Age-related Changes in Human Lens Crystallins Identified by Two-dimensional Electrophoresis and Mass Spectrometry

Lampi, Kirsten J.; Ma, Zijuan; Hanson, Stacy R.A.; Azuma, Mitsuyoshi; Shih, M.; Shearer, Thomas R.; Smith, David L.; Smith, Jean B.; David, Larry L.

doi:10.1006/exer.1998.0481

Cited by 242 publications

(240 citation statements)

References 43 publications

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“…Cleavage of long‐lived proteins on the N‐terminal side of Ser has been reported in proteins from a number of tissues (Table 1) (Smith et al ., 1992; Takemoto, 1995; Lampi et al ., 1998; Santhoshkumar et al ., 2008; Su et al ., 2010, 2012; Friedrich et al ., 2012) although this process has been most well documented in the lens. In the human lens, an abundant peptide is derived from the age‐related truncation of alpha B crystallin between residues 18 (His) and 19(Ser) (Santhoshkumar et al ., 2008).…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage next to these residues has been often observed in long‐lived proteins (Smith et al ., 1992; Takemoto, 1995; Lampi et al ., 1998; Santhoshkumar et al ., 2008; Su et al ., 2010, 2012; Friedrich et al ., 2012). Our work extends a publication on dipeptides by Yahiro et al .…”

Section: Discussionmentioning

confidence: 99%

“…However, the mechanisms that underpin some of these processes are still poorly understood. One such modification, which is commonly observed in long‐lived proteins, is truncation at the N‐terminal side of Ser and Thr residues (Lampi et al ., 1998; Friedrich et al ., 2012, 2000; Sergeant et al ., 2003; Santhoshkumar et al ., 2008; Vlad et al ., 2011; Su et al ., 2012). In some tissues, this process could be enzymically mediated; however, proteases with specificity for cleavage next to Ser/Thr are rare.…”

Section: Introductionmentioning

confidence: 99%

“…In the adult human lens, no active proteases remain in the centre (Zhu et al ., 2010) due to the particular growth pattern of the lens, so this is an excellent tissue to examine protein cleavage processes that occur spontaneously. In this tissue, several cleavage events on the N‐terminal side of Ser and Thr have been documented (Lampi et al ., 1998; Schey et al ., 2000; Santhoshkumar et al ., 2008; Su et al ., 2012). …”

Section: Introductionmentioning

confidence: 99%

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Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

2016

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SummaryOld proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long‐lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N‐terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser‐ and Thr‐containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N‐terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co‐ordinates zinc and that once zinc is co‐ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long‐lived proteins, for example truncation on the N‐terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long‐term stability of cellular proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

2016

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“…Over time, proteins are subject to a wide range of post-translational modifications that can reduce their stability and promote aggregation (Hains and Truscott 2007;Lampi et al 1998;Yanshole et al 2013). The crystallin proteins of the lens are particularly long-lived and any age-related damage they suffer typically accumulates.…”

Section: Introductionmentioning

confidence: 99%

Biophysical chemistry of the ageing eye lens

Ray

2015

Biophys Rev

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This review examines both recent and historical literature related to the biophysical chemistry of the proteins in the ageing eye, with a particular focus on cataract development. The lens is a vital component of the eye, acting as an optical focusing device to form clear images on the retina. The lens maintains the necessary high transparency and refractive index by expressing crystallin proteins in high concentration and eliminating all large cellular structures that may cause light scattering. This has the consequence of eliminating lens fibre cell metabolism and results in mature lens fibre cells having no mechanism for protein expression and a complete absence of protein recycling or turnover. As a result, the crystallins are some of the oldest proteins in the human body. Lack of protein repair or recycling means the lens tends to accumulate damage with age in the form of protein posttranslational modifications. The crystallins can be subject to a wide range of age-related changes, including isomerisation, deamidation and racemisation. Many of these modification are highly correlated with cataract formation and represent a biochemical mechanism for age-related blindness.

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Multiple separations facilitate identification of protein variants by mass spectrometry

Zhang

Smith

2001

Proteomics

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Identification of variant proteins from complex biological samples promises to contribute much to our understanding the etiology of pathological states. Characterization of variants, either due to genetic mutations in protein sequences or to post-translational modifications, is considerably more difficult than the simple protein identifications typical of most current proteomic investigations. Identification of a few peptides by database retrieval is not adequate when the goal is to have a complete understanding of the modifications of the protein. Although one advantage of mass spectrometry is its ability to obtain specific responses to several components, the complexity of biological samples is often overwhelming, resulting in spectra lacking useful information. For complex mixtures, isolation procedures before mass spectrometric analysis may need to include a variety of chromatographic and electrophoretic separation techniques. In this report, we illustrate how several preparative steps were essential for obtaining information about modified human lens beta-crystallins. The preparative techniques prior to mass spectrometry included size exclusion chromatography, reversed phase chromatography, two-dimensional gel electrophoresis, in situ digestion of the proteins and peptide trapping and washing before a final reversed phase high performance liquid chromatographic separation on-line to the mass spectrometer. This approach for isolation and analysis, when customized for other proteins, should find application in many studies where protein variants of complex mixtures are to be identified.

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Age-related Changes in Human Lens Crystallins Identified by Two-dimensional Electrophoresis and Mass Spectrometry

Cited by 242 publications

References 43 publications

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

Biophysical chemistry of the ageing eye lens

Multiple separations facilitate identification of protein variants by mass spectrometry

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