DataSheet1_Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.docx

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Amblyomin-X是一种Kunitz型凝血因子Xa（FXa）抑制剂，其通过对饰纹钝眼蜱（Amblyomma sculptum）唾液腺的转录组分析（transcriptome analysis）得以鉴定。该蛋白包含两个大小相当的结构域，可在多种肿瘤细胞系中诱导细胞凋亡（apoptosis），并能促进肿瘤生长消退、降低转移发生率。为研究Amblyomin-X N端（N-ter）与C端（C-ter）结构域的结构特性与功能作用，我们采用固相肽合成（solid-phase peptide synthesis）法对二者进行了合成，解析了N端结构域的X射线晶体结构（X-Ray crystallographic structure），证实其具有Kunitz型结构特征，并对其生物学特性开展了研究。本研究证实，C端结构域是Amblyomin-X被肿瘤细胞摄取的关键功能区域；同时我们揭示了该结构域介导胞内递送的能力：将其与细胞摄取效率（cellular-uptake efficiency）低下的分子（如p15）偶联后，可显著增强这些分子的胞内检出信号。与之相反，Amblyomin-X的N端Kunitz结构域无法穿透细胞膜，但当将其显微注射（microinjected）入细胞内或与TAT细胞穿透肽（TAT cell-penetrating peptide）融合时，可引发肿瘤细胞毒性。此外，我们鉴定出一段最小长度的C端结构域F2C，其可进入SK-MEL-28细胞，并能诱导动力蛋白链（dynein chains）的基因表达调控——动力蛋白链是一种分子马达（molecular motor），在Amblyomin-X的摄取与胞内转运（intracellular trafficking）过程中发挥重要作用。