Cytidine 5'-triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography-mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters Vmax and Km of CTPS and verified that an inhibition of the enzyme activity was induced after 3-deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis-Menten kinetics for lysates of resting cells were Km =280±310 μmol/L for UTP, Vmax =83±20 pmol/min and, for lysates of activated PBMCs, Km =230±280 μmol/L for UTP, Vmax =379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.

中文翻译：

---

液相色谱串联质谱法在多反应监测模式下测定CTP合成酶活性。

已知胞苷5'-三磷酸合成酶（CTPS）是CTP从头合成的核心酶。我们最近证明，CTPS1的缺乏与活化淋巴细胞增殖能力受损有关，从而导致合并的免疫缺陷疾病。为了更好地证明其在免疫调节中的作用，我们开发了一种测量人淋巴细胞中CTPS活性的方法。使用液相色谱-质谱联用，我们通过测量细胞裂解液中的CTP来定量CTPS活性。CTP的稳定同位素类似物用作内标。我们表征了CTPS的动力学参数Vmax和Km，并验证了在已知的CTPS抑制剂3-deazauridine（3DAU）处理后诱导了酶活性的抑制。然后，我们确定了健康志愿者中CTPS的活动，一个孩子的CTPS1基因显示纯合突变的家庭，以及在肺移植后出现或未患慢性肺同种异体功能障碍（CLAD）的患者中。观察到CTP测定的线性度高达451μmol/ L，准确度在15％的公差范围内。静止细胞裂解物的Michaelis-Menten动力学对于UTP为Km = 280±310μmol/ L，Vmax = 83±20 pmol / min，对于活化的PBMC裂解物，对于UTP Km = 230±280μmol/ L，Vmax = 379±90 pmol /分钟。通过3DAU和CTPS1基因纯合突变的治疗，取消了与细胞刺激相关的CTPS活性的诱导，并且在发生CLAD的患者中CTPS活性显着降低。我们得出的结论是，该测试适合揭示CTPS改变与免疫缺陷有关。