N-GLYCOSYLATION SITES OF INFLUENZA A VIRUS HEMAGGLUTININ AND SURFACTANT PROTEIN

甲型流感病毒血凝素和表面活性蛋白的 N-糖基化位点

• 批准号: 8365550
• 负责人: JOSEPH ZAIA
• 金额: $ 5.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365550
• 关键词: Accounting; Binding Proteins; Biology; Buffers; Carbohydrates; Cessation of life; Complex; Consensus; Data; Deuterium Oxide; Digestion; Enzymes; Family suidae; Funding; Glycopeptides; Grant; Health Care Costs; Hemagglutinin; Hospitalization; Hybrids; Infection; Influenza; Influenza A virus; Ions; Link; Literature; Location; Mannose; Manuals; Mass Spectrum Analysis; Medicine; Methods; N-Glycosylation Site; National Center for Research Resources; Nature; Parents; Peptide N-glycohydrolase F; Peptides; Phase; Philippines; Polysaccharides; Principal Investigator; Proteins; Public Health; Pulmonary Surfactant-Associated Protein D; Pulmonary Surfactant-Associated Proteins; Relative (related person); Reporting; Research; Research Infrastructure; Resolution; Resources; Sampling; Sialic Acids; Site; Source; Trypsin; United States National Institutes of Health; Viral Proteins; Water; carbohydrate structure; cost; glycosylation; interest; mass spectrometer; meetings; programs; protein aminoacid sequence; reversed phase chromatography; sulfation; surfactant

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Introduction The influenza A virus is a significant concern of public health. It accounts for 36,000 deaths, 200,000 hospitalizations per year resulting in $10 billion in health care costs in the US. Although remarkable advances have been achieved in medicine in the past century, many questions remain regarding infection with influenza. One aspect of influenza infection is the inhibition of the activity of viral protein hemagglutinin (HA) by binding proteins such as lung surfactant proteins (SP-D or A). Although a strong consensus in the literature concerning the interaction of HA and SP involves their N-glycosylation sites, limited structural information concerning the glycans have been reported. We explored a comprehensive mass spectrometry strategy to study HA and SP-D in terms of identification of N-glycosylation sites and the carbohydrate structures in order to meet the need for a detailed understanding of influenza pathobiology. Methods HA from H3N2 strain Philippines 1982 and the truncated pig SP-D protein containing the N-glycosylation site of interest were studied. Trypsin digestion was conducted at 37¿C overnight with 1:50 enzyme-protein (w/w) ratio in 50mM Hepes buffer (pH 8) and desalted with micro-reversed phase chromatography. A PNGase-F digestion was then conducted overnight in water or heavy water to allow for relative quantification of N-linked sites. All samples were analyzed by LC-normal phase-MS/MS in positive ion mode on an LTQ-Orbitrap XL. In order to determine the maximum structural information on the carbohydrates and parent peptides, the glycosyled peptides were identified using the Glycomod program with confirmation using manual interpretation of tandem MS data. Premilary results From the comparison of the LC-MS data obtained from the enzymatic deglycosylation in O18 water, the number, the specific location and the relative site occupancy of glycosylation were clearly identified. In total, 5 of the 13 theoretical N-glycosylation sites were confirmed in HA, and a single site was observed for pig SP-D truncated protein. Site occupancy of 100% was observed for almost all of the sites. The high resolution (60,000) and the mass accuracy (less than one 2ppm) provided by the high resolution Orbitrap mass spectrometer was essential to remove ambiguities concerning the identification of the site and the sequence of the peptides involved. Depending on the glycosylated site, different carbohydrate structures were observed, including high mannose and complex N-glycans on HA. At this point of our interpretation, sulfation and the presence of sialic acid have not been identified for H3N2 HA. Hybrid complex carbohydrates were clearly demonstrated to be present on the site of glycosylation of the pig SP-D protein. The complementarily nature of different modes of fragmentation (ETD, CID, HCD) have been exploited to collect enhanced structural information of the glycopeptides given the complexity of the data. An example for illustrating our results is the glycosylation of site N165 of HA. The MS and MS/MS data indicated the presence of N-glycans at this site as predominantly composed of high mannose carbohydrates: Hex2to 7 -Man3GlcNAc2. Harshtorm et al (2008) and Hartley et al (1997) support the implication of high mannoses carnohydrates of N165 in the inhibition of the HA activity.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 引言 甲型流感病毒是一个重大的公共卫生问题。在美国，每年有36,000人死亡，200,000人住院，导致100亿美元的医疗成本。尽管在过去的一个世纪里，医学取得了显著的进步，但有关流感感染的许多问题仍然存在。 流感感染的一个方面是病毒蛋白血凝素(HA)的活性受到肺表面活性蛋白(SP-D或A)等结合蛋白的抑制。尽管文献中关于HA和SP的相互作用涉及到它们的N-糖基化位点，但关于这些多糖的结构信息有限。我们探索了一种全面的质谱学方法来研究HA和SP-D，从鉴定N-糖基化位点和糖类结构的角度来满足对流感病理生物学详细了解的需要。 方法 对1982年H3N2毒株菲律宾株的血凝素和含有感兴趣的N-糖基化位点的猪SP-D截短蛋白进行了研究。胰酶在37℃、酶蛋白比为1：50的50 mM HEPES缓冲液(pH=8)中消化过夜，用微量反相色谱脱盐。然后在水或重水中进行PNGase-F消化过夜，以允许N-连接位点的相对定量。所有样品在LTQ-Orbitrap XL上以正离子模式进行LC-正相-MS/MS分析。为了确定碳水化合物和母体多肽的最大结构信息，使用Glycomod程序鉴定糖基化多肽，并使用串联MS数据的手动解释进行确认。 早期结果 通过对O18水中酶促脱糖的LC-MS数据的比较，明确了糖基化的数目、特定位置和相对占有率。总体而言，13个理论N-糖基化位点中有5个在HA中得到证实，而猪SP-D截短蛋白只有一个位点。几乎所有网站的网站占有率都达到了100%。高分辨率Orbitrap质谱仪提供的高分辨率(60,000)和质量精确度(不到2ppm)对于消除有关识别所涉及的多肽的位置和序列的歧义至关重要。 根据糖基化部位的不同，观察到不同的碳水化合物结构，包括高甘露糖和HA上的复杂N-糖链。在我们的解释中，H3N2HA的硫酸盐化和唾液酸的存在还没有被确定。杂交复合碳水化合物明显存在于猪SP-D蛋白的糖基化位点。考虑到数据的复杂性，不同的碎片模式(ETD、CID、HCD)的互补性被用来收集糖肽的增强结构信息。 一个解释我们结果的例子是HA的N165位的糖基化。MS和MS/MS数据表明，该部位存在N-葡聚糖，主要由高甘露糖碳水化合物组成：Hex2到7-Man3GlcNAc2。Harshtorm等人(2008)和Hartley等人(1997)支持N165的高甘露糖水合物对HA活性的抑制作用。