Antibodies against human muscle enolase recognize a 45-kDa bacterial cell wall outer membrane enolase-like protein

Abstract

Enolase, is a glycolytic enzyme ubiquitous in higher organisms, where it forms tissue specific dimers of isoforms, also found in the cytoplasm of fermentative bacteria. The aim of this work was to identify enolase-like proteins in the cell wall of some Gram-negative bacteria using antibodies against human beta-enolase, an isoenzyme specific to skeletal and heart muscles. Cell wall outer membrane protein (OMP) preparations were obtained from 9 strains of Enterobacteriaceae and one of Pseudomonas aeruginosa. Specific enzymatic enolase activity was detected in the supernatant fractions of cytosolic and inner membrane material, but not in purified OMP preparations. Rabbit polyclonal antibodies specific against human beta-enolase were prepared and purified using immobilized human beta-enolase in affinity chromatography. In SDS-polyacrylamide gel electrophoresis and immunoblotting assay of purified OMP preparations, rabbit anti-enolase antibody interacted specifically with a few OMPs, of which a 45-kDa band also interacted with human sera of patients presenting Buerger disease and atherosclerosis. The most distinct interaction of human sera was observed with a 45-kDa OMP of Klebsiella pneumoniae. This protein was further isolated from K. pneumoniae cell mass in two ways, namely preparative SDS-polyacrylamide gel electrophoresis and specific affinity chromatography using immobilized affinity-purified rabbit antibody raised against human beta-enolase. The data obtained from tandem mass spectrometry tryptic peptide analysis and sequence comparison of human and bacterial enolases using protein databases, could reveal the similarity in the epitopes between membrane enolase-like protein from Klebsiella and human beta-enolase. The results show that the protein present in all studied strains has a common epitope on human beta-enolase. These data raise the question whether such a bacterial protein might be a marker for detecting and monitoring damage to skeletal and heart muscles.

Mining the Moraxella catarrhalis Genome: Identification of Potential Vaccine Antigens Expressed during Human Infection

“…However, there are numerous examples of enzymes that are located in the outer membrane of gram-negative bacteria, including a phosphomonoesterase (P4) and a glycerol-3-phosphodiester phosphodiesterase (protein D) in H. influenzae, an enolase in Pseudomonas aeruginosa, the lipid kinase YegS in Salmonella, and the lipid A acylase PagP and deacylase PagL found in Pseudomonas, Bordetella, and Salmonella (24,32,39,41,42,49). Additionally, the M. catarrhalis BRO-1 ␤-lactamase is in the outer membrane (12).…”

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

“…Enolase from human skeletal muscle was prepared by a procedure reported previously [33] and stored in stabilizing 15 mM imidazol -HCl buffer, pH 7.0, with 50 mM NaCl, 3 mM MgSO 4 , and 25% glycerol. For the experiments the enzyme samples were dialyzed against PBS buffer pH 7.34 or against 15 mM Tris-HCl pH 7.0 containing 3 mM MgSO 4 .…”

“…The amount of micromoles PEP was calculated from DA 240 /min using value 1520 M 21 cm 21 of the molar absorbtion coefficient for PEP [34]. One unit of enolase activity is defined as the amount of protein which catalyzes the synthesis of 1 micromole of PEP/min under these conditions [33,35]. The specific activity was expressed in units per milligram of enzyme.…”

“…The protein bands were fixed, stained with 0.025% Coomassie Brilliant Blue R-250 and analyzed for the molecular masses according to Witkowska et al [33].…”

Distribution of beta-enolase in normal and tumor rat cells.

“…Purification of enolase from the human striated muscle, preparation of rabbit polyclonal serum against human β-enolase and isolation of specific antibodies from the rabbit serum by affinity chromatography were preformed according to our previous report [9]. The specificity of the obtained antibodies was confirmed on homogenates of human and rat striated muscles and β-enolase purified from human and rat tissue.…”