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

Abstract

Background and AimsThe 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 ResultsEpitope 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.ConclusionsOur 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.

Carnosinase concentration, activity, and CNDP1 genotype in patients with type 2 diabetes with and without nephropathy

“…This observation was not only limited to the homozygous CNDP1 (CTG) 5 genotype. Although the RYSK173 proportion of CNDP1, a CNDP1 conformation with allegedly low enzyme activity (Zhang et al 2016), was decreased in the patients with DN it only accounts for less than 2% of the total CNDP1 concentration and therefore not significantly contributing to the overall CNDP1 enzyme activity.…”

“…It is influenced by competing substrates such as homocarnosine (Peters et al 2010) and it seems that the conformation of CNDP1 also affects CNDP1 activity (Adelmann et al 2012). With respect to the latter we have shown that our in-house-made anti-CNDP1 monoclonal antibody RYSK173 can distinguish between different CNDP1 conformations, presumably due to the presence or absence of divalent metal ions that associate with the dinuclear zinc-binding site of CNDP1 at His132 (Zhang et al 2016). While assessment of CNDP1 with the RYSK173 monoclonal antibody reveals an inverse relation with enzyme activity, with the commercially available polyclonal anti-CNDP1 antibody ATLAS a good fit with enzyme activity is observed.…”

“…Hence, renal clearance of CNDP1 alone cannot explain low serum CNDP1 concentrations in these patients. A recent study also demonstrated that CNDP1 concentrations are increased directly after hemodialysis, making a relevant removal of CNDP1 via filtration during dialysis unlikely (Zhang et al 2016). Uremia-induced alterations in protein metabolism and gastrointestinal tract function can result in poor nutritional status.…”