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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM090270-05
Application #
8628408
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Marino, Pamela
Project Start
2009-09-30
Project End
2017-12-31
Budget Start
2014-02-01
Budget End
2014-12-31
Support Year
5
Fiscal Year
2014
Total Cost
$291,608
Indirect Cost
$96,608
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
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
Wu, Zhimeng; Guo, Xueqing; Gao, Jian et al. (2013) Sortase A-mediated chemoenzymatic synthesis of complex glycosylphosphatidylinositol-anchored protein. Chem Commun (Camb) 49:11689-91
Wu, Zhimeng; Guo, Xueqing; Gu, Guofeng et al. (2013) Chemoenzymatic synthesis of the human CD52 and CD24 antigen analogues. Org Lett 15:5906-8
Guo, Xueqing; Wu, Zhimeng; Guo, Zhongwu (2012) New method for site-specific modification of liposomes with proteins using sortase A-mediated transpeptidation. Bioconjug Chem 23:650-5
Burgula, Srinivas; Swarts, Benjamin M; Guo, Zhongwu (2012) Total synthesis of a glycosylphosphatidylinositol anchor of the human lymphocyte CD52 antigen. Chemistry 18:1194-201
Wu, Zhimeng; Guo, Zhongwu (2012) Sortase-Mediated Transpeptidation for Site-Specific Modification of Peptides, Glycopeptides, and Proteins. J Carbohydr Chem 31:48-66
Wu, Zhimeng; Guo, Xueqing; Guo, Zhongwu (2011) Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides. Chem Commun (Camb) 47:9218-20
Swarts, Benjamin M; Guo, Zhongwu (2011) Chemical synthesis and functionalization of clickable glycosylphosphatidylinositol anchors. Chem Sci 2:2342-2352
Swarts, Benjamin M; Guo, Zhongwu (2010) Synthesis of a glycosylphosphatidylinositol anchor bearing unsaturated lipid chains. J Am Chem Soc 132:6648-50