New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics Glycosylphosphatidylinositol (GPI) attachment to the protein and glycoprotein C-terminus is an important and ubiquitous posttranslational modification in eukaryotic species, which helps anchor proteins and glycoproteins to the extracellular membrane. GPI-anchored proteins and glycoproteins play a pivotal role in various biological and pathological processes. However, currently, detailed studies on these molecules and their functions are limited, mainly because of the difficulty to access them in pure form and sufficient quantity and the lack of proper tools to analyze these diverse, complex, and amphipathic molecules. Therefore, it is highly desirable to have strategies that can facilitate access to and investigation of GPI-anchored proteins and glycoproteins. The ultimate goals of this research project are to develop strategies that enable access to homogeneous and structurally defined natural GPI-anchored proteins and glycoproteins and strategies that enable rapid, effective isolation and analysis of GPI-anchored proteins and glycoproteins. Accordingly, this proposal has three specific aims.
Aim 1 is to prepare both the recombinant catalytic subunit GPI8 of GPI transamidase (GPI-T), the natural enzyme used by eukaryotic cells to attach GPIs to proteins, and membrane-associated intact GPI-T derived from the cell endoplasmic reticulum (ER) and use them to create a potentially general method for enzymatic synthesis of natural GPI-anchored proteins and glycoproteins.
Aim 2 is to develop a practical strategy for the study of GPI- anchored proteins expressed by cells via metabolic engineering of GPI-anchored protein biosynthetic pathways, namely, to give cells or isolated ERs a tagged synthetic GPI analog that can be used by GPI-T to add to proteins bound for GPI attachment. This will result in the specific labeling of GPI-anchored proteins to enable their rapid isolation and then MS-based proteomics analysis.
Aim 3 is to develop a practical strategy for the study of cell surface GPI-anchored proteomics by using CAPM factor, a bacterial toxin that has a high-affinity binding to GPI anchors, to facilitate the isolation of GPI-linked proteins and glycoproteins released from cells upon treatment with phosphatidylinositol-specific phospholipase C enzyme and subsequent GPI-anchored proteomics analysis. Both the strategy for natural GPI-anchored protein and glycoprotein synthesis and the two strategies for GPI- anchored proteomics study are original and innovative, because currently there is no method for the synthesis of truly natural GPI-anchored proteins/glycoproteins and no proper method for systematic study of GPI-anchored proteomics. The proposed research will have a broad and significant impact. A practical synthetic method for GPI-anchored proteins and glycoproteins will allow access to these important molecules and their functionalized analogs in pure and defined forms for various biological and biophysical studies. Strategies allowing systematic study of GPI-anchored proteomics will help reveal the relationships between GPI-anchored proteins and diseases, as well as other important information, and help identify new protein markers. The results will be widely useful for the development of new diagnostic and therapeutic strategies with modulated activity, targeting ability, etc.

Public Health Relevance

Many surface proteins and glycoproteins are anchored to cell membranes by glycosylphosphatidylinositols (GPIs), a class of complex glycolipids, while GPI-anchored proteins and glycoproteins play an important role in various biological processes. However, detailed studies on these molecules are currently limited, because of the difficulty to access them in pure form and sufficient quantity and lack of proper tools to analyze them. This project aims to develop a novel enzymatic method for the synthesis of pure and structurally well-defined natural GPI-anchored proteins and glycoproteins and develop new strategies for the isolation and proteomics analysis of cell surface GPI-anchored proteins and glycoproteins. The results of this research will be widely useful for understanding GPI anchorage, discovery of new protein markers related to diseases, development of new diagnostic and therapeutic strategies for diseases, and so on.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synthetic and Biological Chemistry A Study Section (SBCA)
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Marino, Pamela
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Wayne State University
Schools of Arts and Sciences
United States
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Gao, Jian; Guo, Zhongwu (2018) Progress in the synthesis and biological evaluation of lipid A and its derivatives. Med Res Rev 38:556-601
Zhu, Sanyong; Guo, Zhongwu (2017) Chemical Synthesis of GPI Glycan-Peptide Conjugates by Traceless Staudinger Ligation. Org Lett 19:3063-3066
Gao, Jian; Zhou, Zhifang; Guo, Jiatong et al. (2017) Synthesis of biotin-labelled core glycans of GPI anchors and their application in the study of GPI interaction with pore-forming bacterial toxins. Chem Commun (Camb) 53:6227-6230
Lu, Lili; Gao, Jian; Guo, Zhongwu (2015) Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives. Angew Chem Int Ed Engl 54:9679-9682
Raghupathy, Riya; Anilkumar, Anupama Ambika; Polley, Anirban et al. (2015) Transbilayer lipid interactions mediate nanoclustering of lipid-anchored proteins. Cell 161:581-594
Gao, Jian; Liao, Guochao; Wang, Lizhen et al. (2014) Synthesis of a miniature lipoarabinomannan. Org Lett 16:988-91
Yu, Shichong; Guo, Zhongwu; Johnson, Charlie et al. (2013) Recent progress in synthetic and biological studies of GPI anchors and GPI-anchored proteins. Curr Opin Chem Biol 17:1006-13
Guo, Zhongwu (2013) Synthetic Studies of Glycosylphosphatidylinositol (GPI) Anchors and GPI-Anchored Peptides, Glycopeptides, and Proteins. Curr Org Synth 10:366-383
Wu, Zhimeng; Guo, Xueqing; Gu, Guofeng et al. (2013) Chemoenzymatic synthesis of the human CD52 and CD24 antigen analogues. Org Lett 15:5906-8
Gao, Jian; Guo, Zhongwu (2013) Synthesis of a tristearoyl lipomannan via preactivation-based iterative one-pot glycosylation. J Org Chem 78:12717-25

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