alpha A-crystallin (alpha A) and alpha B-crystallin (alpha B) are among the predominant proteins of the vertebrate eye lens. In vitro, the alpha-crystallins, which are isolated together as a high molecular mass aggregate, exhibit a number of properties, the most interesting of which is their ability to function as molecular chaperones for other proteins. Here we begin to examine the in vivo functions of alpha-crystallin by generating mice with a targeted disruption of the alpha A gene. Mice that are homozygous for the disrupted allele produce no detectable alpha A in their lenses, based on protein gel electrophoresis and immunoblot analysis. Initially, the alpha A-deficient lenses appear structurally normal, but they are smaller than the lenses of wild-type littermates. alpha A-/- lenses develop an opacification that starts in the nucleus and progresses to a general opacification with age. Light and transmission electron microscopy reveal the presence of dense inclusion bodies in the central lens fiber cells. The inclusions react strongly with antibodies to alpha B but not significantly with antibodies to beta- or gamma-crystallins. In addition, immunoblot analyses demonstrate that a significant portion of the alpha B in alpha A-/- lenses shifts into the insoluble fraction. These studies suggest that alpha A is essential for maintaining lens transparency, possibly by ensuring that alpha B or proteins closely associated with this small heat shock protein remain soluble.

No 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.

No abstract