Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2^−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

组织转谷氨酰胺酶 (TG2) 是转谷氨酰胺酶家族的一种多功能蛋白，在与衰老相关的血管硬化和功能障碍中具有推定的与转酰胺作用无关的功能。开发临床前模型对于充分了解 TG2 的转酰胺基独立活性的生理相关性以及确定 TG2 在治疗靶向方面的特定功能至关重要。因此，在本研究中，我们利用 CRISPR-Cas9 基因编辑技术在小鼠Tgm2基因活性位点的半胱氨酸 277 处引入突变。杂合子和纯合子Tgm2 -C277S 小鼠表型正常，以预期的孟德尔频率出生。TG2 蛋白在Tgm2 中普遍表达-C277S 小鼠的水平与野生型 (WT) 小鼠相似。在Tgm2 -C277S 小鼠中，TG2 转谷氨酰胺酶功能被成功消除，但归因于 GTP、纤连蛋白和整联蛋白结合的转酰胺独立功能得以保留。在体外，重塑刺激导致 WT 小鼠血管顺应性的显着丧失，但在Tgm2 -C277S 或 TG2 ^-/-小鼠中没有。根据脉搏波速度和拉伸试验测量，WT 小鼠的血管硬度随着年龄的增长而增加。TGm2-C277S 小鼠免受与年龄相关的血管硬化的影响，并且 TG2 敲除产生了进一步的保护。总之，这些研究表明，TG2 对整体血管模量和血管反应性有显着贡献，与其转酰胺功能无关，但转酰胺活性是老化过程中血管基质硬化的重要原因。最后，Tgm2 -C277S 小鼠可用于体内研究，以探索 TG2 在生理学和病理生理学中的转酰胺独立作用。