Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1)
Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1)
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DOI：
10.1007/s10719-012-9408-z
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发表时间：
2012-06
影响因子：
3
通讯作者：
Yifan Qian;Xingwang Zhang;Lei Zhou;Xiao-jing Yun;Jianhui Xie;Jiejie Xu;Yuanyuan Ruan;S. Ren
中科院分区：
文献类型：
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作者：
Yifan Qian;Xingwang Zhang;Lei Zhou;Xiao-jing Yun;Jianhui Xie;Jiejie Xu;Yuanyuan Ruan;S. Ren
人类LOX-1/OLR 1在动脉粥样硬化和内皮功能障碍中起关键作用。LOX-1的n -糖基化已被证明在体内影响其生物学功能并调节动脉粥样硬化的发病机制。然而，LOX-1的n -糖基化模式尚未被描述。本研究旨在阐明重组人LOX-1的n -糖基化特征和n -糖基化位点。本研究采用非特异性蛋白酶（Pronase E）酶切、MALDI-QIT-TOF质谱和多阶段质谱（MS3）分析的方法来获得重组人LOX-1的位点特异性n -糖基化信息。结果表明，通过对非特异性蛋白酶消化产生的糖肽进行直接质谱分析，可以同时确定n-聚糖的结构及其对应的LOX-1的附着位点。利用这种方法，很容易地鉴定出重组人LOX-1在asn139上的一个潜在糖基化位点，并发现该位点携带异质络合物型n -聚糖。此外，利用诊断离子对多阶段质谱数据的手动注释被发现在定义糖肽和聚糖的结构方面特别有用，以便进行适当的光谱解释。本文的研究结果将为进一步研究LOX-1 n -聚糖的结构-功能关系提供新的思路。
Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functionsin vivoand modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinant human LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS3) analysis is explored to obtain site-specific N-glycosylation information of recombinant human LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinant human LOX-1 on Asn139is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1 N-glycan.