Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation.
Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation.
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DOI：
10.1111/jth.14785
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发表时间：
2020-06
期刊：
影响因子：
--
通讯作者：
O'Sullivan JM
中科院分区：
文献类型：
--
作者：
Fazavana J;Brophy TM;Chion A;Cooke N;Terraube V;Cohen J;Parng C;Pittman D;Cunningham O;Lambert M;O'Donnell JS;O'Sullivan JM
先前的研究表明，VWF的A1A2A3结构域在体内调节巨噬细胞介导的清除中起关键作用。特别是，a1结构域已被证明可调节与巨噬细胞LRP1清除受体的相互作用。此外，a2结构域内的n -链聚糖已被证明可保护VWF免受lrp1介导的过早清除。然而，重要的是，A1A2A3中使巨噬细胞结合的特定区域尚未确定。为了解决这个问题，我们利用了位点定向聚乙二醇化，并在A1A2A3-VWF中引入了新的靶向n -链糖基化，随后检测了VWF的清除。与40 kda PEG片段结合后，以位点特异性方式显著延长了A1A2A3-VWF在VWF−/−小鼠中的半衰期。例如，与野生型A1A2A3-VWF相比，a1结构域（S1286）和a3结构域（V1803, S1807）内特定位点的PEGylation在体内降低了VWF的清除率。此外，在这些特定位点的聚乙二醇化可以在体外消除与分化的THP-1巨噬细胞和LRP1簇II和簇IV的结合。相反，其他位置（q1353 - a1结构域和m1545 - a2结构域）的PEGylation对VWF清除或与LRP1结合的影响有限。在a3结构域的N1803和N1807处引入了新的n链聚糖链。与这些位点的聚乙二醇化相反，这些n -聚糖变体没有观察到半衰期的显著延长。这些新数据支持了A1A2A3结构域调节巨噬细胞介导的VWF清除的假设，并强调了A1-和a3结构域内的特定区域可能是促进与LRP1相互作用的关键。这些结果也为巨噬细胞lrp1介导的VWF清除如何通过位点定向聚乙二醇化而减弱提供了见解。
Previous studies have demonstrated that the A1A2A3 domains of VWF play a key role in regulating macrophage-mediated clearance in-vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage LRP1 clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. To address this, we utilised site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance. Conjugation with a 40-kDa PEG moiety significantly extended the half-life of A1A2A3-VWF in VWF−/− mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in-vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants. These novel data support the hypothesis that the A1A2A3 domains regulate macrophage-mediated clearance of VWF and highlight that specific regions within the A1- and A3-domains may be key in facilitating interaction with LRP1. These results also provide insights into how macrophage-LRP1 mediated VWF clearance may be attenuated using site-directed PEGylation.
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影响因子：
20.3
作者：
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通讯作者：
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10.4
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