Role of N-glycosylation in activation of proMMP-9. A molecular dynamics simulations study

Human matrix metalloproteinase proMMP-9 is secreted as latent zymogen, which requires two-steps proteolytic activation. The secreted proMMP-9 is glycosylated at two positions: Asn38 and Asn120 located in the prodomain and catalytic domain, respectively. It has been demonstrated that glycosylation at Asn120 is required for secretion of the enzyme, while the role of Asn38 glycosylation is not well understood, but is usually linked to the activation process. One hypothesis stated that the Asn38 glycosylation might protect against proteolytic activation. However, the activation process occurs with or without the presence of this glycosylation. We conducted molecular dynamics (MD) simulations on the glycosylated and non-glycosylated proMMP-9 to elucidate the effect of Asn38 glycosylation on this two-step activation process. The simulation results suggest that Asn38 glycosylation does not hinder the activation process directly, but induces conformational changes in the vicinity of the first proteolytic region in such a way that E59-M60 cleavage is processed before R106-F107. These results correlate with analysis provided by Boon et al. and experimental data from Ogata et al. who attempted to determine the order of events in activation of proMMP-9. Results from additional MD simulations for the model of glycosylated proMMP-9 bound to galectin-8 N-domain suggest that Gal-8 by interacting with Asn38 glycan might further facilitate processing of the first cleavage between E59-M60. Thus, our simulation results suggest that both Asn38 glycosylation and interaction with Gal-8N may be involved in facilitating and the temporal order of the activation process of pro-MMP9. The aim of this report is to provide an inspiration for future detailed experiments aimed at explaining the role of N-glycosylation in the activation process of prodomain of MMP-9.

人基质金属蛋白酶proMMP-9（human matrix metalloproteinase proMMP-9）以潜伏酶原的形式被分泌，其激活需经历两步蛋白水解过程。分泌产生的proMMP-9存在两个糖基化位点：分别位于前结构域（prodomain）与催化结构域（catalytic domain）的Asn38和Asn120。已有研究证实，Asn120位点的糖基化是该酶分泌过程的必需条件，而Asn38糖基化的功能尚未完全阐明，但通常认为其与酶原激活过程存在关联。曾有假说提出，Asn38糖基化可能对蛋白水解激活起到保护作用，但实际情况是，无论该糖基化是否存在，激活过程均可正常发生。我们针对糖基化与非糖基化形式的proMMP-9开展了分子动力学（molecular dynamics, MD）模拟，以期阐明Asn38糖基化对其两步激活过程的影响。模拟结果显示，Asn38糖基化并未直接阻碍激活进程，而是通过改变首个蛋白水解区域附近的构象，使得E59-M60位点的切割先于R106-F107位点发生。该结果与Boon等人的分析及Ogata等人的实验数据相吻合，后者曾尝试解析proMMP-9激活过程中的事件时序。针对结合半乳糖凝集素-8 N结构域（galectin-8 N-domain）的糖基化proMMP-9模型开展的补充分子动力学模拟结果表明，Gal-8可通过与Asn38糖链相互作用，进一步促进E59-M60位点的首次切割。综上，本研究的模拟结果显示，Asn38糖基化以及与Gal-8N的相互作用，均可能参与调控proMMP-9激活过程的时序并促进其发生。本研究旨在为未来开展深入实验提供思路，以阐明N-糖基化在MMP-9前结构域激活过程中的具体作用。