The N1317H Substitution Associated with Leber Congenital Amaurosis Results in Impaired Interdomain Packing in Human CRB1 Epidermal Growth Factor-like (EGF) Domains

Abstract

The calcium-binding epidermal growth factor-like (cbEGF) domain is a widely occurring module in proteins of diverse function. Amino acid substitutions that disrupt its structure or calcium affinity have been associated with various disorders. The extracellular portion of CRB1, the human homologue of Drosophila Crumbs, exhibits a modular domain organization that includes EGF and cbEGF domains. The N1317H substitution in the 19th cbEGF domain of CRB1 is associated with the serious visual disorder Leber congenital amaurosis. We have investigated the structure and Ca 2؉ binding of recombinant wild-type and N1317H CRB1 fragments (EGF18-cbEGF19) using NMR and find that Ca 2؉ binding is altered, resulting in disruption of long range interactions between adjacent EGF domains in CRB1. From these observations, we propose that this substitution affects the structural integrity of CRB1 in the inter-photoreceptor matrix of the retina, where it is expressed. Furthermore, we identify disease-causing substitutions in other cbEGF-containing proteins that are likely to result in similar disruption of interdomain packing, supporting the hypothesis that the tandem cbEGF domain linkages are critical for the structure and function of proteins containing cbEGF domains.CRB1 is the human homologue of Drosophila Crumbs, a well conserved gene with homologues across multiple phyla (1). In Drosophila, the Crumbs protein is expressed in all ectodermally derived epithelial cells, which includes photoreceptors, and its disruption results in a discontinuous cuticle and extensive cell death (2, 3). In higher organisms, the orthologues CRB2 and CRB3 have a similarly broad expression profile (4, 5), but CRB1 is restricted to brain and retina (6). The expression of CRB1 in retina has been localized to the subapical region adjacent to the outer limiting membrane, a region analogous to the zonula adherens in Drosophila where a similar localization of the protein is observed (7-9).Unsurprisingly from its localization in humans, mutations in CRB1 have been associated with a number of visual disorders including retinitis pigmentosa with (10, 11) or without (11, 12) preserved para-arteriolar retinal pigment epithelium, paravenous pigmented chorioretinal atrophy (13), and Leber congenital amaurosis (LCA) 3 (14, 15). Of these disorders, LCA is the most severe form of inherited retinal dystrophy and is characterized by severe visual impairment from birth or very early life and a decreased or absent electroretinogram response (16). LCA is generally inherited in an autosomal recessive manner (16). Mutations in CRB1 represent a significant cause (10 -13%) of LCA among the identified LCA loci; missense, nonsense, and splice site mutations together with insertions and deletions have been identified (17, 18). The majority of disease-associated missense mutations in CRB1 have been localized to the extracellular region of the protein, and substitutions associated with the various disorders are approximately evenly distributed along its length, with no unambiguous g...

Biophysical Characterisation of Fibulin-5 Proteins Associated with Disease

“…Addition of Ca 2+ resulted in numerous peak shifts and intensity changes, consistent with Ca 2+ -dependent conformational changes. These features have been observed in other natively folded cbEGF constructs 25,29,31,33,35 and thus confirm that these constructs have the correct disulphide-bonded structure. The spectra for the G202R construct looked strikingly similar to those for the wild type, while some minor differences between the spectra of wild-type cbEGF2-4 and the spectra of I169T-substituted cbEGF2-4 were seen.…”

Mutations inCRB1are a Relatively Common Cause of Autosomal Recessive Early-Onset Retinal Degeneration in the Israeli and Palestinian Populations

“…The intracellular region that has a highly conserved role in organizing a macromolecular protein scaffold contains a conserved FERM binding domain, which is involved in localizing proteins to the plasma membrane, and a PDZ binding motif (PBM), used in the process of anchoring to the cytoskeleton. 2,3,7,16,18,19 Although no binding partners for the extracellular region have been identified so far to our knowledge, almost all identified CRB1 mutations affect this region. 1,[3][4][5]8,14 Missense mutations affecting cysteine residues are predicted to affect the secondary structure of the protein due to disruption of disulfide bridges.…”

“…For epithelial cells and photoreceptors, separation of their apical and basal compartments is critical for proper development and function of the cells and the tissue, including adhesion and signaling between and within cells. 2,7,13,[16][17][18][19] Jacobson et al suggested that CRB1 mutations underlie developmental defects in LCA, including thickening of the retina and lack of distinct layering in the fully developed adult retina. 13 Rashbass et al postulated that CRB1 has a role in localizing phototransduction proteins to the apical membrane of the photoreceptors.…”

“…Thus, missense mutations in those conserved residues might affect protein interaction. 20 In addition, missense mutations in the laminin AG-like and EGFlike domains are predicted to affect protein-protein interactions, calcium binding, and protein folding 2,16,19 and, thus, may affect proper retinal formation and/or function.…”

“…[3][4][5]7,13,15 CRB1 is a human homologue of the drosophila transmembrane crumbs protein, and is expressed in the brain and in the inner segments of mammalian photoreceptors. 2,3,7,[16][17][18][19] The Crumbs protein is implicated in mechanisms that control cellcell adhesion, intracellular communication and apicobasal cell polarity. For epithelial cells and photoreceptors, separation of their apical and basal compartments is critical for proper development and function of the cells and the tissue, including adhesion and signaling between and within cells.…”