Laboratory of Immunohematology and Glycobiology, Josep Carreras Leukaemia Research Institute, Ctra. de Can Ruti, Cami de Les Escoles S/N, 08916, Badalona, Spain.
Sci Rep. 2023 Aug 26;13(1):13996. doi: 10.1038/s41598-023-40900-4.
Some stem region mutants of human blood group A transferase (hAT) possess Forssman synthase (FS) activity, but very little is known about the mechanisms responsible for this enzymatic crosstalk. We performed confocal microscopy and image analysis to determine whether different intra-Golgi localization was accountable for this acquired activity. We also performed structural modeling and mutational and normal mode analyses. We introduced new mutations in the stem region and tested its FS and AT activities. No differences in subcellular localization were found between hAT and FS-positive mutants. AlphaFold models of hAT and mFS (mouse Forssman synthase) showed that the hAT stem region has a tether-like stem region, while in mFS, it encircles its catalytic domain. In silico analysis of FS-positive mutants indicated that stem region mutations induced structural changes, decreasing interatomic interactions and mobility of hAT that correlated with FS activity. Several additional mutations introduced in that region also bestowed FS activity without altering the AT activity: hAT 37-55 aa substitution by mFS 34-52, 37-55 aa deletion, and missense mutations: S46P, Q278Y, and Q286M. Stem region structure, mobility, and interactions are crucial for hAT specificity. Moreover, stem region mutations can lead to heterologous Forssman activity without changes in the catalytic machinery.
一些人血型 A 转移酶（hAT）的茎区突变体能表现出福斯曼合成酶（FS）活性，但对于导致这种酶交叉对话的机制知之甚少。我们通过共聚焦显微镜和图像分析来确定是否不同的高尔基体内定位是造成这种获得性活性的原因。我们还进行了结构建模以及突变和正常模式分析。我们在茎区引入新的突变，并测试其 FS 和 AT 活性。hAT 和 FS 阳性突变体之间的亚细胞定位没有差异。hAT 和 mFS（鼠福斯曼合成酶）的 AlphaFold 模型表明，hAT 的茎区具有类似系绳的茎区，而在 mFS 中，它环绕其催化结构域。FS 阳性突变体的计算机分析表明，茎区突变诱导结构变化，降低 hAT 的原子间相互作用和流动性，与 FS 活性相关。该区域中引入的其他几个突变也赋予了 FS 活性，而不改变 AT 活性：hAT 的 37-55aa 被 mFS 的 34-52,37-55aa 取代、缺失和错义突变：S46P、Q278Y 和 Q286M。茎区结构、流动性和相互作用对 hAT 的特异性至关重要。此外，茎区突变可导致异源福斯曼活性，而催化机制不变。
