Structural and functional characterization of recombinant human cellular retinaldehyde‐binding protein

Crabb, John W.; Carlson, Anne; Chen, Yang; Goldflam, Steve; Intres, Richard; West, Kenneth A.; Hulmes, Jeffrey D.; Kapron, James T.; Luck, Linda A.; Horwitz, Joseph; Bok, Dean

doi:10.1002/pro.5560070324

Cited by 43 publications

(54 citation statements)

References 38 publications

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“…Production of rCRALBP-Wild-type human recombinant CRALBP (rCRALBP) was expressed in Escherichia coli strain BL21(DE3)LysS with an N-terminal His tag and apo-rCRALBP was purified to apparent homogeneity by nickel affinity chromatography (10,18). Unless otherwise noted, protein in the first five nickel affinity chromatography fractions (0.5 ml each) was pooled for the present structure-function study.…”

Section: Methodsmentioning

confidence: 99%

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

Hasan

Liu

et al. 2004

Journal of Biological Chemistry

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Cellular retinaldehyde-binding protein (CRALBP) 1 is a 36-kDa water soluble protein with a high affinity binding pocket for retinoids uniquely associated with vision, namely 11-cisretinal and 11-cis-retinol (1). Human CRALBP gene defects can either tighten or abolish retinoid interactions, which in turn can compromise substrate carrier interactions with 11-cis-retinol dehydrogenase and lead to several retinal pathologies (2). Targeted disruption of the CRALBP gene in mice impairs regeneration of both rod and cone visual pigments (3). These and other studies establish multiple functions for CRALBP in the retinal pigment epithelium (RPE), including roles as a major acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a facilitator of oxidation of 11-cis-retinol to 11-cis-retinal by 11-cis-retinol dehydrogenase (2-6). Ongoing proteomic studies support the existence of a RPE retinoid processing protein complex containing CRALBP (7). Direct CRALBP interactions have been demonstrated in vitro with 11-cis-retinol dehydrogenase (2) and with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), also known as sodium hydrogen exchanger regulator factor type 1 (NHERF-1) (8). Interactions with EBP50 have been suggested as a mechanism for localizing CRALBP to the apical RPE plasma membrane for export of 11-cis-retinal to the adjacent rod photoreceptor cells for visual pigment regeneration (8). The functions of CRALBP in tissues other than the RPE remain to be determined (i.e. in retinal Mü ller cells, ciliary epithelium, iris, cornea, pineal gland, and a subset of oligodendrocytes of the optic nerve and brain).To better understand CRALBP visual cycle functions, which require rapid association and dissociation of retinoid, we are characterizing the structure of the ligand binding pocket. Ligand interactions in CRALBP are non-covalent and previous structure-function studies (2, 9, 10) have implicated eight residues as possibly interacting with retinoid (Fig. 1). In this report, the interaction between human recombinant CRALBP and retinoid were characterized by mass spectrometry using hydrogen/deuterium exchange and photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal (DK-11-cis-retinal). This photoaffinity analogue of 11-cis-retinal has previously been used to map the movement of ligand within rhodopsin following sensi-* This study was supported in part by National Institutes of Health Grants EY6603, EY14239, GM63020, a Research Center Grant from The Foundation Fighting Blindness, and funds from the Cleveland Clinic Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Tables I and II. ¶ This work was submitted in partial fulfillment of the requirements for a doctoral degree in chemistry from Cleveland State University. 1 The abbreviations used are: CRALBP, cellular retinaldehyde-binding protein; DK...

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Section: Methodsmentioning

confidence: 99%

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

Hasan

Liu

et al. 2004

Journal of Biological Chemistry

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“…A value of 4.1 kcal/mol was further added to the calculated value for peptide xiv of fetuin due to likely salt bridge formation of this peptide (36). A value of 2 kcal/mol was further added to the calculated values for peptides xxiv-xxv due to losses of NH 3 at C-terminal, respectively (35,38 …”

Section: Ms Analysis Of Tryptic Peptide Mixtures From Complex Proteinmentioning

confidence: 99%

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Ding

Nothaft

Szymanski

et al. 2009

Molecular & Cellular Proteomics

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Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between nonglycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of nonglycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. Olinked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wildtype and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics. Molecular & Cellular Proteomics 8:2170 -2185, 2009.Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1-4). In fact, clinical biomarkers and therapeutic targets are often glycoproteins (5-9). Comprehensive glycoprotein characterization, involving glycosylation site identification, glycan structure determination, site occupancy, and glycan isoform distribution, is a technical challenge particularly for quantitative profiling of complex protein mixtures (10 -13). Both N-and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS 1 signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11-13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them. However, when peptides and glycopeptides coelute, parent ion scanning is required to selectively detect the glycopeptides (14). This can be problematic in terms of sensitivity, especially for detecting glycopep...

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“…Structure-function studies have defined ligand stereoselectivity and photosensitivity and a topological and epitope map and established in vitro evidence for a substrate carrier function in the RPE (6,7,20). Human and bovine CRALBP cDNAs have been cloned, and human recombinant CRALBP has been produced and used in functional domain analyses (21,29,30). This study has explored the molecular mechanisms controlling expression of the human CRALBP gene (RLBP1) in the RPE.…”

Section: Pce In the Cralbp Proximal Promoter Interacts With Proteins mentioning

confidence: 99%

Transcriptional Regulation of Cellular Retinaldehyde-binding Protein in the Retinal Pigment Epithelium

Kennedy¹,

Goldflam²,

Chang³

et al. 1998

Journal of Biological Chemistry

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Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Mü ller cells of the retina, where it is thoughtto function in retinoid metabolism and visual pigment regeneration. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. To identify the DNA elements that regulate expression of the human CRALBP gene in the RPE, transient transfection studies were carried out with three CRALBP-expressing human RPE cell culture systems. The regions from ؊2089 to ؊1539 base pairs and from ؊243 to ؉80 base pairs demonstrated positive regulatory activity. Similar activity was not observed with cultured human breast, liver, or skin cells. Since sequence analysis of the ؊243 to ؉80 region identified the presence of two photoreceptor consensus element-1 (PCE-1) sites, elements that have been implicated in photoreceptor gene regulation, the role of these sequences in RPE expression was examined. Mutation of either PCE-1 site significantly reduced reporter activity, and mutation or deletion of both sites dramatically reduced activity. Electrophoretic mobility shift analysis with RPE nuclear extracts revealed two complexes that required intact PCE-1 sites. These studies also identified two identical sequences (GCAGGA) flanking PCE-1, termed the binding CRALBP element (BCE), that are also important for complex formation. Southwestern analysis with PCE-1/BCEcontaining probes identified species with apparent masses near 90 -100 and 31 kDa. These results begin to identify the regulatory regions required for RPE expression of CRALBP and suggest that PCE-1-binding factor(s) may play a role in regulating RPE as well as photoreceptor gene expression. The retinal pigment epithelium (RPE)1 is a polarized monolayer of cells between the photoreceptors and choroid of the eye. Its primary function is maintaining and nourishing the rod and cone photoreceptor cells, including the synthesis of 11-cis-retinal for phototransduction (1). The critical importance of the RPE to the health of photoreceptors has been further illustrated by recent findings that mutations in either of two genes differentially and abundantly expressed in the RPE, namely the cellular retinaldehyde-binding protein (CRALBP) and RPE-65, can cause early onset autosomal recessive retinitis pigmentosa (2-4). The in vivo functions of CRALBP and RPE-65 have yet to be clarified; however, they are both thought to be involved in vitamin A metabolism. A retinal degeneration possibly associated with gene regulation in the RPE has been described for the vitiligo mouse; the vitiligo mouse exhibits genetic defects in transcription factor Mi and may also suffer from abnormal vitamin A metabolism (5).CRALBP appears to serve as a substrate carrier protein in the mammalian visual cycle, modulating whether 11-cis-retinol is stored as an ester in the RPE or oxidized by 11-cis-retinol dehydrogenase to 11-cis-retinal for visual pigment regeneration (6, 7). The protein carries 11-cis...

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Structural and functional characterization of recombinant human cellular retinaldehyde‐binding protein

Cited by 43 publications

References 38 publications

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Transcriptional Regulation of Cellular Retinaldehyde-binding Protein in the Retinal Pigment Epithelium

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