Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173

PLoS One. 2016 Jan 22;11(1):e0146831. doi: 10.1371/journal.pone.0146831. eCollection 2016.

Abstract

Background and aims: The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis.

Methods and results: Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn2+ and Cu2+ concentrations after dialysis.

Conclusions: Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / pharmacology
  • Binding Sites / immunology
  • COS Cells
  • Catalytic Domain / physiology
  • Cell Line
  • Chlorocebus aethiops
  • Copper / blood
  • Copper / metabolism*
  • Dipeptidases / blood
  • Dipeptidases / genetics
  • Dipeptidases / immunology*
  • Epitope Mapping
  • Epitopes / immunology*
  • Female
  • Genetic Variation / genetics
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Kidney Failure, Chronic / pathology
  • Male
  • Middle Aged
  • Renal Dialysis
  • Zinc / blood
  • Zinc / immunology
  • Zinc / metabolism*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • monoclonal antibody RYSK173
  • Copper
  • CNDP1 protein, human
  • Dipeptidases
  • Zinc

Grants and funding

This study was supported by Deutsche Forschungsgemeinschaft (DFG, URL: http://www.dfg.de/) SA 2143/1-1 to S.H. This author read and revised this manuscript. It was also supported by Graduiertenkolleg (GRK, URL: http://www.umm.uni-heidelberg.de/ag/grk1874/) 1874/1 to B.Y. This author designed, prepared, and decided to publish this manuscript.