Abstract
The small heat shock protein, alpha-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of alphaA-crystallin involving the truncation of 5 C-terminal residues (alphaA(1-168)). Using novel mass spectrometry approaches and established biophysical techniques, we show that alphaA(1-168) forms oligomeric assemblies with a lower average molecular mass than wild-type alphaA-crystallin (alphaA(WT)). Also apparent from the mass spectra of both alphaA(WT) and alphaA(1-168) assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for alphaA(1-168). To examine the rate of exchange of subunits between assemblies, we mixed alphaB crystallin with either alphaA(WT) or alphaA(1-168) and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when alphaA(1-168) is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both alphaA(WT) and alphaA(1-168). Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian alphaA-crystallin.
Highlights
The small heat shock protein, ␣-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins
Using novel mass spectrometry approaches and established biophysical techniques, we show that ␣A1–168 forms oligomeric assemblies with a lower average molecular mass than wild-type ␣A-crystallin (␣AWT)
Effect of C-terminal Truncation on Oligomeric Size of ␣ACrystallin—Two separate approaches were used to investigate the oligomeric size of ␣AWT and ␣A1–168: size exclusion chromatography/multiangle light scattering (SEC-MALS) and nanoelectrospray-mass spectrometry (MS) under non-dissociating conditions
Summary
The small heat shock protein, ␣-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. As a member of the sHSP family of proteins, it is thought that, aside from contributing to the refractive index of the lens, ␣-crystallin provides protection to other resident proteins that undergo unfolding events [5] Such destabilization may be initiated by any number of stresses during the lifetime of the lens, such as UV light exposure, oxidation, chemical modification, and post-translational modifications [6]. The production of full-length ␣A and ␣B is under direct genetic control [9]; a multitude of post-translational modifications to these proteins, including phosphorylation, deamidation, and truncation, have been reported in the healthy lens (4, 10 –12) The impact of these modifications on the chaperone function of ␣-crystallin has been studied for many years with, in many cases, conflicting conclusions reported. We have recently reported that an S45D phosphorylation mimic of recombinant ␣B-crystallin leads to changes in subunit organization within the oligomer and consequent loss of effective chaperone function [17]
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