Human Molecular Genetics, 2000, Vol. 9, No. 12 1779-1786
© 2000 Oxford University Press
Link between a novel human 
D-crystallin allele and a unique cataract phenotype explained by protein crystallography
ka2
ezá
ová1ek1Institute of Inherited Metabolic Diseases, Division B, Building D, Ke Karlovu 2, 12808 Prague 2, Czech Republic, 1Department of Gene Manipulation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 2Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic, 3Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic and 4Ophthalmologic Clinic, Charles University, 1st Faculty of Medicine and University Hospital, Prague, Czech Republic
Received 10 March 2000; Revised and Accepted 12 May 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We describe a 5-year-old boy with a unique congenital cataract caused by deposition of numerous birefringent, pleiochroic and macroscopically prismatic crystals. Crystal analysis with subsequent automatic Edman degradation and matrix-associated laser desorption ionization time-of-flight mass spectrometry have identified the crystal-forming protein as
D-crystallin (CRYGD) lacking the N-terminal methionine. Sequencing of the CRYGD gene has shown a heterozygous C
A transversion in position 109 of the inferred cDNA (36RS transversion of the processed, N-terminal methionine-lacking CRYGD). The lens protein crystals were X-ray diffracting, and our crystal structure solution at 2.25 Å suggests that mutant R36S CRYGD has an unaltered protein fold. In contrast, the observed crystal packing is possible only with the mutant protein molecules that lack the bulky Arg36 side chain. This is the first described case of human cataract caused by crystallization of a protein in the lens. It involves the third known mutation in the CRYGD gene but offers, for the first time, a causative explanation of the phenotype. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cataract (lens opacification) is an etiologically and genetically heterogeneous process, the common denominator of which is loss of lens mass physical homogeneity leading to abrupt changes of the refractive index resulting in light scattering and loss of transparency.
Cataractogenesis is associated with a long list of metabolic disorders either inherited or acquired, leading to breakdown of the lens supramolecular architecture mainly through loss of osmoregulation or oxidative damage (1,2). As the major proteins maintaining the lens transparency are crystallines (representing up to 90% of the soluble lens proteins) it is reasonable to consider them as a frequent final target of the pathologic events leading to their precipitation and formation of high molecular weight aggregates. Besides that, a number of structural alterations at the microscopic level have been described encompassing vacuoles, globular and membranous formations together with a number of alterations defined solely at the macroscopic level without microscopic correlation (3).
Recently it was shown that cataractogenesis can be primarily induced by mutations in the family of genes coding for crystallines, which in the mutant form are prone to aggregation. The disorders are congenital and are transmitted as an autosomal dominant trait. In laboratory animals the process was associated with mutations in the ß and crystallin genes (4–7). In humans, cataracts of various appearances were found to be associated with mutations in the CRYBB2 (8), CRYBA1 (9) and CRYAA (10) genes. Reactivation of the CRYGE pseudogene mutated in the promotor region and overexpressing the improperly folded incomplete single motif protein was found in a family with the hereditary Coppock-like cataract (11), which was later determined to be due to mutation in the CRYGC (12). Finally, cataracts with either dust-like opacities or with aculeiform phenotype were determined to be caused by mutation in the CRYGD gene (13,14). In some of the above-mentioned cases, the problem of causative explanation of the cataract phenotype seems to depend much, if not entirely, on understanding the propensity of the respective mutant crystallin to form deposits. A need for understanding the protein structural basis that underlies formation of protein aggregates has already been recognized and attempts have been made to accumulate relevant information. Up to now, the most advanced results in this direction were obtained by molecular modeling of the R14C CRYGD structure (13). These results confirm a substantial role for protein surface residues but, however plausible in general, do not unequivocally solve the problem as to how the altered primary structure may project itself into macroscopic properties of the mutated protein. We believe that our present study further establishes the structural basis of pathogenicity of mutant crystallines. In this report we describe a novel allele of the CRYGD gene that confers R36S mutation and is associated with unique deposition of crystals of the mutant gene product in the eye lens. As detailed below, X-ray diffraction data were obtained with isolated crystals of R36S mutated CRYGD and the solved crystal structure has directly shown that the mutation may fundamentally promote the crystal packing.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Microscopic analysis and histochemistry of the aspirated lens material
The lens extract smears showed crystals slightly stained by the Giemsa–May–Gruewald technique, resistant to Sudan dyes, to 0.1 N HCl, to 0.1 N NaOH and to chloroform–methanol extraction. There was no detectable acid phosphatase activity either around the crystals or in the rest of the smeared lens fibers. When suspended in phosphate-buffered saline (PBS) the crystals were macroscopically prismatic, pleiochroic and birefringent (Fig. 1b and c).
|
Ultrastructure
Many of the lens fiber cells displayed ultrastructure which could be classified as normal. There was a uniformly fine granular appearance of the cytoplasm and varied outline of the cell borders which were either even and almost linear, or highly undulating (ball-and-socket arrangement). Some were present as invaginations, which were either small (vermiform) or large (globular) up to 1 µM in diameter, from the neighboring cells. The invaginations, especially those of highly dense cytoplasms, could be distinguished from residential cell inclusions by the presence of gap junctions which were frequently seen as focal around 0.5 µM long plaque-like structures marking the cell membranes. Similar structures with highly irregular outlines were seen without apparent gap junction structures. There were marked differences in density of individual fiber cells ranging from very light to very dense. There were foci of massive globular cytoplasmic transformation with cytoplasmic globules 0.1–0.6 µM in diameter. The surface of the globules was covered by either a single or multilayered periodic membrane system of uncertain origin. The crystals were seen in a small number of cells of the sample processed for electron microscopy. They were either deposited free in the cytoplasm (Fig. 2a) or surrounded by concentrically arranged membranes (Fig. 2b and c) in foci rich in globular transformation.
|
Characterization of crystals
Resistance of the crystals to the above-mentioned procedures suggested their protein nature. To determine their protein identity, isolated crystals were analyzed on a 10–20% gradient SDS–PAGE gel. The gel was stained with Coomassie blue. The resulting predominant protein (20 kDa band) (Fig. 3b) was either transferred on polyvinylidene difluoride (PVDF) membrane and sequenced using automatic Edman degradation or cut from the gel, trypsin digested and subjected to matrix-associated laser desorption ionization time-of-flight (MALDI–TOF) mass spectrometry analysis. Both the 17 amino acid N-terminal sequence (Fig. 3b) and the 40% sequence coverage (for further increase, see below and Fig. 3d) showed the protein to be human
D-crystallin (CRYGD) (NCBI Protein Database accession no. I77413).
|
DNA sequencing and mutation analysis
Sequence analysis of the propositus CRYGD gene showed a heterozygous transversion in the coding region of exon 2 [109C
A in the corresponding cDNA (Fig. 3a)]. This mutation predicts an arginine to serine change at position 36 (R36S) of the processed, initiation-methionine-lacking protein. The predicted R36S replacement in the processed CRYGD was confirmed at the protein level by mass spectrometry of tryptic peptides of the crystal-forming protein (Fig. 3d) showing the expected mass difference in the fragment spanning amino acids 32–58 as well as perfect CRYGD identity in the remaining coverage of ~75% of the polypeptide length (see above). No mutation was identified either in the mother or in the healthy sibling of the propositus and in the 100 independent control alleles, using PCR–RFLP analysis (Fig. 3c). No DNA was available for analysis from the father, who died accidentally at age 33 years. He suffered from easily correctable myopia and there were no signs of an overt cataract on ophthalmologic examination of his eye fundus carried out by the ophthalmologist who examined the proband.
Protein crystallography
A striking feature of the R36S D-crystallin-composed crystals obtained from the lens deposits was their ability to diffract X-rays so that they provided fully measurable sets of reflections. From the viewpoint of protein crystallography, the crystals were of suboptimal quality (mosaicity: 0.9°, B value from the Wilson plot: 55 Å2), but allowed us to solve the crystal structure (Fig. 4). We solved the structure of human R36S-mutated CRYGD by molecular replacement using the known structure of bovine CRYGD (15) as a search model. The unit cell dimensions at 100K are: a = 54.24 Å, b = 81.98 Å and c = 105.54 Å. The space group is P212121 with two molecules of CRYGD per asymmetric unit (termed A and B). In the refinement of the crystal structure, 22 930 reflections were used (working set 21 753, test set 1177). The structure is presently refined to R/Rfree 26.4%/28.6% (by slow-cooling protocol of simulated annealing with non-crystallographic symmetry applied; no 
cut-off). A total of 342 residues plus 25 solvent molecules were refined (total 2823 atoms). The model lacks the C-terminal amino acids 172 and 173, where the electron density is poor. The stereochemistry of the final model shows that 259 residues (85.8% of the total 302 non-end, non-glycine and non-proline residues) have most favored main-chain dihedral angles, and 43 residues (14.2%) have additional allowed angles. Omit density maps clearly indicate the presence of serine side chains in both molecules at amino acid position 36, and make it obvious that the bulky arginine side chains cannot be accommodated there in any rotamer position. Although the protein fold of the mutated human CRYGD (Fig. 4d) is almost identical to that of bovine CRYGD, the respective molecular contacts (Fig. 4a, b and c) differ substantially. In crystals of bovine CRYGD (15) the side chains of arginine 36 do not point towards neighboring protein molecules. The structure determined for R36S human CRYGD shows that the Ser36 side chain of molecule A interacts with Asn24 of molecule B and the Ser36 side chain of molecule B points to Asn24 of the symmetry-related molecule A' (SerO to AsnN
distances being 3.0 and 2.7 Å, respectively) (Fig. 4b and c).
|
It is believed that many structural features determine the relative orientation of molecules in crystal lattices, the distribution of the surface charges being prominent among them. The absence of the Arg36 charge (Fig. 4e) could be seen as the factor promoting the resultant orientation of the molecules and, in turn, the crystal formation (and pathogenesis). At least, with all caveats of the complicated theory of mechanisms and kinetics of crystal formation, the missing charge can be seen as the feature that decreases the solubility of the R36S-mutated protein. Protein crystallography, however, gives us the most solid and clear-cut clue to the pathogenic crystal formation: the crystals cannot form with wild-type protein (either as the major or as an admixed component) because of steric hindrances imposed by the bulky Arg36 side chains. It is tempting to extend this line of reasoning to explain at least qualitatively the dominance of the mutant allele: the molecules of the protein product of the normal allele cannot, because of steric hindrance, enter (and perturb) the crystals that form with the pathogenic product.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Our case belongs to an extremely rare group of cataracts associated with crystal deposition. Exhaustive survey of the literary data showed, however, that the crystals described so far were composed of cholesterol, amino acids or calcium salts (16). The lens crystals strongly resembling those of our case were described in a case of crystalline cataract associated with uncombable hair (17). However, the crystals were suggested to be composed of cystine due to their high sulphur content. To the best of our knowledge, our case represents the first cataract caused by deposition of defined crystallized protein, proved to be CRYGD. CRYGD gene analysis identified a single transversion in heterozygous state (cDNA 109C
A) predicting R37S substitution (of unprocessed protein) at the protein level. The mutation was absent in both the patient’s mother and brother and in the control series. The father’s DNA sample was not available for analysis; however, his vision was reported by an ophthalmologist to be unaffected and no signs of a cataract were present. As hereditary congenital cataracts tend to be fully penetrative (18), we think that the mutation of the propositus arose de novo (cave! pater semper incertus).
The predicted R36S replacement in the processed CRYGD was confirmed at the protein level by mass spectrometry of tryptic peptides of the crystal-forming protein (Fig. 3d) showing the expected mass difference in the fragment spanning amino acids 32–58 as well as perfect CRYGD identity in the remaining coverage of ~75% of the polypeptide length. This finding, taken together with high conservation of the said arginine residue (scoring 35 among 36 CRYGD homologs cloned from various species, MAXHOM alignment; http://dodo.cpmc.columbia.edu ) provides conclusive evidence for the pathogenic nature of the product of the mutant allele. Mutations in CRYGD were recently proved to be cataractogenic, but leading to non-crystalline deposits. The R14C mutation led to non-crystalline punctate opacities (13), whereas the R58H mutation was associated with the aculeiform phenotype (14). Our R36S mutation was uniquely associated with formation of protein crystals in situ, whereas all other crystal-forming lens deposits described so far proved to be of uncertain (17) or non-protein (16) nature. The X-ray diffraction measurements with crystals of human mutant R36S CRYGD extracted from the patient’s lens enabled us to solve the crystal structure. This structure is the second ever solved for a member of the CRYGD family [the first being that of non-mutated bovine CRYGD (15), used by us as the search model in molecular replacement]. For comparative purposes, results of the protein crystallography study can be divided into two categories: (i) those elucidating the three-dimensional build-up of the mutated human CRYGD molecule; and (ii) those showing the arrangement of the molecules within the crystal lattice. Regarding the former, protein folds in R36S CRYGD and in the related wild-type molecule bear the closest resemblance, as could be expected also from the high homology of primary structures. The distinguishing feature is the crystal packing: in the structure of R36S CRYGD different contacts appear, and this is impossible with wild-type molecules. These contacts, inter alia, make the crystals of the mutant protein somewhat denser. Further, even though there is nothing that would prevent the molecules of mutant R36S CRYGD from forming the same contacts as the related wild-type molecules (15), such crystal packing has not actually occurred in the pathogenesis. All this taken together with consideration of the possible effects of elimination of the R36 surface charge, points to an inherent tendency of the mutant protein molecules to form permanent mutual contacts more easily than their wild-type counterparts. Needless to say, even with solid protein structural knowledge a broad variety of modalities are to be expected to take part in the triggering and progression of crystal growth. Thus, what remains to be explained are the epigenetic phenomena that govern the non-uniform pattern of deposition of the crystals within the cell population (suggesting the importance of local conditions) and the pathogenesis of the accompanying cell degradation. It is highly probable that the mutant CRYGD also exists in an alternative aggregate state represented by the dense amorphous globules, corresponding to its coprecipitate with both 
- and ß-crystallins in cold-induced experimental cataract (19).
Apart from this, this specific case permitted an unexpectedly straightforward protein crystallographic interpretation. Such ease may, in a more general context, only strengthen the rationale for the current trend to use X-ray crystallography in conjunction with structural genomics (20).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Patient
A 5-year-old boy suffering from photophobia and decreased visual acuity (symbol test chart 0.17 in his right eye and 0.15 in his left eye) due to symmetrical crystal deposition and greyish opacities in both lenses (Fig. 1a) underwent uncomplicated bilateral extracapsular cataract extraction by simple aspiration with intraocular lens implantation. There were no other clinical, pathological or biochemical findings, including hair abnormalities. On slit lamp examination there was a normal finding on the conjunctiva, cornea, anterior chamber and on the iris. In both lenses there were numerous transparent longitudinal crystals, some of them reaching 1 mm length with axes randomly oriented. The crystals were evenly distributed throughout the lens with a slightly higher density in the central area. The lens substance in between the crystals and the opacities was clear. The ophthalmoscopic examination of the fundus revealed normal finding. The child underwent uncomplicated successive extracapsular cataract extraction with anterior and posterior circular capsulotomy, anterior vitrectomy and intraocular lens implantation in both eyes, 2 and 5 months after the initial examination. The lens material was aspirated by canula, the anterior chamber being maintained by irrigation with Ringer’s solution. Subsequent examinations were carried out 4 and 7 months after the surgery showed quiet eyes with visual acuity 0.66. There were no other clinical findings, including hair abnormalities (see Discussion). Routine biochemical examinations, serum and urinary lactate, pyruvate and 3-OH butyrate concentrations, amino acid and urinary organic acid profiles, and serum galactitol concentration were within control range.
Staining procedures and tissue processing
The smeared lens extract was stained with Giemsa–May–Gruewald, examined under fluorescence and polarization microscopy. Acid phosphatase activity was examined using naphthol ASBI phosphate and hexazonium pararosaniline as coupler (21). Solubility was tested using dried acetone (15 min at room temparature) and chloroform:methanol 2:1 (1 h at room temparature) and 0.1 N HCl (15 min at room temparature). Lipid nature was tested with Sudan Black B. Part of the extracted lens was fixed with 10% paraformaldehyde, dehydrated and embedded into araldite–epon mixture. Semi-thin sections were stained with alkaline Toluidine blue, the thin sections were double contrasted and examined with a JEM 100B microscope.
Crystal isolation
The lens aspirate was methanol-fixed on the microscope slide and the crystals were picked up under an inverse microscope using a Pasteur pipette and collected in an Eppendorf tube. They were washed with distilled water, solubilized in SDS-containing buffer and analyzed by 10–20% gradient SDS–PAGE. The predominant 20 kDa protein band was electroblotted on a PVDF membrane and microsequenced using Edman degradation on an LF3600D Protein Sequencer (Beckman Instruments, Fullerton, CA). Search in sequence databases was done using the BLAST algorithm (http:// www.ncbi.nlm.nih.gov ). For mass spectrometry analysis, the band was excised from the SDS–PAGE gel and digested by trypsin. The resulting peptides were extracted into 40% acetonitrile in 1% TFA and analyzed on a MALDI–TOF Bruker Biflexmass spectrometer (Bruker-Franzen, Bremen, Germany), as described (22). Protein identity was elucidated using online resources (http://prowl.rockefeller.edu/cgi-bin/ProFound and http://falcon.ludwig.ucl.ac.uk/mshome3.2.htm ).
CRYGD gene amplification and sequencing
The genomic sequence of CRYGD gene was obtained from GenBank database (accession nos K03006 and K03005). The following primer pairs containing universal sequencing overhangs (underlined) were used for PCR amplification of two genomic fragments:
exons 1 and 2:
upper primer: 5'-GAAACAGCTATGACCATGGCCCCTTTTGTGCGGTTCTTGC-3';
lower primer: 5'-ATACGACTCACTATAGGGCGACTGATCGCTACTTCTAATGT-3'
exon 3:
upper primer: 5'-GAAACAGCTATGACCATGCACACTTGCTTTTCTTCTCTTT-3';
lower primer: 5'-ATACGACTCACTATAGGGCAAGACACAAGCAAATCAGTGCC-3'.
DNA templates were amplified using KlenTaq DNA polymerase (Ab Peptides, St Louis, MO) and standard PCR conditions (94°C for 5 min; 35 cycles of 94°C for 20 s, 60°C for 30 s, 74°C for 30 s; and final extension at 74°C for 10 min). Both fragments were subsequently gel purified and cycle sequenced on both strands using Cy5 5'-labeled T7 and reverse primers (Generi Biotech, Hradec Králové, Czech Republic). The sequences were analyzed on AlfExpress sequencer and read using the ALFWIN software (Pharmacia, Uppsala, Sweden). The sequence analysis was performed on a control, mother, brother and propositus genomic DNA samples isolated from blood (Qiagen, Hilden, Germany).
To confirm the mutation found in the propositus and to be able analyze the family members and controls, the analysis of the mutation destroying restriction site for Bsh1236I (MBI, Vilnius, Lithuania) was performed. PCR products of exons 1 and 2 were digested for 3 h according to the manufacturer’s instructions. Resulting fragments were analyzed on 12% denaturing polyacrylamide gel and visualized using the ethidium bromide staining (Fig. 3c).
Protein crystallography
Several crystals, ~0.15 x 0.15 x 0.5 mm from the lens tissue extract were transferred to PBS buffer with 25% (v/v) glycerol and flash cooled in 100 K nitrogen stream for measurements. Data were collected to 2.25 Å resolution at ESRF (Grenoble, France), beamline ID 14 EH3 [Rmerge 6.2% (23% for highest resolution shell); data completeness 99.6% (99.0%)]. The space group was determined from the systematic extinction of axis reflections and confirmed by Patterson synthesis and molecular replacement. Raw data were processed using the programs Denzo (23) and Scalepack (24) and converted into a unique set of structure factor amplitudes. The initial model was obtained by molecular replacement using the program Epmr (25) and one molecule of bovine CRYGD (26) (PDB code 1elp). Mutations of the bovine to the human amino acid residues and the choice of the side chain rotamers were done in all 23 differing positions: the requirement for substitution was in most cases visible also from the differential electron density map. The structure was refined using the CNS package (27) and Shelxl (28), and non-crystallographic symmetry was included through restraints; manual model building was done using the program O (29).
|
ACKNOWLEDGEMENTS |
|---|
We thank Julien Lescar (ESRF, Grenoble) for help with synchrotron data collection, Rolf Hilgenfeld (Institute of Molecular Biotechnology, Jena) and David Brooks (University of Pennsylvania) for critical reading of the manuscript. This work was supported by grant 203/98/K023 from the grant agency of the Czech Republic to J.S., by the Volkswagen Foundation grant I-74679 to K.B. and by projects of the ministry of education of the Czech Republic VS 96127 to M.E. and VS 96141 to K.B.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +420 2 2491 8283; Fax: +420 2 2491 9392; Email: melleder@beba.cesnet.cz
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Hejtmancik, J.F., Kaiser, M.I. and Piatigorsky, J. (1995) Molecular biology and inherited disorders of the eye lens. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Bases of Inherited Disease, 7th edn. McGraw-Hill, New York, NY, pp.4325–4349.
2 Hejtmancik, J.F. (1998) The genetics of cataract: our vision becomes clearer. Am. J. Hum. Genet., 62, 520–525.[Web of Science][Medline]
3 Yanoff, M. and Fine, B.S. (1996) Ocular Pathology, 4th edn. Mosby–Wolfe, London, UK/Baltimore, MD.
4 Chambers, C. and Russell, P. (1991) Deletion mutation in an eye lens beta-crystallin. An animal model for inherited cataracts. J. Biol. Chem., 266, 6742–6746.
5 Rodriguez, I.R., Gonzalez, P., Zigler Jr, J.S. and Borras, T. (1992) A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene. Biochim. Biophys. Acta, 1180, 44–52.[Medline]
6 Cartier, M., Breitman, M.L. and Tsui, L.C. (1992) A frameshift mutation in the gamma E-crystallin gene of the Elo mouse. Nature Genet., 2, 42–45.[Web of Science][Medline]
7 Klopp, N., Favor, J., Loster, J., Lutz, R.B., Neuhauser-Klaus, A., Prescott, A., Pretsch, W., Quinlan, R.A., Sandilands, A., Vrensen, G.F. and Graw, J. (1998) Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes. Genomics, 52, 152–158.[Web of Science][Medline]
8 Litt, M., Carrero-Valenzuela, R., LaMorticella, D.M., Schultz, D.W., Mitchell, T.N., Kramer, P. and Maumenee, I.H. (1997) Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human beta-crystallin gene CRYBB2. Hum. Mol. Genet., 6, 665–668.
9 Kannabiran, C., Rogan, P.K., Olmos, L., Basti, S., Rao, G.N., Kaiser-Kupfer, M. and Hejtmancik, J.F.C. (1998) Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene. Mol. Vis., 4, 21–26.[Medline]
10 Litt, M., Kramer, P., LaMorticella, D.M., Murphey, W., Lovrien, E.W. and Weleber, R.G. (1998) Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum. Mol. Genet., 7, 471–474.
11 Brakenhoff, R.H., Henskens, H.A., van Rossum, M.W., Lubsen, N.H. and Schoenmakers, J.G. (1994) Activation of the gamma E-crystallin pseudogene in the human hereditary Coppock-like cataract. Hum. Mol. Genet., 3, 279–283.
12 Heon, E., Priston, M., Billingsley, G.D., Lubsen, N. and Munier, F.L. (1999) Clarifying the role of gamma crystallin in congenital CCL cataract. Am. J. Hum. Genet., 65 (suppl.), A19.
13 Stephan, D.A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A.D., Robbins, C.M., VanAuken, A., Quesenberry, M.I., Bailey-Wilson, J. et al. (1999) Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene. Proc. Natl Acad. Sci. USA, 96, 1008–1012.
14 Heon, E., Priston, M., Schorderet, D.F., Billingsley, G.D., Girard, P.O., Lubsen, N. and Munier, F.L. (1999) The gamma-crystallins and human cataracts: a puzzle made clearer. Am. J. Hum. Genet., 65, 1261–1267.[Web of Science][Medline]
15 Chirgadze, Y.N. and Tabolina, O.Yu. (1996) Alternating charge clusters of side chains: new surface structural invariants observed in calf eye lens gamma-crystallins. Protein Eng., 9, 745–754.
16 Duke-Elder, S. (ed.) (1969) Diseases of the Lens and Vitreous; Glaucoma and Hypotony. System of Ophthalmology, vol. XI. Henry Kimpton, London, UK, p. 134.
17 de Jong, P.T., Bleeker-Wagemakers, E.M., Vrensen, G.F., Broekhuyse, R.M., Peereboom-Wynia, J.D. and Delleman, J.W. (1990) Crystalline cataract and uncombable hair. Ultrastructural and biochemical findings. Ophthalmology, 97, 1181–1187.[Web of Science][Medline]
18 Francis, P.J., Berry, V., Moore, A.T. and Bhattacharya, S. (1999) Lens biology: development and human cataractogenesis. Trends Genet., 15, 191–196.[Web of Science][Medline]
19 Lo, W.K. (1989) Visualization of crystallin droplets associated with cold cataract formation in young intact rat lens. Proc. Natl Acad. Sci. USA, 86, 9926–9930.
20 Burley, S.K., Almo, S.C., Bonanno, J.B., Capel, M., Chance, M.R., Gaasterland, T., Lin, D., Sali, A., Studier, F.W. and Swaminathan, S. (1999) Structural genomics: beyond the Human Genome Project. Nature Genet., 23, 151–157.[Web of Science][Medline]
21 Lojda, Z., Gossaru, R. and Schiebler, T.H. (1979) Enzyme Histochemistry. A Laboratory Manual. Springer, Berlin/Heidelberg/New York.
22 Bezouska, K., Sklenar, J., Novak, P., Halada, P., Havlicek, V., Kraus, M., Ticha, M. and Jonakova, V. (1999) Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin resistant boar seminal O-glycoprotein related to pB1 protein. Protein Sci., 8, 1551–1556.[Web of Science][Medline]
23 Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol., 276, 307–326. [Web of Science]
24. Collaborative Computational Project No. 4 (1994) The CCP4 Suite: programs for protein crystallography. Acta Crystallogr., D50, 760–763.
25. Kissinger, C.R., Gehlhaar, D.K. and Fogel, D.B. (1999) Rapid automated molecular replacement by evolutionary search. Acta Crystallogr., D55, 484–491.
26 Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr., D50, 760–763.
27 Kissinger, C.R., Gehlhaar, D.K. and Fogel, D.B. (1999) Rapid automated molecular replacement by evolutionary search. Acta Crystallogr., D55, 484–491.
28 Chirgadze, Y.N., Driessen, H.P.C., Wright, G., Slingsby, C., Hay, R.E. and Lindley, P.F. (1996) Structure of the bovine eye lens gammaD (gamma-Iiib)-crystallin at 1.95 Angstroms. Acta Crystallogr., D52, 712–721.
29 Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S. et al. (1998) Crystallography and NMR system (CNS): a new software system for macromolecular structure determination. Acta Crystallogr. D. Biol. Crystallogr., D54, 905–921.[Medline]
30 Sheldrick, G.M. and Schneider, T.R. (1997) SHELXL: high-resolution refinement. Methods Enzymol., 277, 319–343.
31 Jones, T.A., Zou, J.-Y., Cowan, S.W. and Kjeldgaard, M. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr., A47, 110–119.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Talla, N. Srinivasan, and D. Balasubramanian Visualization of In Situ Intracellular Aggregation of Two Cataract-Associated Human {gamma}-Crystallin Mutants: Lose a Tail, Lose Transparency Invest. Ophthalmol. Vis. Sci., August 1, 2008; 49(8): 3483 - 3490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. McManus, A. Lomakin, O. Ogun, A. Pande, M. Basan, J. Pande, and G. B. Benedek From the Cover: Altered phase diagram due to a single point mutation in human {gamma}D-crystallin PNAS, October 23, 2007; 104(43): 16856 - 16861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Wang, C. Cheng, L. Li, H. Liu, Q. Huang, C.-h. Xia, K. Yao, P. Sun, J. Horwitz, and X. Gong {gamma}D-Crystallin Associated Protein Aggregation and Lens Fiber Cell Denucleation Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3719 - 3728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Talla, C. Narayanan, N. Srinivasan, and D. Balasubramanian Mutation Causing Self-Aggregation in Human {gamma}C-Crystallin Leading to Congenital Cataract Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5212 - 5217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Flaugh, I. A. Mills, and J. King Glutamine Deamidation Destabilizes Human {gamma}D-Crystallin and Lowers the Kinetic Barrier to Unfolding J. Biol. Chem., October 13, 2006; 281(41): 30782 - 30793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Liu, X. Zhang, L. Luo, M. Wu, R. Zeng, G. Cheng, B. Hu, B. Liu, J. J. Liang, and F. Shang A Novel {alpha}B-Crystallin Mutation Associated with Autosomal Dominant Congenital Lamellar Cataract. Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 1069 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. V. Sergeev, L. V. Soustov, E. V. Chelnokov, N. M. Bityurin, P. S. Backlund Jr, P. T. Wingfield, M. A. Ostrovsky, and J. F. Hejtmancik Increased Sensitivity of Amino-Arm Truncated {beta}A3-Crystallin to UV-Light-Induced Photoaggregation Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3263 - 3273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Liu, X. Du, M. Wang, Q. Huang, L. Ding, H. W. McDonald, J. R. Yates III, B. Beutler, J. Horwitz, and X. Gong Crystallin {gamma}B-I4F Mutant Protein Binds to {alpha}-Crystallin and Affects Lens Transparency J. Biol. Chem., July 1, 2005; 280(26): 25071 - 25078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Santhiya, S. M. Manisastry, D. Rawlley, R. Malathi, S. Anishetty, P. M. Gopinath, P. Vijayalakshmi, P. Namperumalsamy, J. Adamski, and J. Graw Mutation Analysis of Congenital Cataracts in Indian Families: Identification of SNPs and a New Causative Allele in CRYBB2 Gene Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3599 - 3607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Ferrini, D. F. Schorderet, P. Othenin-Girard, S. Uffer, E. Heon, and F. L. Munier CRYBA3/A1 Gene Mutation Associated with Suture-Sparing Autosomal Dominant Congenital Nuclear Cataract: A Novel Phenotype Invest. Ophthalmol. Vis. Sci., May 1, 2004; 45(5): 1436 - 1441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.A. Reddy, O.A. Bateman, C. Chakarova, J. Ferris, V. Berry, E. Lomas, R. Sarra, M.A. Smith, A.T. Moore, S.S. Bhattacharya, et al. Characterization of the G91del CRYBA1/3-crystallin protein: a cause of human inherited cataract Hum. Mol. Genet., May 1, 2004; 13(9): 945 - 953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Graw, A. Neuhauser-Klaus, N. Klopp, P. B. Selby, J. Loster, and J. Favor Genetic and Allelic Heterogeneity of Cryg Mutations in Eight Distinct Forms of Dominant Cataract in the Mouse Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1202 - 1213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A T Moore Understanding the molecular genetics of congenital cataract may have wider implications for age related cataract Br J Ophthalmol, January 1, 2004; 88(1): 2 - 3. [Full Text]
|
|
|
|
|
|
J. Graw, N. Klopp, A. Neuhauser-Klaus, J. Favor, and J. Loster CrygfRop: The First Mutation in the Crygf Gene Causing a Unique Radial Lens Opacity Invest. Ophthalmol. Vis. Sci., September 1, 2002; 43(9): 2998 - 3002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Graw, A. Neuhauser-Klaus, J. Loster, N. Klopp, and J. Favor Ethylnitrosourea-Induced Base Pair Substitution Affects Splicing of the Mouse {gamma}E-Crystallin Encoding Gene Leading to the Expression of a Hybrid Protein and to a Cataract Genetics, August 1, 2002; 161(4): 1633 - 1640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S T Santhiya, M Shyam Manohar, D Rawlley, P Vijayalakshmi, P Namperumalsamy, P M Gopinath, J Loster, and J Graw Novel mutations in the {gamma}-crystallin genes cause autosomal dominant congenital cataracts J. Med. Genet., May 1, 2002; 39(5): 352 - 358. [Full Text] [PDF]
|
|
|
|
|
|
D. G. Brooks, K. Manova-Todorova, J. Farmer, L. Lobmayr, R. B. Wilson, R. C. Eagle Jr, T. G. St. Pierre, and D. Stambolian Ferritin Crystal Cataracts in Hereditary Hyperferritinemia Cataract Syndrome Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 1121 - 1126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Graw, A. Neuhauser-Klaus, J. Loster, and J. Favor A 6-bp Deletion in the Crygc Gene Leading to a Nuclear and Radial Cataract in the Mouse Invest. Ophthalmol. Vis. Sci., January 1, 2002; 43(1): 236 - 240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Graw, J. Löster, D. Soewarto, H. Fuchs, A. Reis, E. Wolf, R. Balling, and M. H. de Angelis Aey2, a New Mutation in the {beta}B2-Crystallin-Encoding Gene of the Mouse Invest. Ophthalmol. Vis. Sci., June 1, 2001; 42(7): 1574 - 1580. [Abstract] [Full Text]
|
|
|
|
|
|
A. Pande, J. Pande, N. Asherie, A. Lomakin, O. Ogun, J. King, and G. B. Benedek Crystal cataracts: Human genetic cataract caused by protein crystallization PNAS, May 22, 2001; 98(11): 6116 - 6120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Graw, N. Klopp, J. Löster, D. Soewarto, H. Fuchs, J. Becker-Follmann, A. Reis, E. Wolf, R. Balling, and M. H. de Angelis Ethylnitrosourea-Induced Mutation in Mice Leads to the Expression of a Novel Protein in the Eye and to Dominant Cataracts Genetics, March 1, 2001; 157(3): 1313 - 1320. [Abstract] [Full Text]
|
|
|
|
|
|
N. Klopp, J. Löster, and J. Graw Characterization of a 1-bp Deletion in the {{gamma}}E-Crystallin Gene Leading to a Nuclear and Zonular Cataract in the Mouse Invest. Ophthalmol. Vis. Sci., January 1, 2001; 42(1): 183 - 187. [Abstract] [Full Text]
|
|
|
|
|
|
D. Sinha, M. K. Wyatt, R. Sarra, C. Jaworski, C. Slingsby, C. Thaung, L. Pannell, W. G. Robison, J. Favor, M. Lyon, et al. A Temperature-sensitive Mutation of Crygs in the Murine Opj Cataract J. Biol. Chem., March 16, 2001; 276(12): 9308 - 9315. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||