-soluble protein which is found only in retina and pineal gland and which carries 11-cis- retinaldehyde or 11-cis-retinal as physiologic ligands. The RLBP1 gene was investigated computationally for single nucleotide polymorphisms. 116 polymorphisms were identified within this gene in which 3 were non-synonymous SNPs (nsSNPs) and only one was in synonymous region. Non-coding region was comprised of 7 SNPs in UTR and 98 were in the intronic region. The three nsSNPs were found within transcription factor binding sites. These included a G/A SNP, which resulted in a Arginine to Glutamine substitution (Arg151Gln), C/T SNP resulted in a Arginine to Tryptophan (Arg234Trp) and C/G/A SNP resulted in a Histidine to Glutamine substitution (His269Gln) in the mature retinaldehyde binding protein 1. Human RLBP1 model was build by Homology Modeling. The three reported SNPs on position 151, 234 and 269 were shown and mutated and the changes in the protein structure were analyzed. The models for these SNPs (p.151R>Q, p.234R>W and p.269H>Q) were constructed and analyzed for hydrogen bonding, ion pairs, accessibility and other parameters. The analysis enhances knowledge of RLBP1 structure function relationships, important for understanding associations of RLBP1 SNPs with genetic predisposition to night vision blindness, Bothnia retinal dystrophy (BRD), Newfoundland rod-cone dystrophy (NFRCD) and Fundus Albipunctatus (FA). Mutations in the domain could decrease the interactions between the residues causing unstability.
Cellular retinaldehyde binding protein (CRALBP) is a 36-kD water-soluble protein which is found only in retina and pineal gland and which carries 11-cis- retinaldehyde or 11-cis-retinal as physiologic ligands (Victor, 1991). Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Muller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration (John, et al. 1998). Human RLBP1 has molecular weights of 36,347 (36 kDa) (John, et al.1988) and its chromosomal location is 15q26. The retinoid-binding domain is located within the C-terminal part, between residues 120 to 313 (Chen et al. 1994; Crabb et al. 1998). Eight different RLBP1 (chromosome 15q26) mutations have been reported, including 4 missense, 2 frameshift, and 2 splice site alterations (Franceschett, François and Babel. 1974; Gränse et al. 2001; Morimura, Bersona and Dryja. 1999.). In Human Rlbp1 the retinoid-binding domain contains more nonpolar than polar residues (John W. 1998).
It is predicted from the RLBP1 cDNA nucleotide sequence that there are 317 residues (Crabb, et al. 1988). Mutation of protein sequence i.e. replacement of Glycine with Arginine results a change in structure as well as function of protein.
Retinitis pigmentosa is an inherited disorder, and therefore not caused by injury, infection or any other external or environmental factors. People suffering from RP are born with the disorder already programmed into their cells. Retinitis pigmentosa is characterized by constriction of the visual fields, night blindness, and fundus changes, including 'bone corpuscle' lumps of pigment. RP unassociated with other abnormalities is inherited most frequently (84%) as an autosomal recessive, next as an autosomal dominant (10%), and least frequently (6%) as an X-linked recessive (Boughman. 1980).
The complete x-ray crystallographic structure of the human Retinaldehyde binding protein is not known, but the sequence and basic protein structure is well understood. The 3-D structure of Rlbp1 is known and well characterized (Irina. 2003).
The analysis enhances knowledge of RLBP1 structure function relationships, important for understanding associations of RLBP1 SNPs with genetic predisposition to night vision blindness [MIM:268000], Bothnia retinal dystrophy (BRD) [MIM:607475], Newfoundland rod-cone dystrophy (NFRCD) [MIM:607476] and Fundus Albipunctatus (FA) [MIM:136880].
MATERIALS AND METHODS
Database Mining for SNP's
The detailed information of RLBP1 gene was obtained from the Online Mendelian Inheritance in Man (OMIM) http://www.ncbi.nlm.nih.gov/omim. We used National Center for Biotechnology Information (NCBI) database dbSNP for retrieval of SNP's causing Bothnia dystrophy (nonsyndromic autosomal recessive retinitis pigmentosa) and retinitis punctata albescens.
Functional Significant of SNP
Functional effects of the SNP's on protein were predicted by using freely available web servers. SIFT (http://blocks.fhcrc.org/sift/SIFT.html) was used to distinction between functional and non-functional coding mutations and its phenotypic effect. SIFT scores â‰¤ 0.05 are predicted by the algorithm to be intolerant or deleterious amino acid substitutions, whereas scores >0.05 are considered as tolerant (Ng and Henikoff, 2003). Higher the tolerance index of a particular amino acid substitution, lesser is its likely impact. Another server used for identification of potentially functional nsSNPs was PolyPhen (genetics.bwh.harvard.edu/pph). Predictions are based on a combination of phylogenetic, structural and sequence annotation information characterizing a substitution and its position in the protein. PolyPhen results were classified as 'benign', 'possibly damaging', or 'probably damaging' (Xi, et al.2004). The higher a position-specific independent counts (PSIC) score difference, the higher functional impact a particular amino acid substitution is likely to have. A PSIC score difference of 1.5 and above is considered to be damaging.
Modeling of SNP's location on protein structure
Primary sequence of human RLBP1 (Accession No: P12271) was retrieved from the SWISSPROT (Bairoch and Apweiler. 1997) data bank. Sequence homology searches of the Protein Data Bank, PDB (Berman,et al. 2000). Two homology models of Rlbp1 were obtained based on two different X-ray coordinates (PDB ID: 1xgg.pdb, 1xgh.pdb). The crystal structure coordinates were used as template for constructing the homology model of human Rlbp1. The 3D coordinates of the template were extracted from the Protein Data Bank, PDB.
The automated homology model building was performed using the protein structure-modeling program Deepveiwer / Swiss PDB. Reliability of the predicted homology models was assessed. To validate our models, Ramachandran plots were created and the structures were analyzed by PROCHECK (Laskowski. 1993). The model figures were prepared with Chimera (Pettersen, et al.2004).
We confirmed the mutation positions and the mutation residues from dbSNP server. These mutation positions and residues were in complete agreement with the results obtained with SIFT and PolyPhen programs. The mutations (p.151R>Q, p.234R>W and p.269H>Q) were performed using SWISSPDB viewer (Guex and Peitsch. 1997).
To study the diversity of RLBP1 among different species, a phylogenetic tree has been constructed. The multiple alignment used contains protein sequences of randomly selected animals (Fig-3). The phylogenetic tree has been constructed using the PHYLIP 3.5 package program (Felsenstein. 1981) and Treeview (Eisen. 1998) program has been used to visualize it. The method chosen for the construction of the tree was Most-likelihood (Baum. 1989).
Out of 116 SNPs, 3 were non-synonymous SNPs (nsSNPs) and only one was observed as synonymous. Non-coding region is comprised of 7 SNPs in UTR and 98 were in the intronic region. SIFT algorithm was used to predict whether an amino acid substitution may have an impact on protein function by aligning similar proteins, and calculating a score which is used to determine the evolutionary conservation status of the amino acid of interest. Three nsSNPs retrieved from RLBP1 were submitted independently to the SIFT program to check its tolerance index. SIFT scores (Xi. 2004) were classified as intolerant (0.00-0.05), potentially intolerant (0.051-0.10), borderline (0.101-0.20), or tolerant (0.201-1.00). The higher the tolerance index, the less functional impact a particular amino acid substitution is likely to have, and vice versa. Table-1 shows that two nsSNPs exhibit SIFT scores of 0.95, and are classified as 'Not tolerant' that could affect the protein function in the RLBP1 genes.
The structural levels of variation were determined by applying the PolyPhen program. It predicts the functional effect of amino acid changes by considering evolutionary conservation, the physiochemical differences, and the proximity of the substitution to predicted functional domains and/or structural features. All the three nsSNPs from RLBP1 submitted to SIFT were also submitted as input to the PolyPhen server. Table-1 presents the distribution of the variants by PolyPhen score. PolyPhen scores of >2.0, scores expected to be "Probably damaging" to protein structure and function (Sunyaev. 2000). Amino acid variants can impact the folding, interaction sites, solubility or stability of proteins. To understand the relationship between genetic and phenotypic variation, it is essential to assess the structural consequences of the respective non-synonymous mutations in proteins. To identify how often a disease phenotype can be explained by a destructive effect on protein structures or functions, we have mapped known disease mutations onto known three-dimensional structures of proteins based on PolyPhen score. The nsSNPs with accession numbers namely rs62640017, rs28933990 and rs28933989 showed a PSIC score 2.69, 2.65 and 1.7 at positions H269Q, R234W and R151Q respectively in RLBP1 gene were selected for modeling analysis. The nsSNPs which were predicted to be deleterious in causing an effect in the structure and function of the protein by SIFT and PolyPhen correlated well experimental studies (Maw.1997; Marie, 1999; Katsanis, et al. 2001; Nakamura, et al. 2005).
3D Structure Modeling and mutation studies
Single amino acid mutations can significantly change the stability of a protein structure. So, the knowledge of a protein's three-dimensional (3D) structure is essential for a full understanding of its functionality. Two homology models of RLBP1 were obtained based on two different X-ray coordinates (PDB ID: 1xgg.pdb, 1xgh.pdb). The crystal structure coordinates were used as template for constructing the homology model of human RLBP1. The 3D coordinates of the template were extracted from the Protein Data Bank (PDB). Mutation analysis was performed based on the results obtained from highest PolyPhen scores. The mutations at their corresponding positions were performed by SWISS-PDB viewer independently to achieve modeled structures. Then, energy minimizations were performed. The PolyPhen scores of SNPs in RLBP1 gene with ids namely rs62640017, rs28933990 and rs28933989 were 2.696, 2.654 and 1.778 respectively. It can be seen that the total energy for mutant type structure H269Q, R234W and R151Q were found to be -8663.721, -8048.969, -8369.965 Kcal/mol respectively.
Based on the SIFT, PolyPhen, and total energy values of the mutant proteins, solvent accessibility and secondary structure of all the residues in the normal protein and mutant protein H269Q, R234W and R151Q of Rlbp1 were computed with NetASA (Ahmad and Gromiha. 2002) and WHAT IF (Vriend. 1990). Solvent accessibilities and secondary structures of amino acid residues give a useful insight into the structure and function of a protein (Eyal, et al. 2004; Totrov. 2003). The prediction of residue solvent accessibility can help in better understanding the relationship between sequence and structure. The residue SER(150) showed a change in solvent accessibility from an buried to exposed state in the mutant protein R151Q and GLY(291) showed a change in solvent accessibility from an exposed to buried state in the mutant protein R151Q. Many studies have suggested that hydrophobic core residues are likely sites of deleterious mutations. Hence, change in solvent accessibility from an exposed to buried state could be considered functionally significant in the mutant protein at structural level (Chen and Zhou. 2005).
We used PHYLIP for constructing the phylogenetic tree. It was observed from the tree that RLBP1 gene of Human is more similar to the Pan troglodytes and is a little bit different from the Macaca mulatta. Bos Taurus and cattle were found similar near the primates. Rattus norvegicus and Mus musculus are from the same family Muridae that's why they were observe a same branch of the tree and showed a great similarity. Variation was observed between the Rlbp1 gene of Canis famillaris and Equus caballus, besides this all the other species were very much different from each other (Fig-3).
The retinaldehyde-binding protein 1 was investigated for single nucleotide polymorphisms (SNP). Out of 116 SNPs, 3 were non-synonymous SNPs (nsSNPs) of the RLBP1 gene. Which were submitted to the SIFT and PolyPhen algorithms. Sorting Intolerant from Tolerant (SIFT) classified 2 of 3 variants (66%) as "Not Tolerant." Polymorphism Phenotyping (PolyPhen) classed 2 amino acid substitutions as "probably damaging" and one as Possibly Damaging. Based on the PolyPhen scores and availability of 3D structures, structure analysis was carried out with the major mutation that occurred in the native protein coded by RLBP1 gene. Based on the SIFT, PolyPhen and total energy values of the mutant proteins, solvent accessibility and secondary structure of all the residues in the native protein and mutant protein (p.151R>Q, p.234R>W and p.269H>Q) of RLBP1 gene were computed with NetASA and WHATIF. Solvent accessibilities and secondary structures of amino acid residues give a useful insight into the structure and function of a protein. Based on this approach, we have shown that two nsSNPs, which were predicted to have functional consequences, were already found to be associated with disease risk.
In this study a G/A SNP, which resulted in an Arginine to Glutamine substitution change at position 151 in the mature protein. To examine the effect of the (Arg151Gln) substitution on protein structure a 3D model of the protein was generated (Fig-1 (B)). The neighbor residues of Arg151 were more exposed to the outer surface. The change is likely to disturb the hydrophobic core thus decrease in stability of this mutated protein is expected. The overall change is not significant in case of this mutation, as the H-bonding pattern was also similar in both wild and mutated protein. According to Maw.1997, mutant rCRALBP was purified from the soluble cell lysate and the protein structure was verified by mass spectrometry. The mutant protein lacked the ability to bind 11-cis-retinaldehyde, which leading to disruption of retinal vitamin-A metabolism.
Other SNPs of interest included a C/T SNP resulted in an Arginine to Tryptophan substitution (Arg234Trp). Replacement of Arg with Trp did not alter the accessibility significantly. The accessibility of Arg was found to be 14Å2 and that of mutated Trp was 12.7Å2. There was also no significant change occurs in the vicinity of Arg234Trp. It was also observed that the Hydrogen bonds of Arginine with its neighbor residues break up and new bonds have been observed by mutating the Arginine residue to Tryptophan (Fig-2). There are currently no data available to demonstrate a functional role for this amino acid, but the conservation of the residue in a family of related proteins suggests that it has a functional significance (Marie, et al. 1999).
Another exciting amino acid change identified is the His269Gln (Fig-1 (D)). Introduction of polar but uncharged glutamine residue is expected to cause relatively large structural changes then the Histidine mutant. According to this results obtained in our study, alteration in the amino acid accessibilities were observed; His=9.7Å2 ; Gln=15.5 Å2 . Some of the amino acids moved to slightly more buried positions as compared to the original model. These include His239, Ile241, Phe267, Val268, Phe276 and Glu279. Gly270 and Gly275 were moved to slightly exposed positions.
We would like to express our thanks to our colleagues and friends in Center of Excellence in Molecular Biology, Lahore and in Korean Bioinformatics Center, South Korea, who encouraged us by showing interest in our work. They generously provided reading materials and shared their knowledge with us.