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Age-related cleavages of crystallins in human lens cortical fibre cells generate a plethora of low molecular weight peptides and cross-linked crystallin complexes

ABSTRACT: A large variety of low molecular weight (LMW) peptides, derived from the breakdown of crystallins, have been reported in middle to old age human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilisation of crystallins – these being hall marks of cataractogenesis. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of human lenses aged 16, 44, 75 and 83 years. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fibre cells. Selected reaction monitoring (SRM) analysis of a prominent ?A-crystallin peptide (?A57-65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of ?A57-65 formation escalated significantly. Using 2-D gel electrophoresis and nanoLC-ESI-MS/MS, 13 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterised from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilise degraded crystallins by sequestration into water soluble complexes.

INSTRUMENT(S): QSTAR

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Xiaomin Song

PROVIDER: MSV000081536 | MassIVE |

SECONDARY ACCESSION(S): PXD001593

REPOSITORIES: MassIVE

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Age-related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes.

Su Shih-Ping SP Song Xiaomin X Xavier Dylan D Aquilina J Andrew JA

Proteins 20150819 10

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old-age hum ...[more]

PMID: 26238763

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Project description:Purpose: Measurement of crystallin abundances in human lens has previous relied on poorly reproducible electrophoretic separations or only semi-quantitative mass spectrometry. Therefore, isotope labeled standards were created for all major human lens crystallins so that their absolute abundances could be accurately measured in lens from birth to adulthood. Methods: All major human crystallins (αA, αB, βA2, βA3/A1, βA4, βB1, βB2, βB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as a standard. Soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses was then spiked with these mixed 15N labeled crystallin standards, proteins trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data-independent MS2 scans and data analysis using Skyline software. Human and mouse αA was also measured in the transgenic mouse αA N101D cataract model. Results: Crystallin abundances were accurately measured with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 5/1 to 2/1. γS-crystallin also increased 2-nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while βB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins βA2, βB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. Conclusions: The high content of αA and the transient abundance of βA2, βB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. βB1 becoming the most abundant crystallin may result from its role in promoting higher order β-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for crystallin purification and study. The 15N labeled crystallin standards will allow accurate quantitation of crystallins in lens anatomic regions, as well as measuring the composition of insoluble light scattering crystallin aggregates in cataractous lenses. They can also be used to measure the abundance of crystallins expressed in transgenic animal models.