An Atypical Form of αB-crystallin Is Present in High Concentration in Some Human Cataractous Lenses

Abstract

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, ␣B-crystallin. The concentration of total ␣B-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as ␣B g . Mass spectrometric analyses of tryptic and Asp-N digests showed ␣B g is ␣B-crystallin minus the C-terminal lysine. ␣B g constituted 10 -90% of the total ␣B-crystallin in these cataracts and was preferentially phosphorylated over the typical form of ␣B-crystallin. Human ␣B g and ␣B-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human ␣B g was comparable to that of recombinant human ␣B-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating ␣B g is not known, but a premature termination of the ␣B-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the ␣B-crystallin gene from lenses containing ␣B g . The 16.4-kDa protein was an N-terminally truncated fragment of ␣B g . The high concentration of ␣B-crystallin in these cataracts is the first observation of this kind in human lenses.

Tracking pathology with proteomics: Identification ofin vivo degradation products of αB-crystallin

“…Figure 3 shows that spot 5 is present in high concentrations in the 90-and 69-year individuals and, though less pronounced, is also elevated in the Soemmerring rings of the 51-and 79-year individuals. A high concentration of aB 1±174 , aB g , in some cataracts has been previously reported [9]. While aB 1±174 is not present in clear lens tissue at visually detectable levels using Coomassie blue stain, it has been detected in clear lenses by MS and is shown here to be detected when brightness and contrast are optimized in Fig.…”

“…Spot 5 consists of aB-crystallin residues 1±174, aB 1±174 , previously called aB g [9]. Figure 3 shows that spot 5 is present in high concentrations in the 90-and 69-year individuals and, though less pronounced, is also elevated in the Soemmerring rings of the 51-and 79-year individuals.…”

Mechanism of Asparagine Deamidation During Human Senile Cataractogenesis

“…Numerous studies have demonstrated that it is possible to induce in vitro, deamidation of asparagine residues (Robinson and Rudd, 1974;Tyler-Cross and Schirch, 1991;Fujii et al, 1999). Furthermore, it has been shown that lens proteins undergo oxidation and truncation during human senile cataractogenesis (Takemoto, 1997;Jimenez-Asensio et al, 1999). By simulating these two process in vitro, it may be possible to induce structural changes in the gammaS crystallin molecule that will facilitate deamidation of asparagine-143 via a molecular mechanism that is analogous to the deamidation described in this report.…”

Shotgun identification of protein modifications from protein complexes and lens tissue

“…The described modifications to this family of proteins include: *The proteases trypsin, subtilisin, and elastase were chosen because they consistently produced peptides with different specificity resulting in high total sequence coverage by tandem mass spectrometry. truncation of N and C termini (30)(31)(32)(33)(34), deamidation (35)(36)(37), racemization (38), phosphorylation (33,35,39), oxidation (33,37,38,40), acetylation (41,42), carbamylation (43), disulfide formation (35,44), and glycation (42). Finally, because these proteins are long lived, they are prone to adventitious modification; therefore, it is necessary to have the ability to survey many possible modifications during a single experiment.…”