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, alphaB-crystallin. The concentration of total alphaB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as alphaB(g). Mass spectrometric analyses of tryptic and Asp-N digests showed alphaB(g) is alphaB-crystallin minus the C-terminal lysine. alphaB(g) constituted 10-90% of the total alphaB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of alphaB-crystallin. Human alphaB(g) and alphaB-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 alphaB(g) was comparable to that of recombinant human alphaB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating alphaB(g) is not known, but a premature termination of the alphaB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the alphaB-crystallin gene from lenses containing alphaB(g). The 16.4-kDa protein was an N-terminally truncated fragment of alphaB(g). The high concentration of alphaB-crystallin in these cataracts is the first observation of this kind in human lenses.
Highlights
From the NEI and the §NICHHD, National Institutes of Health, Bethesda, Maryland 20892, the ¶University of Pisa, 56100 Pisa, Italy, the ʈCenter for Cellular and Molecular Biology, 500 007 Hyderabad, India, and the **L.V
Human cataractous material was obtained from intracapsular cataract surgery done in India and from extracapsular extractions done at the National Eye Institute
The second spot, indicated by the white arrow migrated at a Mr of 16,400. Both of these proteins reacted with antibodies made against recombinant human ␣B-crystallin (Fig. 1B) suggesting that the proteins were related to ␣B-crystallin
Summary
From the NEI and the §NICHHD, National Institutes of Health, Bethesda, Maryland 20892, the ¶University of Pisa, 56100 Pisa, Italy, the ʈCenter for Cellular and Molecular Biology, 500 007 Hyderabad, India, and the **L.V. 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. On the other hand, ␣-crystallins are members of the family of small heat shock proteins and are thought to provide protection against cellular stresses (10 –12) Members of this family are structurally related via the ␣-crystallin domain, they form large aggregates, are phospho-proteins, and have chaperonelike activity (12–19). It is not known whether the primary function of ␣B-crystallin is that of a stress protein and related to its chaperone-like function or if its major role is structural. It is possible its role in the lens may change depending on the stage of development and location in the lens
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