A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating lupus aggravation in mice

Abstract

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1) mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409)A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409)A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409)A+Leader pep or K(409)A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409)A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1) mice were inoculated with Hsp60 or K(409)A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409)A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.

Figure 1. Characterization of K⁴⁰⁹A pep synthetic peptide.

A) Purification of K⁴⁰⁹A pep synthetic peptide by reversed-phase chromatography. B) Mass spectrometry analysis of pure K⁴⁰⁹A pep; mz 2,323.26. C) Amino acid sequence of Leader pep and K⁴⁰⁹A pep synthetic peptides.

Figure 2. Effect of the Leader pep and K⁴⁰⁹A pep peptides and autologous Hsp60 inoculation on SLE.

A) Survival of [NZBxNZW]F₁ female mice [n = 5 to 15/group] inoculated with synthetic peptides at 45 day-old. §Estimate was limited to the largest censored time [315 day-old]. ^#Data from . MST = Mean survival time. *p<0.05: K⁴⁰⁹A pep versus Leader pep; **p<0.01: Leader pep versus control group; ***p<0.001: WT versus control group. Results are representative of 2 independent experiments. B) Amino acid alignment of M. leprae Hsp65 and mouse Hsp60. Amino acid homology colors: Identical: red; strongly similar: green; different: black. C) Survival of [NZBxNZW]F₁ 45-day-old female mice [n = 5 to 6] inoculated with mouse Hsp60 [♦]; control group [Δ]. Results are representative of 2 independent experiments.

Figure 3. Anti-Hsp65 antibody production in H_III mice.

Time-course production of anti-Hsp65 IgG1 [A] and IgG2a [B] antibodies in H_III mice [n = 3 to 5/group] immunized with Leader pep or K⁴⁰⁹A pep. Antibody titers were evaluated at 7, 14, and 25 days after immunization with synthetic peptides. Results are representative of 2 independent experiments. Dashed lines represent basal levels. Data are expressed as means ± SD.

Figure 4. The neutralizing potential of previous inoculation of K⁴⁰⁹A molecule and anti-K⁴⁰⁹A antibodies on SLE suppression.

A) Combined effect of K⁴⁰⁹A and WT in survival time. Forty-five-day-old female [NZBxNZW]F₁ mice [n = 4 to 6/group] were inoculated with K⁴⁰⁹A pep or K⁴⁰⁹A, and after 7 days received WT rHsp65 [K⁴⁰⁹A pep+WT group: ▪] or Leader pep [K⁴⁰⁹A+Leader pep group: ◊]. ^#Data from . ***p<0.001: K⁴⁰⁹A pep+WT and K⁴⁰⁹A+Leader pep versus WT+K⁴⁰⁹A group. B) Survival analysis of [NZBxNZW]F₁ 45-day-old mice [n = 4 to 7/group] inoculated with Leader pep [•]; Leader pep incubated with H_III normal mice sera [○]; or Leader pep incubated with H_III K⁴⁰⁹A-immunized mice [▾]. Leader pep group was the reference group for unpaired t-test analysis; **p<0.01. Results are representative of 2 independent experiments.

Figure 5. Structural modeling of M. leprae WT and K⁴⁰⁹A Hsp65 and mouse Hsp60.

A) Superposition of the WT and K⁴⁰⁹A M. leprae Hsp65 overall structures showing the three characteristic domains: apical [yellow], intermediate [red], and equatorial [green], with the ADP evidenced in spheres. The peptide region [352 to 371 residues in WT Hsp65] is evidenced in orange and regions with high B-factor described by are shown in red. The inset table shows the results of the structural alignment of the proteins. B, C, and D) Main differences in the polar contacts performed by the peptide region and that affect the electrostatic potential in the WT Hsp65, Hsp65 K⁴⁰⁹A, and mouse Hsp60 proteins, respectively. Red color shows negative and blue positive charges.

Figure 6. Analysis of the prediction of peptide-epitope binding to MHC class I and II molecules for WT and K⁴⁰⁹A M. leprae Hsp65, focusing in 352–370 amino acid region.

Theoretical amino acid sequence identification of peptide-epitopes of the WT (A) and K⁴⁰⁹A (B) Hsp65 and the homology percentage of consensus peptide binders to MHC H2 alleles. Primary structure identification of peptide-epitopes of the WT (C) and K⁴⁰⁹A (D) binding to mice and human MHC class II molecules.