Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types

Nemeth, Peter; Small, William; Evans, Claudia T.; Wang, Zhi; Persson, Lars; Srere, Paul A.

doi:10.1002/jmr.300040206

Cited by 13 publications

(6 citation statements)

References 30 publications

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“…Cut‐off values were calculated from the average of measured optical density (OD) 492 nm data. All measurements were standardized using a monoclonal anti‐citrate synthase antibody (clone 4H3‐E5) produced previously .…”

Section: Methodsmentioning

confidence: 99%

Correlation of natural autoantibodies and cardiovascular disease-related anti-bacterial antibodies in pericardial fluid of cardiac surgery patients

Simon

Gilicze

Farkas

et al. 2018

Clinical and Experimental Immunology

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Our previous studies showed that anti-citrate synthase (anti-CS) immunoglobulin (Ig)M natural autoantibodies are present in healthy individuals without previous antigen stimulation, but no studies have investigated their presence in the pericardial fluid (PF). Therefore, we detected the natural anti-CS IgG/M autoantibody levels in plasma and PF of cardiac surgery patients and investigated their relationship with cardiovascular disease-associated bacterial pathogens. PF and blood samples of 22 coronary artery bypass graft (CABG) and 10 aortic valve replacement (AVR) patients were tested for total Ig levels, natural autoantibodies and infection-related antibodies using enzyme-linked immunosorbent assay (ELISA) and Luminex methods. The B cell subsets were measured by flow cytometry. The total Ig subclass levels were four to eight times lower in PF than in plasma, but the natural anti-CS IgM autoantibodies showed a relative increase in PF. The frequency of CD19 B lymphocytes was significantly lower in PF than in blood (P = 0·01), with a significant relative increase of B1 cells (P = 0·005). Mycoplasma pneumoniae antibody-positive patients had significantly higher anti-CS IgM levels. In CABG patients we found a correlation between anti-CS IgG levels and M. pneumoniae, Chlamydia pneumoniae and Borrelia burgdorferi antibody titres. Our results provide the first evidence that natural autoantibodies are present in the PF, and they show a significant correlation with certain anti-bacterial antibody titres in a disease-specific manner.

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Section: Methodsmentioning

confidence: 99%

Correlation of natural autoantibodies and cardiovascular disease-related anti-bacterial antibodies in pericardial fluid of cardiac surgery patients

Simon

Gilicze

Farkas

et al. 2018

Clinical and Experimental Immunology

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“…Two different biochemical measures were utilized to determine the mechanism of increased gall-bladder microsomal prostanoid synthesis after BDL. A kinetic enzyme analysis [14][15] and the technique of protein blot analysis [16][17][18] were used to define the specific arachidonic acid metabolic pathway that leads to increased prostaglandin content in the inflamed gall-bladder [14,15]. BDL stimulated a quantitative change in the enzymes involved with gall-bladder conversion of arachidonic acid into 6oxo-PGF1.…”

Section: Discussionmentioning

confidence: 99%

“…Total microsomal proteins (15,ug) from control and 3DBDL rabbit gall-bladders were separated by 7 %-PAGE in the presence of SDS and transferred on to nitrocellulose filters [16][17][18]. The filters were blocked for 1 h at room temperature in 100 ml of PBS containing 0.05 % Tween 20, 0.5 M-NaCl and 1 % BSA (Buffer A) and incubated overnight at room temperature in the same solution containing a rabbit a-prostacyclin synthase IgG Filters were washed twice for 20 min in Buffer A at room temperature, then once for 20 min in PBS containing 0.5 M-NaCl and 1 % BSA (buffer B).…”

Section: Immunoblot Analysismentioning

confidence: 99%

“…The mechanism underlying exaggerated gall-bladder microsomal synthesis of PGI2 and PGE2 described after rabbit BDL is the primary focus of the present study. The methodologies utilized to investigate this question will include the kinetic analysis described by Eisenthal & Cornish-Bowden and Merino [14,15] and the method of protein blot analysis [16][17][18]. A secondary focus of the present paper is to utilize intact gall-bladder tissue slices to determine if BDL in the rabbit stimulates both increased prostanoid synthesis and release.…”

Section: Introductionmentioning

confidence: 99%

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Increased gall-bladder prostanoid synthesis after bile-duct ligation in the rabbit is secondary to new enzyme formation

Myers

Evans

Bartula

et al. 1992

Biochemical Journal

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Ligation of the common bile duct (BDL) in the male rabbit resulted in increased gall-bladder microsomal total cyclo-oxygenase activity with prostaglandin E2 (PGE2) and 6-oxoprostaglandin F1 alpha [6-oxo-PGF1 alpha, stable metabolite of prostaglandin I2 (PGI2; prostacyclin)] as the major prostanoids synthesized after 24 and 72 h. Kinetic analysis of gallbladder microsomal membrane fractions incubated with increasing levels of [14C]arachidonic acid indicated that BDL for 24 and 72 h did not change substrate affinity (apparent Km) but markedly increased the rate of conversion (apparent Vmax.) suggesting the presence of more total enzyme responsible for synthesis of 6-oxo-PGF1 alpha and PGE2. BDL for 24 and 72 h significantly increased gall-bladder tissue slice basal release of 6-oxo-PGF1 alpha, but not PGE2, when compared with the controls. Gall-bladder slice release of PGE2 was 3-fold less than 6-oxo-PGF1 alpha in the control gall-bladder slices. Immunoblot analysis of 72 h BDL gall-bladder microsomal membrane fractions showed a slight increase in cyclo-oxygenase content and a 5-fold increase in the content of prostacyclin synthase as compared with the control. These data suggest that the BDL-stimulated total gall-bladder cyclo-oxygenase activity was the result of an increase in the level of specific prostaglandin-synthetic enzymes, in particular prostacyclin synthase, and not from a change in enzyme affinity.

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“…Mitochondrial inner-membrane enzymes are among the oldest proteins with conserved structures and possess basic biological functions. Citrate synthase (CS) is a well-conserved molecule with a very similar amino acid (aa) sequence and the same function in both prokaryotic and eukaryotic cells [12,13]. Low-affinity IgM isotype autoantibodies are present in the human circulation against these structures to protect them from a targeting immune response [14].…”

mentioning

confidence: 99%

Detection of citrate synthase-reacting autoantibodies after heart transplantation: an epitope mapping study

Petrohai¹,

Nagy²,

Bősze³

et al. 2005

Transpl Int

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Autoimmune mechanisms play an important role in the pathogenesis of allograft vasculopathy following heart transplantation, but the autoantigens involved have been only sparsely studied. Citrate synthase (CS) enzyme is a conserved molecule, and, as an important mitochondrial autoantigen, it is protected by the "immunological homunculus". Tissue destruction and alteration of the immune regulatory mechanisms can induce pathological immune response against CS in other autoimmune diseases. In our present study we aimed to detect CS-specific autoantibodies in heart transplant patients, therefore, prospective, randomised clinical tests were conducted on 33 heart transplant patients and compared with 130 healthy blood donors. The level and isotype of CS antibodies were detected by simple binding indirect enzyme-linked immunosorbent assay (ELISA). The epitope specificities of the autoantibodies were measured on synthetic overlapping peptide sequences of CS enzyme by an indirect multi-pin ELISA method. Mainly IgM isotype CS autoantibodies were found in healthy controls, while IgG was found at higher levels and frequency (four-times higher) in heart transplant patients. Autoantibodies of IgG isotype recognise different epitopes than do autoantibodies of IgM isotype, even within the same group and individual. New epitope-specific IgG and IgM isotype autoantibodies appeared in heart transplant patients when compared with the controls. Our findings suggest a possible role of CS-specific autoantibodies in the pathomechanism of allograft vasculopathy.

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Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types

Cited by 13 publications

References 30 publications

Correlation of natural autoantibodies and cardiovascular disease-related anti-bacterial antibodies in pericardial fluid of cardiac surgery patients

Correlation of natural autoantibodies and cardiovascular disease-related anti-bacterial antibodies in pericardial fluid of cardiac surgery patients

Increased gall-bladder prostanoid synthesis after bile-duct ligation in the rabbit is secondary to new enzyme formation

Detection of citrate synthase-reacting autoantibodies after heart transplantation: an epitope mapping study

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