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首页> 外文学位 >Structural and functional characterization of N-glycans from recombinant human lactoferrin expressed in Aspergillus awamori.
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Structural and functional characterization of N-glycans from recombinant human lactoferrin expressed in Aspergillus awamori.

机译：泡盛曲霉中表达的重组人乳铁蛋白的N-聚糖的结构和功能表征。

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摘要

Human lactoferrin (h-LF) is widely reported to be involved in numerous biological functions like iron transport, anti-microbial defense, anti-tumor mechanisms and immune system regulation. Currently there is an extensive interest in recombinant sources of this glycoprotein for pharmaceutical and neutraceutical purposes. Recombinant human lactoferrin from Aspergillus awamori (Rh-LF) is a major scalable source presently undergoing several clinical trials. In this study, we comprehensively characterized the structure and also studied the functions of N-glycans in Rh-LF.;Lys-C peptide map LC-MS analysis of Rh-LF localized N-linked oligosaccharides to Asn138, Asn479 and Asn624. The third site at Asn624 was partially occupied with high-mannose oligosaccharides unlike native human lactoferrin, wherein Asn624 is typically vacant. N -glycan profiling using NP-HPLC coupled with ESI-TOF mass spectrometry analysis revealed that high mannose glycans of Rh-LF were composed of 6 - 14 mannose moieties. SPR assays showed that presence or absence of glycans had no effect on binding affinity towards Intelectin , a major in vivo lactoferrin receptor and the binding affinity of deglycosyalted Rh-LF was observed to be close to that of Rh-LF and native human Lactoferrin. SEC analysis revealed deglycosylation did not resulted in increased aggregation of Rh-LF, signifying lack of role of glycans in Rh-LF stability post secretion.
机译：人乳铁蛋白（h-LF）被广泛报道涉及许多生物学功能，如铁转运，抗微生物防御，抗肿瘤机制和免疫系统调节。目前，对于用于药物和中性营养目的的这种糖蛋白的重组来源引起了广泛的关注。来自泡盛曲霉（Rh-LF）的重组人乳铁蛋白是目前正在进行数项临床试验的主要可扩展来源。在本研究中，我们全面表征了Rh-LF中N-聚糖的结构，并研究了其功能。; Lys-C肽图LC-MS分析Rh-LF定位的N-连接的寡糖为Asn138，Asn479和Asn624。与天然人乳铁蛋白不同，Asn624的第三个位点部分被高甘露糖寡糖占据，其中Asn624通常是空的。使用NP-HPLC和ESI-TOF质谱分析对N-聚糖进行分析，发现Rh-LF的高甘露糖聚糖由6-14个甘露糖部分组成。 SPR分析表明，聚糖的存在或不存在对与Intelectin（一种主要的体内乳铁蛋白受体）的结合亲和力没有影响，观察到去糖基化的Rh-LF的结合亲和力接近Rh-LF和天然人乳铁蛋白。 SEC分析显示，去糖基化不会导致Rh-LF的聚集增加，这表明聚糖在分泌后缺乏在Rh-LF稳定性中的作用。

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  • 作者

    Paliwal, Nirmal S.;

  • 作者单位

    University of Massachusetts Lowell.;

  • 授予单位 University of Massachusetts Lowell.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 64 p.
  • 总页数 64
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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