Creation of a semi-synthetic γS-crystallin to support studies of age-related modifications in cataract

Abstract

Purpose : To develop a semi-synthetic approach using native chemical ligation to introduce specific sites of age-related modifications into crystallins to examine their impact on protein structure and contribution to cataract.

Methods : An N-terminally SUMO tagged human γS-crystallin containing residues 16-177 was expressed containing a Q16C mutation to act as a ligation site. Following removal of the SUMO tag using ulp-1 protease, the resultant γS 16-177 was solubilized in 6M guanidine and ligated to a synthetic γS 1-15 peptide containing a C-terminal peptide thioester using 4-mercaptophenylacetic acid under reducing conditions, followed by dialysis and purification using cation ion exchange chromatography. Full length γS-crystallin was confirmed by mass spectrometry and proper folding of the protein examined by both CD spectroscopy and hydrogen/deuterium exchange to measure the number of surface exposed amides in the whole protein. The ligated product was also compared to full length bacterially expressed γS Q16C, as well as denatured/refolded γS Q16C, and wild type γS.

Results : The semi-synthetic γS crystallin was successfully created and refolded in its native state as supported by an identical CD spectrum and similar numbers of rapidly exchanging amide residues as fully bacterially expressed versions of wild type γS, Q16C γS, and denatured/refolded Q16C γS.

Conclusions : This is the first known successful use of native protein ligation to create a semi-synthetic crystallin in its native state. The methodology will allow introduction of specific isoaspartate residues in γS at residues D12 and N14, two sites containing isoasp modifications in the insoluble protein of human cataractous lenses. Introduction of these age-related modifications in γS will test whether they contribute to cataract or are merely associative. The technique should prove useful to examine other age-related modifications in crystallins and provide more relevant information than previous studies relying on only in-vitro incubation or mutagenesis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.