Human lens gamma-crystallins: isolation, identification, and characterization of the expressed gene products.

Siezen, Roland J.; Thomson, John A.; Kaplan, Elizabeth D.; Benedek, George B.

doi:10.1073/pnas.84.17.6088

Cited by 47 publications

(36 citation statements)

References 48 publications

(97 reference statements)

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“…3, we present the ascending limb of the coexistence curves of HGD and R14C. The critical temperature (T c ; i.e., the maximum temperature on the coexistence curve) of HGD is about 3°C, making it a low-T c protein (6) and not a high-T c protein as has been claimed (1,4). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the past, it has not been possible to conduct detailed studies on human ␥D crystallin because of the difficulty of obtaining sufficient quantities of pure protein from young, normal human lenses (4). Therefore, to characterize the normal protein thoroughly and investigate the mechanism by which the Arg-14 3 Cys mutation in ␥D could lead to cataract, we cloned and expressed human ␥D crystallin and its Arg-14 3 Cys mutant in Escherichia coli.…”

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confidence: 99%

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Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

130

184

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We have expressed recombinant wild-type human ␥D crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiolmediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.I n the hereditary, juvenile-onset cataract described by Stefan et al. (1), the lens, which is clear at birth, develops punctate opacities progressively, such that by two years of age the cataract is readily detectable, and matures by early childhood or adolescence. The punctate opacities seen in this cataract are in the nucleus and inner cortex, regions of the lens that are enriched in the ␥-crystallins. In the human lens, only two members of the ␥-crystallin family, ␥C and ␥D, are expressed in appreciable amounts, and only ␥D crystallin continues to be expressed until late childhood (2, 3). In affected individuals, a single point mutation has been identified in the ␥D crystallin gene that corresponds to the substitution of Arg-14 by a Cys. The identification of this mutation and the parallel between the time course of the pathology and the physiological expression of human ␥D crystallin strongly implicate the Arg-14 3 Cys mutant of ␥D in the development of this cataract. However, the molecular mechanism invoked to explain the observed opacity has been speculative (1).In the past, it has not been possible to conduct detailed studies on human ␥D crystallin because of the difficulty of obtaining sufficient quantities of pure protein from young, normal human lenses (4). Therefore, to characterize the normal protein thoroughly and investigate the mechanism by which the Arg-14 3 Cys mutation in ␥D could lead to cataract, we cloned and expressed human ␥D crystallin and its Arg-14 3 Cys mutant in Escherichia coli. Both the wild-type recombinant ␥D crystallin (HGD) and its Arg-14 3 Cys mutant (R14C) folded efficiently in E. coli and accumulated as soluble proteins. We isolated and purified the HGD and R14C proteins and determined their solution properties. Our results suggest that the disulfidecrosslinked oligomerization of R14C is responsible for the observed cataract. Furthermore, such oligomerization occurs without significant change in protein structure, conformation, and stability. Materials and MethodsCloning, Expression, and Isolation of Proteins. The human ␥D crystallin coding sequence was amplif...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

130

184

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“…In the case of human ␥D, surface properties may be particularly important. This protein belongs to the high critical temperature class of ␥-crystallins (17). High critical temperature proteins are cryoproteins that undergo a reversible phase transition at temperatures above freezing, resulting in opacities known as ''cold cataracts.''…”

Section: Resultsmentioning

confidence: 99%

Progressive juvenile-onset punctate cataracts caused by mutation of the γD-crystallin gene

Stephan

Gillanders

VanderVeen

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

164

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Cataracts are a significant public health problem. Here, we describe the genetic alteration responsible for a progressive form of cataract, segregating as an autosomal dominant trait in a three-generation pedigree. Unlike most autosomal dominant cataracts, these are not clinically apparent at birth but are initially observed in the first year or two of life. The opacification evolves relatively slowly, generally necessitating removal of the lens in childhood or early adolescence. A genome-wide search in our kindred revealed linkage at 2q33-35 where the ␥-crystallin gene cluster resides. A single base alteration resulting in an Arg-14 3 Cys (R14C) substitution in ␥D-crystallin was subsequently identified. Protein modeling suggests that the effect of this mutation is a subtle one, affecting the surface properties of the crystallin molecule rather than its tertiary structure, consistent with the fact that the patients' lenses are normal at birth. This is the first gene defect shown to be responsible for a noncongenital progressive cataract, and studying the defective protein should teach us more about the mechanisms underlying cataract formation.A number of inherited cataracts are known in both humans and animals (1-3). These cataracts are present at birth and generally static, suggesting a structural defect affecting lens development. Some, like the human cerulean (3) and Coppocklike (4) cataracts involve synthesis of grossly misfolded lens crystallins. Others, like the human zonular pulverulent (5) and autosomal dominant congenital cataract (2) cataracts, involve more subtle mutations in other lens proteins that cause congenital opacities. All of the nonsyndromal human cataracts known to be linked to a genetic locus or caused by a specific genetic mutation are congenital. We have identified a threegeneration family with hereditary progressive cataracts transmitted as a fully penetrant autosomal dominant trait (Fig. 1). The lenses of affected individuals appear clear at birth, but with time focal opacities develop, suggesting some progressive alteration in protein structure or interaction. The cataract is always detectable by age 2.5 and consists of grayish-white punctate opacities in the nucleus and surrounding deep cortex, with intervening and peripheral clear lens (Fig. 1 A). The cataracts are 5 to 6 mm in diameter, and the opacities become more dense over time such that lens extraction is ultimately required because of decreased vision. Once the opaque lens is removed and a synthetic lens inserted, vision returns to 20͞20 and the eye functions normally.To find the trait locus in the family, we performed a genome-wide search for linkage, obtained evidence for linkage at 2q33-q35 where the ␥-crystallin genes are located, and looked for mutations in the two ␥-crystallins that are expressed at high levels in the developing lens-␥-crystallins C and D. We discovered a mutation in the ␥D-crystallin (␥D) gene that may alter its interaction with other proteins. MATERIALS AND METHODSThese studies were performed ...

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“…The sequences of human ␣A, ␣B, ␤B1, ␤B2, ␥S, ␥C, and ␥D-crystallins have already been determined and their presence in the human lens has been demonstrated (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Furthermore, the positions where human ␣A, ␣B, ␤B1, ␤B2, and ␥S-crystallins migrate on two-dimensional gels have been determined (12)(13)(14).…”

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confidence: 99%

Sequence Analysis of βA3, βB3, and βA4 Crystallins Completes the Identification of the Major Proteins in Young Human Lens

Lampi

Shih

et al. 1997

Journal of Biological Chemistry

181

152

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A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as ␣A, ␣B, ␤A1, ␤A3, ␤A4, ␤B1, ␤B2, ␤B3, ␥S, ␥C, and ␥D-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human ␤A1, ␤A3, ␤A4, and ␤B3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of ␤A3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.Elucidating the structure and function of crystallins, the major proteins of human lens, is important, because alterations in these proteins may contribute to cataract formation. The goal of our laboratories is to determine what changes occur in human crystallins during aging and cataract formation. To accomplish this goal it is first necessary to perform the following in young, normal human lenses: 1) determine which crystallin subunits are present in the young lens; 2) map the positions where these crystallins migrate on two-dimensional electrophoretic gels; and 3) deduce and confirm the amino acid sequences of these proteins.The sequences of human ␣A, ␣B, ␤B1, ␤B2, ␥S, ␥C, and ␥D-crystallins have already been determined and their presence in the human lens has been demonstrated (1-11). Furthermore, the positions where human ␣A, ␣B, ␤B1, ␤B2, and ␥S-crystallins migrate on two-dimensional gels have been determined (12-14). In the present study, we completed the identification of all major crystallins resolved by two-dimensional electrophoresis, and add ␤A1, ␤A3, ␤B3, and ␤A4 to the list of major ␤-crystallins in young, normal human lens. We also correct the sequence of human ␤A3, complete the sequence determination of human ␤B3, and for the first time, report the sequence of human ␤A4. These deduced amino acid sequences were confirmed by mass spectrometry. MATERIALS AND METHODS Identification of Major HumanCrystallins-The posterior poles of human eyes from organ donors 7-months of age or less were obtained from the Lions Eyebank of Oregon within 48 h post-mortem. Following decapsulation, the lenses were homogenized in 1.0 ml of 20 mM phosphate buffer (pH 7.0), and 0.1 mM EGTA. Water-soluble proteins were isolated by centrifugation at 10,000 ϫ g for 30 min. Protein content was assayed by the BCA assay (Pierce) according to the manufacturer's protocol using bovine serum albumin as a standard. Water-soluble fractions were then dried by vacuum centrifugation and stored at Ϫ70°C prior to electrophoretic or chromatographic separation.Two-dimensional electrophoresis of water-soluble lens proteins, transfer to polyvinylidene difluoride membranes, and direct sequence analysis were carried out as described previously (15). Non-equilibrium pH gradient...

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Human lens gamma-crystallins: isolation, identification, and characterization of the expressed gene products.

Cited by 47 publications

References 48 publications

Molecular basis of a progressive juvenile-onset hereditary cataract

Molecular basis of a progressive juvenile-onset hereditary cataract

Progressive juvenile-onset punctate cataracts caused by mutation of the γD-crystallin gene

Sequence Analysis of βA3, βB3, and βA4 Crystallins Completes the Identification of the Major Proteins in Young Human Lens

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