Bhakta Varsha, Tehrani Negin Chaeichi, Ask Antje, Sheffield William P
Canadian Blood Services, Centre for Innovation, Hamilton, Ontario, Canada.
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
Biochem Biophys Rep. 2025 Jun 15;43:102092. doi: 10.1016/j.bbrep.2025.102092. eCollection 2025 Sep.
Alpha-1 antitrypsin (AAT) is the most abundant member of the serpin superfamily of protease inhibitors found in human plasma. Expression of a broad-spectrum AAT variant (AAT M358R) and an AAT variant (AAT-RC-2) that specifically inhibits coagulation Factor XIa (FXIa) in the methylotrophic yeast were compared. When protein secretion was directed by the 85 amino acid alpha mating factor (AMF) prepro signal sequence 1 (ss1), AAT-RC-2 was purified as a 45 kDa homogeneous polypeptide preparation, but AAT M358R was heterogeneous. Replacement of the ss1 prepro sequence with any of 5 presequences lacking pro sequences eliminated the heterogeneity. The highest yield was obtained with ss3-AAT M358R, where ss3 was the 19 amino acid AMF presequence. Enzymatic deglycosylation of ss1-AAT M358R converted its high molecular weight heterogeneity into a simple combination of 55 kDa and 45 kDa polypeptides consistent with failure to remove the AMF presequence and inhibition of the Kex2 propeptide convertase by AAT M358R. Purified ss3-AAT M358R inhibited FXIa significantly more rapidly than -derived AAT M358R with indistinguishable reaction stoichiometry; purified ss1-AAT-RC-2 did not differ from its -derived counterpart in either kinetic parameter. Both AAT variants formed denaturation-resistant complexes with FXIa. Substitution of the AMF prepro sequence with its constituent presequence eliminated the protein expression problem caused by inhibition of the propeptide processing machinery by AAT M358R and will make feasible comparison of AAT M358R and AAT-RC-2 in animal models of thrombosis and bleeding.
α-1抗胰蛋白酶（AAT）是人类血浆中发现的丝氨酸蛋白酶抑制剂（serpin）超家族中最丰富的成员。比较了广谱AAT变体（AAT M358R）和在甲基营养酵母中特异性抑制凝血因子XIa（FXIa）的AAT变体（AAT-RC-2）的表达。当由85个氨基酸的α交配因子（AMF）前原信号序列1（ss1）指导蛋白质分泌时，AAT-RC-2被纯化为45 kDa的均一多肽制剂，但AAT M358R是异质的。用5个缺乏前导序列的前序列中的任何一个替换ss1前原序列消除了异质性。ss3-AAT M358R的产量最高，其中ss3是19个氨基酸的AMF前导序列。ss1-AAT M358R的酶促去糖基化将其高分子量异质性转化为55 kDa和45 kDa多肽的简单组合，这与未能去除AMF前导序列以及AAT M358R对Kex2前肽转化酶的抑制作用一致。纯化的ss3-AAT M358R比衍生的AAT M358R显著更快地抑制FXIa，反应化学计量学难以区分；纯化的ss1-AAT-RC-2在动力学参数上与其衍生对应物没有差异。两种AAT变体都与FXIa形成抗变性复合物。用其组成前导序列替换AMF前原序列消除了由AAT M358R抑制前肽加工机制引起的蛋白质表达问题，并将使在血栓形成和出血动物模型中比较AAT M358R和AAT-RC-2成为可能。
