Analysis of human lens crystallins and identification of post-translational modifications by mass spectrometry

Laura Jennene Miesbauer, Purdue University

Abstract

Post-translational modification as a result of aging or disease is thought to disrupt the proper close packing of the lens crystallins, causing a change in the refractive index and opacity of the lens. This study is a characterization of the most abundant human crystallins and the in vivo modifications that are present in normal lenses. Data from young normal lenses were used to identify modifications in crystallins from renal failure lenses. Two mass spectrometric techniques, electrospray ionization and fast atom bombardment mass spectrometry, were used to accurately determine the molecular weights of intact proteins, and peptides formed by enzymatic digestion, respectively. The water soluble and guanidine soluble portions of human lenses were fractionated by gel filtration chromatography, ion exchange and reversed-phase HPLC. The molecular weights of the intact proteins were determined by electrospray ionization mass spectrometry. Following enzymatic digestion of the proteins, the molecular weights of the peptides were determined by fast atom bombardment mass spectrometry. Post-translational modifications and errors in proposed amino acid sequences were indicated by molecular weights of proteins and peptides that differed from the calculated molecular weights. The specific sites of modification were determined. Although the majority of the crystallins appeared unmodified, mass spectrometric analysis of human $\alpha$-crystallin indicated an error in the amino acid sequence of $\alpha$A and a number of modifications. Three phosphorylation sites in $\alpha$B and two in $\alpha$A were identified, as well as deamidation and C-terminal degradation. An intramolecular disulfide bond was also observed in $\alpha$A. The $\alpha$-crystallins from renal failure lenses were similar to the normal $\alpha$-crystallins except for the presence of a protein-glutathione mixed disulfide bond and increased deamidation and degradation. The amino acid sequence of the principal $\beta$-crystallin, $\beta$B2, was determined. Mass spectrometric analysis of the $\gamma$-crystallins indicated that only $\gamma$s, $\gamma$C and $\gamma$D were present, and suggested that there were errors in the reported sequences and that the $\gamma$-crystallins were modified. All classes of crystallins were found in the guanidine soluble monomers from normal and renal failure lenses.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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