Alpha-crystallin mutations alter lens metabolites in mouse models of human cataracts

Abstract

Cataracts are a major cause of blindness worldwide and commonly occur in individuals over 70 years old. Cataracts can also appear earlier in life due to genetic mutations. The lens proteins, αA- and αB-crystallins, are chaperone proteins that have important roles maintaining protein solubility to prevent cataract formation. Mutations in the CRYAA and CRYAB crystallin genes are associated with autosomal dominant early onset human cataracts. Although studies about the proteomic and genomic changes that occur in cataracts have been reported, metabolomics studies are very limited. Here, we directly investigated cataract metabolism using gas-chromatography-mass spectrometry (GC-MS) to analyze the metabolites in adult Cryaa-R49C and Cryab-R120G knock-in mouse lenses. The most abundant metabolites were myo-inositol, L-(+)-lactic acid, cholesterol, phosphate, glycerol phosphate, palmitic and 9-octadecenoic acids, α-D-mannopyranose, and β-D-glucopyranose. Cryaa-R49C knock-in mouse lenses had a significant decrease in the number of sugars and minor sterols, which occurred in concert with an increase in lactic acid. Cholesterol composition was unchanged. In contrast, Cryab-R120G knock-in lenses exhibited increased total amino acid content including valine, alanine, serine, leucine, isoleucine, glycine, and aspartic acid. Minor sterols, including cholest-7-en-3-ol and glycerol phosphate were decreased. These studies indicate that lenses from Cryaa-R49C and Cryab-R120G knock-in mice, which are models for human cataracts, have unique amino acid and metabolite profiles.

Fig 1

Representative chromatogram of a lens from a 143-day-old WT mouse showing the elution of amino acids, carbohydrates, and sterols (top). The peaks in the chromatogram were identified and are shown in the table (bottom).

Fig 2. Total number of compounds and average total peak areas in mouse lenses.

Data were analyzed using the MPP software. (A) The total number of compounds and (B) average total peak areas in lenses from WT, Cryaa-R49C-het, Cryaa-R49C-homo, Cryab-R120G-het, and Cryab-R120G-homo mice are shown. The mice were 222 ± 73 days old, and the total number of lenses was 34.

Fig 3

(A) Representative chromatograms displaying changes in sugars and sterols in mouse lenses. Sugars (B) and sterols (C) in lenses from WT and Cryaa-R49C-het mice are shown. The sugar and sterol content in the mutant lenses were compared to lenses from WT mice. The match factors were 927 and 946 for α-D-mannopyranose and β-D-glucopyranose, respectively. (D) Lactic acid content in cryaa-R49C-het mouse lenses were compared to levels in WT mouse lenses. Data are presented as the means ± S.D. (P < 0.05).

Fig 4. Representative chromatograms of long-chain fatty acids in mouse lenses.

Several compounds in the lenses from Cryaa-R49C-homo mice were unchanged in abundance compared to lenses from WT mice (arrow). The inset displays an enlarged view of chromatogram in the beige circle. WT, blue; Cryaa-R49C-homo, red. (1), 2-palmitoylglycerol 2 TMS; (2), 1-monopalmitin 2TMS; (3), 2-monostearin 2TMS; (4), glycerol monostearate 2TMS.

Fig 5. Major and minor sterols in Cryab-R120G mouse lenses are compared to WT lenses.

Four or six lenses from mice of each genotype were individually analyzed, and the average percentage was determined. Data are presented as the means ± S.D. (*P < 0.05).

Fig 6. Amino acid content in Cryab-R120G mouse lenses are compared to WT mouse lenses.

The lenses were first derivatized with TMS, and the amino acids were detected as single- or double-derivatized species. Four or six lenses from mice of each genotype were individually analyzed, and the average percent area of each amino acid was determined. Data are presented as the means ± S.D. (*P < 0.05).