The identification and characterization of IbpA, a novel α-crystallin-type heat shock protein from mycoplasma

Abstract

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immuneelectron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.

N- and C-terminal regions of the small heat shock protein IbpA from Acholeplasma laidlawii competitively govern its oligomerization pattern and chaperone-like activity

“…† pET15b-IbpA that provides the expression of N terminally His 6 -tagged AlIbpA was obtained previously. 40 The truncated and mutant ibpA genes were amplied from A. laidlawii genomic DNA by using primer pairs as shown in Table S1 † to obtain a given plasmid. Sequences of primers used are shown in Table S2.…”

“…The reaction was performed at 25 C for 20 minutes and then stopped by the addition of 100 mM Tris pH 7.4. The samples were further analyzed by immunoblotting with AlIbpA-specic antibodies obtained previously 40 and developed by using anti-rabbit antibodies conjugated with peroxidase (SigmaAldrich cat. no A0545).…”

“…It has been shown previously that AlIbpA demonstrates the chaperone-like activity in vitro by preventing the temperature-induced denaturation of the bovine insulin. 40 Taking into account the fact that the denaturation temperature (T m 50 ) of ADH and other proteins exceeded that of AlIbpA, the bovine insulin has been chosen as a model substrate for the chaperone-like activity assays (see Table S4 † for values). For that, the T m 50 of insulin in the presence of either full-length or truncated AlIbpA proteins was determined by using the uorescent SYPRO-orange dye as described in Materials and methods (see Table S4 † for values).…”

“…The phytopathogenic mycoplasma Acholeplasma laidlawii contains only one single gene encoding sHSP. 40,41 Under heat shock conditions, the amount of AlIbpA protein in A. laidlawii cells increases up to 7% of the total cellular proteins and could appear a key factor that determines the temperature adaptation capacity of this bacterium. 40,41 In vitro AlIbpA was shown to exhibit the chaperone-like activity by preventing the DTT-or heat-induced aggregation of insulin 40 as well as of various cellular proteins from E. coli and A. laidlawii itself.…”

“…40,41 Under heat shock conditions, the amount of AlIbpA protein in A. laidlawii cells increases up to 7% of the total cellular proteins and could appear a key factor that determines the temperature adaptation capacity of this bacterium. 40,41 In vitro AlIbpA was shown to exhibit the chaperone-like activity by preventing the DTT-or heat-induced aggregation of insulin 40 as well as of various cellular proteins from E. coli and A. laidlawii itself. 42,43 In the following we unravel the roles of both N-and C-terminal regions of AlIbpA for self-oligomerization, substrate binding and the chaperone function regulation.…”