Chemistry Department, University of Massachusetts-Amherst, 240 Thatcher Way, Amherst, Massachusetts 01003, United States.
Biochemistry and Molecular Biology Department, University of Massachusetts-Amherst, 240 Thatcher Way, Amherst, Massachusetts 01003, United States.
Anal Chem. 2020 Jun 2;92(11):7565-7573. doi: 10.1021/acs.analchem.0c00115. Epub 2020 May 13.
Understanding molecular mechanisms governing interactions of glycosaminoglycans (such as heparin) with proteins remains challenging due to their enormous structural heterogeneity. Commonly accepted approaches seek to reduce the structural complexity by searching for "binding epitopes" within the limited subsets of short heparin oligomers produced either enzymatically or synthetically. A top-down approach presented in this work seeks to preserve the chemical diversity displayed by heparin by allowing the longer and structurally diverse chains to interact with the client protein. Enzymatic lysis of the protein-bound heparin chains followed by the product analysis using size exclusion chromatography with online mass spectrometry detection (SEC/MS) reveals the oligomers that are protected from lysis due to their tight association with the protein, and enables their characterization (both the oligomer length, and the number of incorporated sulfate and acetyl groups). When applied to a paradigmatic heparin/antithrombin system, the new method generates a series of oligomers with surprisingly distinct sulfation levels. The extent of sulfation of the minimal-length binder (hexamer) is relatively modest yet persistent, consistent with the notion of six sulfate groups being both essential and sufficient for antithrombin binding. However, the masses of longer surviving chains indicate complete sulfation of disaccharides beyond the hexasaccharide core. Molecular dynamics simulations confirm the existence of favorable electrostatic interactions between the high charge-density saccharide residues flanking the "canonical" antithrombin-binding hexasaccharide and the positive patch on the surface of the overall negatively charged protein. Furthermore, electrostatics may rescue the heparin/protein interaction in the absence of the canonical binding element.
由于糖胺聚糖（如肝素）与蛋白质相互作用的分子机制极其复杂，因此理解这一机制具有一定挑战性。通常采用的方法是通过寻找酶解或化学合成产生的短肝素寡聚物的有限子集内的“结合表位”，来降低结构的复杂性。本文提出的自上而下的方法旨在通过允许较长和结构多样的链与客户蛋白质相互作用，来保留肝素所展示的化学多样性。用酶裂解与蛋白质结合的肝素链，然后使用带有在线质谱检测的排阻色谱法（SEC/MS）对产物进行分析，揭示了由于与蛋白质紧密结合而免受裂解的寡聚物，并能够对其进行表征（包括寡聚物的长度以及掺入的硫酸根和乙酰基的数量）。当将该新方法应用于肝素/抗凝血酶系统这一范例时，会生成一系列具有惊人不同硫酸化水平的寡聚物。最小长度结合物（六聚体）的硫酸化程度相对适中但很稳定，这与六个硫酸根基团对于抗凝血酶结合既必要又充分的观点一致。然而，较长链的存在表明，在六糖核心之外的二糖上存在完全硫酸化。分子动力学模拟证实了在缺乏“典型”抗凝血酶结合六糖的情况下，高电荷密度糖残基与整体带负电荷蛋白质表面上的正斑块之间存在有利的静电相互作用。此外，在没有典型结合元件的情况下，静电作用可能会挽救肝素/蛋白质相互作用。
