Many surface proteins and glycoproteins are anchored to cell membranes by glycosylphosphatidylinositols (GPIs), and GPI-anchored proteins and glycoproteins play an important role in various biological processes. To investigate these processes and to explore GPI anchorage, it is necessary to have access to homogenous and structurally defined GPIs, GPI derivatives, and GPI-anchored proteins and glycoproteins. This proposal aims to develop a practical method for the synthesis of these important molecules and study GPI-anchored proteomics by means of synthetic GPIs. The proposal has three specific aims.
Specific Aim 1 is to develop a general strategy for the synthesis of natural GPIs and various important GPI derivatives bearing functionalities such as alkene, alkyne, ester, thiol, and sulfide. This research plans to use the p-methoxybenzyl (PMB) group as a global and permanent protecting group in the synthesis to assure that the final deprotection can be achieved selectively in the presence of a variety of functionalities as mentioned.
Specific Aim 2 is to develop a practical method for the chemical synthesis of natively linked GPI-anchored peptides/glycopeptides or proteins/glycoproteins. The fundamental synthetic design is to prepare the GPI and peptide/glycopeptide segments separately and then link them together via a mild and chemoselective reaction. For this purpose, synthetic GPIs will be modified to bear a hydroxylamine or methoxylamine group at the GPI glycan non-reducing end, and peptides will be modified to bear an 1-ketoacid functionality at the C-terminus. Subsequently, the two segments will be coupled by Bode's ligation method under mild conditions.
Specific Aim 3 is to develop a feasible method for rapid isolation and identification of GPI-anchored proteins and glycoproteins and use it to study surface GPI anchorage. For this purpose, the endoplasmic reticulum (ER) membranes of mutant cells lacking GPI biosynthesis functionality will be isolated and used to react with synthetic biotinylated GPIs. Since these ER membranes contain both GPI transamidases, the enzymes that catalyze GPI addition to proteins, and all nascent proteins destined for GPI anchorage, their reaction with biotinylated GPIs will result in the biotinylation of all GPI-anchored proteins. This will create a cell-free system for obtaining the entire complement of GPI-anchored proteins in the biotinylated form. Thereafter, GPI-anchored proteins will be easily isolated with a streptavidin column and analyzed by MS and other methods established for proteomic studies. The broad impact of this project: An effective and widely useful method for the synthesis of homogenous and structurally defined GPIs and GPI-anchored peptides/glycopeptides/proteins is essential for the study and understanding of GPI anchorage and GPI-anchored molecules. Systematic analysis of GPI-anchored proteins on cells will provide important information about the profiles of cell GPI anchorage. This information can help identify GPI-anchored protein markers associated with certain diseases, which can be used as new molecular targets for the design and development of more effective diagnostic techniques and disease treatments.

Public Health Relevance

This proposal aims to develop a practical synthetic method for glycosylphosphatidylinositols (GPIs) and GPI- anchored peptides/glycopeptides or proteins/glycoproteins that play pivotal roles in many biological processes and to develop a practical method for identifying and analyzing GPI-anchored proteins/glycoproteins expressed by cells. This research is important for the study and understanding of the functions of GPI anchors and GPI- anchored molecules. Systematic analysis of GPI-anchored proteins on cells will provide important information that can help identify disease-associated GPI-anchored protein markers that are useful molecular targets for the design and development of more effective diagnostic techniques and disease treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090270-04
Application #
8324038
Study Section
Special Emphasis Panel (ZGM1-PPBC-3 (CS))
Program Officer
Marino, Pamela
Project Start
2009-09-30
Project End
2014-01-31
Budget Start
2012-09-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$293,699
Indirect Cost
$97,679
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