Veiga-da-Cunha Maria, Chevalier Nathalie, Stroobant Vincent, Vertommen Didier, Van Schaftingen Emile
From the Welbio and the Laboratory of Physiological Chemistry,
From the Welbio and the Laboratory of Physiological Chemistry.
J Biol Chem. 2014 Jul 11;289(28):19726-36. doi: 10.1074/jbc.M114.576579. Epub 2014 Jun 2.
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.
肌肽合成酶是一种依赖ATP的连接酶，分别负责在骨骼肌和大脑中合成肌肽（β-丙氨酰组氨酸）和高肌肽（γ-氨基丁酰组氨酸）。该酶也能以相当高的速率使用赖氨酸、鸟氨酸和精氨酸来替代组氨酸，然而在肌肉或大脑中几乎检测不到由此产生的二肽，这表明它们被一种“代谢物修复”酶清除了。使用放射性标记的底物，我们发现大鼠的骨骼肌、心脏和大脑中含有一种胞质β-丙氨酰赖氨酸二肽酶活性。这种具有金属酶特性的酶从大鼠骨骼肌中纯化了约200倍。对不同纯化阶段获得的组分进行质谱分析表明，PM20D2（一种功能未知的属于金属肽酶20家族的肽酶）平行富集。蛋白质免疫印迹显示PM20D2与β-丙氨酰赖氨酸二肽酶活性共洗脱。重组小鼠PM20D2水解β-丙氨酰赖氨酸、β-丙氨酰鸟氨酸、γ-氨基丁酰赖氨酸和γ-氨基丁酰鸟氨酸的效率最高。它对β-丙氨酰精氨酸和γ-氨基丁酰精氨酸的作用速率较低，但对肌肽或高肌肽几乎没有作用。尽管PM20D2优先作用于源自β-丙氨酸或γ-氨基丁酸的碱性二肽，但它对一些“经典二肽”如α-丙氨酰赖氨酸和α-赖氨酰赖氨酸的作用速率也较低。人PM20D2也表现出相同的活性特征，但该酶对所有测试底物的活性比小鼠酶低约100 -倍。在HEK293T细胞中，小鼠或人PM20D2与肌肽合成酶共转染可防止异常二肽（β-丙氨酰赖氨酸、β-丙氨酰鸟氨酸、γ-氨基丁酰赖氨酸）的积累，从而有利于肌肽和高肌肽的合成，并证实了PM20D2的代谢物修复作用。
