Topology of the outer membrane phospholipase A of Salmonella typhimurium.

The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic beta-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis.

The mutation Asp69-->Ser affects the chaperone-like activity of alpha A-crystallin.

alpha-Crystallins are members of the family of small heat-shock proteins. The conformation and mode of action of these 'junior chaperones' are unknown. To investigate the structure and chaperone-like activity, four mutants of bovine alpha A-crystallin were generated by site-directed mutagenesis. In comparison with wild-type alpha A-crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat-protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat-protective capacity were observed. Although it is assumed that small heat-shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in alpha-crystallin can also influence the efficiency of substrate binding.

Structure and modifications of the junior chaperone alpha-crystallin. From lens transparency to molecular pathology.

alpha-Crystallin is a high-molecular-mass protein that for many decades was thought to be one of the rare real organ-specific proteins. This protein exists as an aggregate of about 800 kDa, but its composition is simple. Only two closely related subunits termed alpha A- and alpha B-crystallin, with molecular masses of approximately 20 kDa, form the building blocks of the aggregate. The idea of organ-specificity had to be abandoned when it was discovered that alpha-crystallin occurs in a great variety of nonlenticular tissues, notably heart, kidney, striated muscle and several tumors. Moreover alpha B-crystallin is a major component of ubiquinated inclusion bodies in human degenerative diseases. An earlier excitement arose when it was found that alpha B-crystallin, due to its very similar structural and functional properties, belongs to the heat-shock protein family. Eventually the chaperone nature of alpha-crystallin could be demonstrated unequivocally. All these unexpected findings make alpha-crystallin a subject of great interest far beyond the lens research field. A survey of structural data about alpha-crystallin is presented here. Since alpha-crystallin has resisted crystallization, only theoretical models of its three-dimensional structure are available. Due to its long life in the eye lens, alpha-crystallin is one of the best studied proteins with respect to post-translational modifications, including age-induced alterations. Because of its similarities with the small heat-shock proteins, the findings about alpha-crystallin are illuminative for the latter proteins as well. This review deals with: structural aspects, post-translational modifications (including deamidation, racemization, phosphorylation, acetylation, glycation, age-dependent truncation), the occurrence outside of the eye lens, the heat-shock relation and the chaperone activity of alpha-crystallin.

Elastase inhibition by the C-terminal domains of alpha-crystallin and small heat-shock protein.

alpha-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of alpha-crystallin, alpha A and alpha B, also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both alpha A-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.

Comparison of the homologous carboxy-terminal domain and tail of alpha-crystallin and small heat shock protein.

The C-terminal domain and tail, which is the most conserved region of the alpha-crystallin/small heat shock protein (HSP) family, was obtained from rat alpha A-crystallin, bovine alpha B-crystallin and mouse HSP25. All three domains have primarily beta-sheet conformation and less than 10% of alpha-helix, like the proteins from which they are derived. Whereas the C-terminal part of alpha A-crystallin forms dimers or tetramers, the corresponding regions of alpha B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the alpha-crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.

Characterization of anti-crystallin autoantibodies in patients with cataract.

Anti-crystallin autoantibodies have often been demonstrated in the serum of healthy persons and, especially, patients with cataract. In no case, however, have the specific crystallin subunits been identified against which such antibodies are directed. This information would be of particular interest in view of the recent finding that several crystallin subunits occur constitutively outside the lens. To fill this gap, we analysed the sera of 15 patients with mature cataract by means of 1- and 2-dimensional immunoblotting. The circulating antibodies turned out to be directed against several beta- and gamma-crystallin subunits. The types of subunits and the intensities of the responses varied considerably between patients. No or only occasional and very weak reactions were observed against the alpha A-, alpha B- and beta B2-crystallin subunits. These are in fact the only crystallins at present known to occur outside the lens in mammals. Our findings thus indicate that anti-crystallin autoantibodies are specifically directed against those crystallins that appear to be lens-restricted, while immunological tolerance would exist for the extra-lenticularly occurring crystallins.

Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones.

alpha-Crystallin, composed of the subunits alpha A and alpha B, is a major vertebrate eye lens protein, accomplishing a structural role in maintaining lens stability and transparency. Both subunits also occur in low amounts outside the lens, where their precise function is unknown. They are structurally related to the small heat-shock proteins (HSPs), and increasing evidence indicates that they have also functional similarities with the small HSPs. To extend our insight into these structural and functional relationships, the mouse small HSP (HSP25) was compared with bovine alpha-crystallin, with respect to several known properties of the latter. We show that alpha-crystallin and HSP25 resemble each other in secondary structure and have similar stability toward urea dissociation at pH 7.0. Mixed polymers can be formed from any combination of alpha A-crystallin, alpha B-crystallin, and HSP25 subunits. Furthermore, we demonstrate that HSP25, like alpha-crystallin, can function as a molecular chaperone, by suppressing heat-induced aggregation of other proteins, and is an efficient inhibitor of elastase. Finally, HSP25 is found to be a substrate for protein cross-linking by tissue-type transglutaminase, like alpha B-crystallin. Our results thus corroborate that alpha-crystallin and the small HSPs have comparable functions, probably being involved in the protection of other proteins under conditions of stress.

Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.

alpha B-crystallin is present in reactive glia in Creutzfeldt-Jakob disease.

alpha-Crystallin is a major eye lens protein, composed of two types of subunits, alpha A and alpha B. The alpha A subunit is restricted to the lens, but alpha B-crystallin has recently also been detected in non-lenticular tissues, including the nervous system. With the use of a polyclonal antiserum directed against a synthetic C-terminal peptide of human alpha B-crystallin, the presence of alpha B-crystallin could be demonstrated immunohistochemically in astrocytes in the brains of patients with Creutzfeldt-Jakob disease (CJD). Most intensive localization was observed in the spongiotic tissue representing abundant progressively changed astrocytes in CJD. In age-matched control brains weak positive reaction was located in individual oligodendroglia cells and subpial astrocytes. Prominent increase of alpha B-crystallin in pathological glia in CJD may represent a response to stress.