Hsp20 is one of the newly described members of the mammalian small heat-shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far-ultraviolet circular dichroism spectra as the most closely related sHsp, AB-crystallin, but was less heat stable, denaturing upon heating to 50°C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43-kDa dimers and 470-kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone-like activity than AB-crystallin, as indicated by its relatively poor capacity to diminish the reduction-induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C-terminus and less polar than other sHsps, but 1 H-NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C-terminal extension, may contribute to the less effective chaperone-like activity.Keywords : small heat-shock protein; dimer ; oligomer; chaperone-like activity ; protein structure.In mammals, six members of the ubiquitous superfamily of small heat-shock proteins (sHsp) are known to occur [1]. These are AA-and AB-crystallin, Hsp25 (depending on species, also indicated as Hsp27 or Hsp28), the more recently discovered p20 [2], (now dubbed Hsp20 [3]), and the latest additions, HSPB2[4] and HspB3 [5]. A-Crystallin is a major eye lens protein, composed of two types of subunits, AA-crystallin and AB-crystallin (for reviews see [6,7]). The latter also occurs at high levels in other tissues, notably in heart and striated muscle [8]. AB-crystallin is stress-inducible [9] and its level is increased in various neurodegenerative disorders and tumors [10Ϫ13]. Hsp25 occurs at low levels in various tissues and is also inducible upon stress (for review see [14]). Both A-crystallins and Hsp25 display ATP-independent chaperone-like properties and confer thermotolerance upon expression in cultured cells. Mammalian A-crystallins and Hsp25 have been studied extensively. In contrast, Hsp20 has been the subject of only four reports [2,3,15,16], while HSPB2 and HspB3 are, as yet, the least known [4,5]. Hsp20 was originally isolated in mixed complexes with AB-crystallin and Hsp25 from rat and human skeletal muscle, and its amino acid sequence is most similar to AB-crystallin [1, 2]. Hsp20 was detected in all rat tissues examined, reaching the highest levels (up to 1.3 % of total protein) in striated muscle, heart and diaphragm, similar to AB-crystallin and Hsp25. Like Hsp25, it is also conspicuously present in smooth muscle of the bladder and rectum. Hsp20 exists in muscle extracts in a multimeric and a disso...

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alphaA-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye lens. In lenses of rodents and some other mammals, there occurs a minor variant of alphaA-crystallin, which has an insertion of 23 amino acid residues. This variant, alphaA(ins)-crystallin, results from differential integration of an optional exon into a small fraction of the mRNA. We have studied whether this alternative splicing is caused by a non-consensus cytosine in the 5' splice site adjacent to the optional exon. After replacement of the aberrant cytosine in the hamster alphaA-crystallin gene by a consensus thymine, and transient transfection of this gene in Chinese Hamster Ovary cells, the optional exon is indeed almost completely spliced into the mature mRNA. In contrast, replacement of the cytosine by adenine or guanine completely abolishes the splicing of the optional exon. Our results confirm that alternative splicing of the alphaA-crystallin primary transcript is mainly due to a non-consensus 5' splice site nucleotide. However, we conclude that the small size of the optional exon is probably an additional contributing factor and therefore it seems that the splicing mechanism is based on recognition of exons rather than introns.