Studies on the structural proteins of the human lens

Clark, R. C.; Zigman, Seymour; Lerman, Sidney

doi:10.1016/s0014-4835(69)80029-1

Cited by 93 publications

(21 citation statements)

References 10 publications

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“…The amount of WI recovered here is higher than that reported in previous studies (4,13), although the procedural methods are similar. However, the trend of increasing insolu- Vertical lines indicate the fractions whose analyses are given in Table 3.…”

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confidence: 62%

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Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.

Masters¹,

Bada²,

Zigler³

1978

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

117

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High D/L aspartic acid ratios are observed in heavy molecular weight aggregates and in water-insoluble protein extracted from whole lenses and nuclear and cortical regions. Purified a-, A-, and -y-crystallins have low D/L ratios. Fractionation of urea-solubilized material from the water-insoluble protein yields four molecular weight classes of proteins. Fractions representing crosslinked material or apparently degraded products have high D/L ratios. Racemization within lens proteins may contribute to formation of the water-insoluble fraction seen in aging lenses and cataracts. The racemization of aspartyl residues has recently been added to the list of age-related changes taking place in the proteins of the human lens (1). D-Aspartyl residues have been shown to accumulate at the rate of 1.25 X 10-3 yr-1 or 0.14% yr-1 in the central nucleus. The extent of racemization in yellow cataracts is equivalent to that observed in age-matched normal lenses, but brunescent cataracts exhibit up to a 2-fold increase in D/L aspartic acid ratios over comparably aged normal lenses or yellow cataracts.The rationale for investigating lens proteins came from our studies of calcified proteins in human teeth (2, 3). Detectable amounts of D-aspartic acid accumulate during the human lifespan as a result of the spontaneous chemical racemization reaction. These findings suggested that the aspartyl residues in any metabolically stable protein maintained at mammalian body temperature for several decades or longer would be subject to racemization. Because the proteins in the nucleus of the human lens are among the most stable in the body, we decided to test our hypothesis with the lens system.Numerous studies have shown that structural changes are taking place in lens proteins during the normal aging process and in cataracts. The proportion of water-insoluble protein increases relative to water-soluble protein (4, 5). The increase of insoluble protein is more marked in cataracts (6). When the water-insoluble residue is extracted with 7 M urea, the ureainsoluble portion is greater in cataracts (6) and is derived primarily from the lens nucleus (7). There is some evidence that more exposed thiol groups occur in cataractous proteins, and these proteins may have a greater susceptibility to tryptic digestion (8). All of these observations indicate that changes in native conformation of the lens proteins are occurring in cataract development and in aging.We have previously suggested that alterations in protein conformation will result from racemization of aspartyl residues (9). If racemization is to any degree responsible for the changes in the properties of lens proteins, then D-aspartic acid should

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contrasting

confidence: 62%

“…The proportion of water-insoluble protein increases relative to water-soluble protein (4,5). The increase of insoluble protein is more marked in cataracts (6).…”

mentioning

confidence: 97%

Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.

Masters¹,

Bada²,

Zigler³

1978

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

117

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“…The complex showed the presence of two tryptic peptide sequences of ␣A-crystallin: TLGPFYPSR (residues 13-21) and HFSPEDLTVK (residues 79 -88), and six tryptic peptides of ␣B-crystallin with sequences of: APSWFDTGLSEMR (oxidized Met residues 57-69), APSWFDTGLSEMRLEK (residue 57-72), HFSPEELK (residues 83-90), DRFSVNLDVK (residues 73-82), QDEHGFISR (residues 108 -116), and IPAD-VDPLTITSSLSSDGVLTVNGPR (residues 124 -149). This spot also contained three ␤A3-crystallin tryptic peptides with the sequences of ITIYDQENFQGKR (residues 33-45), WDAWSG-SNAYHIER (residues 96 -109), and MTIFEKENFIGR (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), two ␤A4-crystallin tryptic peptides with the sequences of MVVWDEDGFQGR (oxidized Met, residues 13-24) and LTIFEQENFLGK (residues 107-118), and four ␤B1 tryptic peptides with the sequence of LVVFELENFQGR (residues 60 -71), VSSGTWVGYQYPGYR (residues 187-201), ISLFE-GANFK (residues 151-160), and GYQYLLEPGDFR (residues 202-213). Additional peptides in the complex were one ␤B2 tryptic peptide with the sequence of VQSGTWVGYQYPGYR (residues 145-159), four ␥S tryptic fragments with the sequence of ITFYEDKNFQGR (residues 7-18), AVHLPSGGQYK (residues 84 -94), QYLLDKK (residues 148 -154), and KPIDW-GAASPAVQSFR (residues 158 -173), and one tryptic peptide of ␥D-crystallin with the sequence of QYLLMPGDYR (with oxidized Met, residues 143-152).…”

Section: Es-ms/ms Analysis Of Components Of Multimeric Complexes Frommentioning

confidence: 99%

“…The proportion of WI proteins increase in human lenses with aging and more so during cataract development (15), and the age-related water insolubilization of crystallins might be mediated via a precursor complex known as water-soluble, high molecular weight (HMW) proteins (16). One approach to deter-mine the relative importance of different modifications in cross-linking of crystallins is to ascertain their presence in the species that form water-insoluble covalent multimers.…”

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confidence: 99%

Characterization of Covalent Multimers of Crystallins in Aging Human Lenses

Srivastava

Kirk

Srivastava

2004

Journal of Biological Chemistry

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The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the waterinsoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25-and 41-yearold subjects showed distinct 5-and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52-and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25-and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. ␣A, ␣B, ␤A3, ␤A4, ␤B1, ␤B2, ␥S, and ␥D), and the second type of ␣-, ␤-, and ␥-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: ␣A-fragment (C-terminally truncated, residues 1-157), ␣B-fragment (residues 83-90), ␤B1-crystallin (residues 60 -71), ␤A3 (residues 33-44), ␤A4 (residues 106 -117), filensin (residues 78 -90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in ␣A-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.Mammalian lens contains three major structural proteins that are known as ␣-, ␤-, and ␥-crystallins. Among these, the ␣-and ␤-crystallins exist as oligomers, whereas the ␥-crystallin as a monomer. The ␣A-and ␣B-crystallins, the two subunits and primary gene products of ␣-crystallin, aggregate to form 800,000-Da oligomers, whereas ␤-and ␥-crystallins originated via gene duplication and form a superfamily. These structural proteins, by virtue of their special structural interactions and high concentrations, contribute to the transparency of the lens and provide refractive index to focus light on to the retina. With aging, crystallins show aggregation, cross-linking, and water insolubilization. Together, these processes contribute to the development of age-related lens opacity. Presently, the sequence of events that lead to development of opacity is not well understood. However, evidence suggests that a variety of posttranslational modifications in crystallins lead to their aggregation, cross-linking, and eventually water insolubilization. Because of several of the post-translational modifications simultaneously occur during aging in the native, aggregated, and water-insoluble crystallins, the identification of a single or combination of potenti...

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“…Only relatively small amounts of this material are present in young lenses (1, 2) but, with aging, this component becomes increasingly prominent (1)(2)(3)(4)(5). In cataract, particularly of the senile type involving the inner regions of the lens, there is a marked increase in this fraction and it may represent as much as 90% of the total protein (5-7).…”

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confidence: 99%

Disulfide-linked high molecular weight protein associated with human cataract.

Spector¹,

Roy²

1978

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

130

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A major high molecular weight disulfide-linked protein has been isolated from cataractous lenses. It is only present in the water-insoluble protein fraction. This species has not been found in normal lenses of comparable age. Upon reduction of this fraction, polypeptides having molecular weights of approximately 60,000, 43,000, and 20,000 as well as a noncharacterized heterogeneous species are released. Similar sized polypeptides have been found in various noncovalently linked agregates in both normal and cataractous lenses. Examination of the disulfide content of the high molecular weight disulfide-linked protein fraction indicates that approximately 70% of the sulfhydryl groups are in the oxidized state. Although little change in the sizes of the other major polypeptides in the water-insoluble fraction is observed upon reduction, these components were also found to contain an appreciable disulfide content. Such results indicate that the only major lens fraction containing disulfide-linked polypeptide is the high molecular weight species and that the disulfides present in the remaining fractions are either intrachain disulfides or link polypeptides to small peptides.One of the least understood protein fractions of the human lens is the water-insoluble protein component. Only relatively small amounts of this material are present in young lenses (1, 2) but, with aging, this component becomes increasingly prominent (1-5). In cataract, particularly of the senile type involving the inner regions of the lens, there is a marked increase in this fraction and it may represent as much as 90% of the total protein (5-7).In the bovine lens the water-insoluble protein is relatively simple, being composed primarily of a crystallin (8). However, in the human lens this is not the case and it would appear that this fraction may contain components arising from possibly all of the major structural lens proteins (9). There are a number of chemical characteristics besides solubility that distinguish the water-insoluble fraction from the remainder of the lens. It probably contains the oldest protein in the tissue. It is normally present in higher concentrations in the older inner region of the lens (7) where little protein synthesis is found (10), it contains a relatively high level of protein-associated nontryptophan fluorescence (2), and it has a markedly higher abundance of D-aspartic acid than does the soluble protein fraction (11,12).It has been suggested that the so-called high molecular weight protein is a precursor to the water-insoluble component because of similarities in its chemistry and its behavior upon differential centrifugation (9). Based upon light scattering theory (13) and the observed relationship between loss of lens transparency and accumulation of these protein species, particularly in the nuclear region, it has been proposed that certain types of cataract may be directly related to the production of these components (14). The present communication reports studies of aspects of the protein of cataract...

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Studies on the structural proteins of the human lens

Cited by 93 publications

References 10 publications

Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.

Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.

Characterization of Covalent Multimers of Crystallins in Aging Human Lenses

Disulfide-linked high molecular weight protein associated with human cataract.

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