Metabolite proofreading in carnosine and homocarnosine synthesis: molecular identification of PM20D2 as β-alanyl-lysine dipeptidase

Abstract

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈ 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.

Keywords: Brain Metabolism; CARNS1; CNDP1; Carnosine; Homocarnosine; M20 Metallopeptidase; Metabolism; Peptidase; Peptides; Skeletal Muscle Metabolism.

FIGURE 1.

Properties of β-Ala-Lys dipeptidase activity in rat tissue extracts. A, distribution of the activity in various rat tissues. B, effect of metal chelators and carnosine on the β-Ala-Lys dipeptidase activity in skeletal muscle extract. Values are the means ± S.E. (n = 3).

FIGURE 2.

Purification of rat muscle β-Ala-Lys dipeptidase on DEAE-Sepharose and Superdex 200 and co-elution of the activity with a protein recognized by anti-PM20D2 antibodies. A, elution of the β-Ala-Lys dipeptidase activity from a DEAE-Sepharose column (see Table 1) with a 0–0.6 m NaCl gradient. B, active fractions from a Q-Sepharose column were further gel-filtered on Superdex-200; the activity peak of the native enzyme was asymmetric, but the peak fraction (F49) co-eluted with marker proteins of ≈220 kDa. C, active fractions F47-F49 from B were gel-filtered in the same Superdex-200 column in the presence of 0.3 KSCN. D, Western blot analysis of the peak fractions from C showing β-Ala-Lys dipeptidase activity co-eluting with a 48-kDa protein that corresponds to PM20D2. nb, number.

FIGURE 3.

Hydrolysis of various β-alanine-containing dipeptides by recombinant mouse and human PM20D2 (β-Ala-Lys dipeptidase) or CNDP1 (carnosinase). Shown are graphic representations of calculated V_max values (see Table 3) for mouse and human PM20D2 (A) and CNDP1 (B) for the hydrolysis of the indicated dipeptides.

FIGURE 4.

Synthesis of carnosine, homocarnosine, β-Ala-Lys, and β-Ala-Orn in HEK293T cells transfected with carnosine synthase; effect of the addition of various amino acids in the medium and of cotransfection with mouse or human PM20D2 or human carnosinase. HEK293T cells were transfected for 48h with plasmids expressing various mouse (m) or human (h) proteins. Panel A illustrates the effect of adding β-alanine, lysine, ornithine, and GABA to the cell culture medium on the accumulation of different dipeptides in the cells. Panels B and C illustrate the effect of cotransfection of human carnosine synthase (hCARNS1) with mouse or human PM20D2 and human carnosinase (hCNDP1) in the indicated combinations. The medium was supplemented with β-alanine (panel B) or GABA (panel C) and lysine to favor the formation of carnosine and homocarnosine, respectively. Results are the means ± S.E. for three independent transfections.