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Long-lived Metabolic Enzymes in the Crystallin Lens Identified by Pulse-labeling of Mice and Mass Spectrometry

ABSTRACT: The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in age-related cataracts, and other lens diseases. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using dietary nitrogen-15 (15N)-labeling of mice between 3 and 15 weeks of age. By performing mass spectrometry we measured the 14N- to 15N-peptide ratios of hundreds of lens proteins, including Crystallin, Aquaporin, Collagen and Laminin of the lens capsule, and enzymes that catalyze glycolysis as well as oxidation and reduction reactions. Direct comparison of lens cortex versus nucleus longevity revealed little or no 15N-protein contents in most proteins in the lens nucleus, while there were a broad range of 14N/15N ratios of proteins in the cortex, including crystallin isoforms. Unexpectedly, like the crystallin proteins, many enzymes were also exceedingly long-lived, including those present at relatively high abundance in the nucleus. The slow replacement of these enzymes in spite of young age of the mice suggests their potential roles in age-related metabolic changes in the lens.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Jing Jin Jeffrey N. Savas, PhD

PROVIDER: MSV000084566 | MassIVE | Fri Nov 08 13:20:00 GMT 2019

SECONDARY ACCESSION(S): PXD016212

REPOSITORIES: MassIVE

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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.

Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants

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Long-lived Metabolic Enzymes in the Crystallin Lens Identified by Pulse-labeling of Mice and Mass Spectrometry

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