July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Deamidations, isomerizations, and cleavages at labile Asn residues in beta/gamma-crystallins associated with age-related cataracts
Author Affiliations & Notes
  • Kirsten J Lampi
    Integrative Biosciences, Oregon Health and Science University, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Kirsten Lampi, None
  • Footnotes
    Support  NEI EY027012 (KJL) and EY027768 (KJL and LLD) and Medical Research Foundation of Oregon (LLD) and Oregon Health & Science University Shared Resources Core Grant (KJL and LLD).
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3044. doi:
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      Kirsten J Lampi; Deamidations, isomerizations, and cleavages at labile Asn residues in beta/gamma-crystallins associated with age-related cataracts. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3044.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To determine if isoasp formation and cleavage occur in association with deamidation at labile Asn residues by simple incubation of crystallins over long periods.

Methods : Recombinantly expressed and purified wildtype and N76D gammaS-crystallin at 1.0 mg/ml concentration was incubated at pH 8 in PBS containing 10 mM imidazole, and 0.003% azide, while non-incubated protein served as a control. Proteins were then digested with trypsin and separated by 4 hour long gradients to resolve deamidated and isomerized peptides, and deamidation detected at high-resolution using 19 mDa mass defect measurements. Release of gamma S peptides due to spontaneous cleavage during incubation was determined by ultrafiltration to isolate low molecular weight peptides, followed by LC/MS analysis.

Results : Four month incubation of gamma S crystallin resulted in over 50% deamidation at N14 and the splitting of gamma S peptide 7-18 into at least 5 distinct peaks. Based on elution profiles, the major deamidated peak contained isoaspartate at residue 14. Analysis of the peptides generated spontaneously during the incubation identified an abundance of peptides containing a C-terminal N14 and D14, suggesting cleavage at the labile N14 proceeded even without deamidation. Similar results were obtained for N15 in betaB1-crystallin.

Conclusions : The susceptibility of N14 in gammaS-crystallin to deamidation, isomerization, and peptide bond cleavage during incubation may be due to its accessibility and the conformational flexibility of the loop region that it is located in. This residue also undergoes similar modifications in aged cataractous lenses supporting the correlation of these modifications with cataracts.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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