Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Dec 1.
Published in final edited form as: Curr Opin Chem Biol. 2013 Oct 12;17(6):10.1016/j.cbpa.2013.09.016. doi: 10.1016/j.cbpa.2013.09.016

Recent progress in synthetic and biological studies of GPI anchors and GPI-anchored proteins

Shichong Yu 1, Zhongwu Guo 2,3,*, Charlie Johnson 3, Guofeng Gu 2, Qiuye Wu 1
PMCID: PMC3874794  NIHMSID: NIHMS531936  PMID: 24128440

Summary

Covalent attachment of glycosylphosphatidylinositols (GPIs) to the protein C-terminus is one of the most common posttranslational modifications in eukaryotic cells. In addition to anchoring surface proteins to the cell membrane, GPIs also have many other important biological functions, determined by their unique structure and property. This account has reviewed the recent progress made in disclosing GPI and GPI-anchored protein biosynthesis, in the chemical and chemoenzymatic synthesis of GPIs and GPI-anchored proteins, and in understanding the conformation, organization, and distribution of GPIs in the lipid membrane.

Keywords: Glycosylphosphatidylinositol, GPI anchor, protein, glycolipid, inositol, carbohydrate

1. Introduction

Glycosylphosphatidylinositols (GPIs) are complex glycolipids. Their attachment to the protein C-terminus is one of the most common posttranslational modifications in eukaryotic cells [1]. To date, many different GPIs have been characterized [2], all of which share the conserved construct: NH2CH2CH2OP(=O)(OH)-6-O-Manα1→2Manα1→6Manα1→4GlcNH2α1→6-myo-inositol-1-O-phosphoglycerolipid (Figure 1) [3]. Proteins always have their C-termini linked to the phosphoethanolamine group on Man-III.

Figure 1.

Figure 1

Open in a new tab

The common core structure and frequently observed modifications of GPI anchors

One of the most apparent functions of GPIs is to anchor surface proteins to the cell membrane (Figure 1). GPIs and GPI-anchored proteins play a vital role in many biological processes. This review summarizes the recent progress made in disclosing GPI and GPI-anchored protein biosynthesis, in the chemical and chemoenzymatic synthesis of GPIs and GPI-anchored proteins, and in understanding the structure and functions of GPIs using synthetic GPI derivatives.

2. GPI and GPI-Anchored Protein Biosynthesis

The common construct of GPIs and GPI-anchored proteins among different species suggests a conserved biosynthetic pathway [4], which was elucidated by using cell-free biosynthetic systems [5]. As depicted in Figure 2A, GPI biosynthesis completes in/on the endoplasmic reticulum (ER). First, a GlcNAc is added to phosphatidylinositol (PI) on the cytoplasmic surface of ER membrane. The resultant GlcNAc-PI is de-N-acetylated and then inositol 2-O-palmitoylated. GlcNAc-de-N-acetylation is important for the subsequent biosynthetic steps [6,7]. Thereafter, the intermediate is translocated to the ER lumen side, where Man-I is added to GlcNH2, followed by sequential addition of Man-II and III. Once Man-I is attached, the inositol residue can be deacylated and re-acylated, suggesting that inositol acylation is reversible [8,9]. For most GPIs, the last biosynthetic step before protein attachment is the addition of phosphoethanolamine to Man-III, although sometimes additional modifications may occur at this stage [8,9].

Figure 2.

Figure 2

Open in a new tab

An outline of the GPI and GPI-anchored protein biosynthetic pathway. (A) GPI biosynthesis: After the addition of a GlcNAc unit to PI and then de-N-acetylation, a palmitoyl group is attached to the inositol 2-O-position. Next, three mannose residues are added sequentially. The final step is to attach a phosphoethanolamine group to the Man-III 6-O-position. The exact pathway may vary slightly in different species or cell types. (B) GPI attachment to proteins to form GPI-anchored proteins: It is realized on the lumen side of the ER membrane via GPI transamidation catalyzed by GPI-T. This reaction is regulated by the GPI attachment signal at the C-terminus of the nascent protein, and the signal peptide is replaced with a GPI anchor during transamidation. The lipids of GPI-anchored proteins are processed further before their delivery onto the cell surface via the Golgi network. (C) Schematic representations of the subunits and construct of T. brucei and human GPI-Ts [19].

GPI attachment to proteins also takes place on the ER lumen side via a process called GPI transamidation (Figure 2B) involving GPI transamidase (GPI-T) [10,11]. Both GPI-T and its substrates, GPI and nascent protein for GPI-attachment, are anchored to the ER membrane. Nascent protein anchor is a hydrophobic peptide at the C-terminus, which also services as the GPI attachment signal [12]. This signal is absolutely necessary for GPI transamidation, but its sequence varies in different proteins [13]. During transamidation, the GPI attachment signal is substituted with a GPI, thus GPI is always linked to the protein C-terminus. Nascent proteins have another signal peptide at their N-termini, which directs proteins to ER but is not removed during GPI transamidation [12]. After GPIs are attached to proteins, the palmitoyl group at the inositol 2-O-position is usually removed for the purpose of product quality control [14]. This may also facilitate GPI-anchored protein transportation from ER to the Golgi apparatus. However, this palmitoyl group can remain in some GPI-anchored proteins, such as T. brucei procyclic acidic repetitive protein [15], human acetylcholinesterase [16] and human CD52 antigen [17]. After GPI-linked proteins are transported to Golgi, GPIs are further modified through a process called “fatty acid remodeling” [18]. Finally, GPI-anchored proteins are delivered onto the cell surface via the trans-Golgi network.

GPI-T is a complex enzyme consisting of five different subunits, a construct conserved among different species [19]. The subunits of Saccharomyces cerevisia GPI-T, Gaa1p, Gpi8p, Gpi17p Gpi16p and Gab1p, are respectively homologous to that of the human GPI-T, GAA1, GPI8, PIG-S, PIG-T and PIG-U (Figure 2C), but some difference was found between certain T. brucei (Figure 2C) and human GPI-T subunits. Bi and coworkers [20] proposed that: (1) Gpi8p is the centerpiece of GPI-T surrounded by and interacting with all other subunits; (2) GPI-T may have two sub-complexes: one containing Gpi8p, Gpi16p and Gaa1p, and the other Gab1p and Gpi17p; (3) the three-component complex is the core and is conserved in all species. The exact role of each subunit is not defined, but it is widely accepted that GPI8 is the catalytic subunit [21], which could hydrolyze a synthetic peptide [22]. For GPI transamidation, Cys at the GPI8 active site cleaves the GPI attachment signal to form a reactive thioester at amino acid ω and transfers its carboxyl group to GPI. Structural modeling results indicated that PIG-T/Gpi16p may mediate protein presentation to GPI8/Gpi8p [23]. PIG-T/Gpi16p may regulate GAA1 and GPI8/Gpi8p expression [24]; GAA1/Gaa1p may assist GPI presentation to GPI8/Gpi8p [25]. In theory, GPI8/Gpi8p, PIG-T/Gpi16p and GAA1/Gaa1p can constitute a catalytically functional complex [20]. Gpi17p is believed to stabilize GPI-T complex and regulate substrate selectivity [23]; Gab1p, which contains a stretch of amino acids of fatty acid elongase, is proposed to mediate GPI presentation to Gpi8p [20].

The GPI and GPI-anchored protein biosynthetic pathway is relatively well established. Despite that, there are still questions that need clarification, such as why the biosynthesis starts on the ER cytoplasmic surface and how the biosynthetic intermediate is translocated onto the ER lumen side. In our opinion, however, a particularly intriguing topic is GPI-T, as it is not only critical for gaining insights into GPI transamidation but also a potentially useful tool in chemical biology. For example, GPI-T may be used to ligate GPIs and proteins in vitro for the synthesis of truly natural GPI-anchored proteins. It may also be used to ligate GPIs and various other molecules engineered to carry the GPI attachment signal. The resultant GPI conjugates should be useful for GPI research or have the targeting ability of GPIs. A major problem, however, is that GPI-T is complex and difficult to study, thus there is no report about its application yet. Any progress in this area should have a big impact.

3. Chemical Synthesis of GPI Anchors

Owing to the presence of different carbohydrates, lipids, and other functional groups, natural GPIs usually exist in heterogeneous forms that are difficult to separate. Thus, access to homogeneous and structurally well-defined GPIs and related derivatives has to rely on chemical synthesis. GPI synthesis is a significant challenge involving multiple disciplines in organic chemistry. Nevertheless, great progress has been made in the area since the first GPI total synthesis [26].

Two general strategies have been developed for GPI synthesis. One starts with GPI skeleton construction and finishes with protecting group manipulation to achieve regioselective phosphate and lipid installation. This strategy can delay the chiral phosphate introduction to a late stage. The other strategy is to install the sterically hindered phosphoglycerolipid early, e.g., following GlcN3, to get phospholipidated intermediate 1 (Figure 3A), and then elongate the glycan. The latter strategy is convergent, so it can be used to rapidly prepare various GPIs and GPI derivatives via coupling proper oligosaccharides to the key intermediate. Moreover, it can also address problems met with late-stage phosphoglycerolipidation in the synthesis of GPIs carrying 2-O-palmitoylated inositol [27]. Both strategies have been used to prepare GPIs. However, due to space limitation and because GPI total synthesis has been covered by several reviews [28–30], here we will focus on recent development in diversity-oriented GPI synthesis.

Figure 3.

Figure 3

Open in a new tab

General strategies for diversity-oriented synthesis of functionalized GPIs and GPI derivatives

Synthesis of GPI Anchors Containing Unsaturated Lipids and Other Useful Functionalities

Many natural GPIs contain unsaturated lipids that are structurally and biologically important [31]. For their synthesis, the traditional tactic to protect hydroxyl groups as benzyl ethers is unsuitable, because its deprotection is incompatible with unsaturated lipids. One attempt to solve the problem was employing benzoyl and silyl protections reported by the Nikolaev group [32,33]. Alternatively, the Guo group [34] described a strategy using the para-methoxybenzyl (PMB) group for global hydroxyl protection (Figure 3A). Thus, after 1 was prepared, its glycan was elongated, which was followed by further modifications. Global deprotection of 3 by a three-step, one-pot protocol gave the target GPI 4 having two unsaturated lipids. Notably, complete removal of PMB groups was accomplished with 10% trifluoroacetic acid (TFA) in < 30 minutes.

The strategy was further showcased in the synthesis of a human lymphocyte CD52 GPI anchor containing a labile polyunsaturated lipid [35]. Using the PMB group to protect hydroxyl groups also allowed for the introduction of other molecular handles. For example, clickable GPIs containing an azido and an alkynyl groups were prepared by this strategy and effectively coupled to a fluorescent tag and an affinity tag via click reaction [36]. These GPI derivatives are valuable tools for GPI research.

Branched GPI Anchor Synthesis

To address the structural diversity of natural GPI anchors, the Seeberger group [37] developed a general synthetic strategy based on protecting group orthogonality. A key feature of this strategy is that the central mannose block, 5 or 6, had several orthogonal protections that enabled regioselective introduction of any branching observed in nature. Its applicability was demonstrated by the synthesis of a Toxoplasma gondii GPI 7 with a disaccharide at the Man-I 4-O-position (Figure 3B) [38].

In the past two decades, numerous schemes have been developed for GPI synthesis. It is currently feasible to chemically secure all kinds of GPIs and GPI derivatives for various biological studies. With that said, GPI chemical synthesis is far from trivial, and each target must be treated as a respectful challenge that needs careful planning of the synthetic scheme and protection tactics. Thus, additional strategies or tools to facilitate GPI synthesis are definitely welcome.

4. Chemical and Chemoenzymatic Synthesis of GPI-Anchored Proteins

As all GPI-anchored proteins identified so far have their C-termini linked to the same position of the GPI core (Figure 1), it is theoretically possible to develop a generally useful method for GPI-anchored protein synthesis through site-specific reactions of GPIs and proteins. In this regard, both chemical and enzymatic methods have been explored.

The Nakahara group [39] was the first to examine GPI-linked peptide synthesis by native chemical ligation (NCL) of Cys-containing GPI analogs and C-terminal thioester of peptides. Later, the Bertozzi group [40] and the Seeberger group [41] reported the use of NCL to couple GPI analogs to proteins, showcasing its potential. Alternatively, the Guo group [42,43] reported GPI-anchored peptide/glycopeptide synthesis by regiospecific coupling of extensively protected peptides/glycopeptides with free C-termini to GPI analogs carrying a free phosphoethanolamine.

Recently, the Guo group [44–46] developed a chemoenzymatic strategy for GPI-anchored peptide/protein synthesis catalyze by sortase A (SrtA), a Staphylococcus aureus-originated transpeptidase [47]. To achieve GPI-protein ligation, the target protein was engineered to bear at its C-terminus a pentapeptide, LPXTG –the sorting signal, recognized by SrtA, while the GPI anchor had a Gly residue attached to the conserved phosphoethanolamine (Figure 4). SrtA can react with the sorting signal, break the peptide bond between T and G to form a reactive thioester, and then attach the carboxyl group of T to the amino group of Gly on the GPI anchor. This reaction was proved to be effective and specific, which has been employed to couple peptides, glycopeptides, and small proteins to GPIs [48].

Figure 4.

Figure 4

Open in a new tab

SrtA-catalyzed synthesis of GPI-anchored proteins. SrtA recognizes the sorting signal at the protein C-terminus, cleaves the peptide bond between T and G to form a reactive thioester with the protein, and then attaches the protein to the Gly residue on the GPI anchor to eventually afford GPI-anchored protein.

In contrast to the great progress in GPI total synthesis, GPI-anchored peptide/protein synthesis remains a formidable challenge. Chemical synthesis of simple GPI-peptide conjugates is feasible, but the strategy is inapplicable to large proteins, as extensive protein protection to secure site-specific GPI-protein coupling is nearly impossible. NCL and SrtA-catalyzed GPI-protein ligations are promising and may be elaborated into useful synthetic methods, but both have limitations and, more significantly, yield unnatural products. For example, NCL gives GPI-linked proteins with a Cys between the GPI and protein, which may affect the structure and function of GPI-anchored proteins. Moreover, the preparation of protein thioesters and Cys-containing GPIs is not trivial. On the other hand, SrtA-mediated GPI-protein ligation gives GPI-linked proteins with a pentapeptide between the GPI and protein; its structural and biological impacts are unclear. Evidently, there is no practical method to access natural and homogeneous GPI-anchored proteins yet, so any development in this respect should be highly desirable.

5. Application of Synthetic GPIs to Biological Studies

As mentioned above, GPIs and GPI-anchored proteins play a critical role in many biological events [49]. However, it is impossible to discuss these findings in detail in this short review. Instead, we will focus on studies about the conformation, organization, and distribution of GPIs in the lipid bilayer, as well as other related general topics. For these studies, synthetic GPIs and derivatives should be particularly helpful.

Computer simulation combined with NMR analysis of synthetic GPIs inserted into lipid micelle suggests that they had extended conformation with a faster relative motion at the terminal mannose and decreased mobility close to the micelle [50]. This motion is oscillation relative to the membrane rather than folding. Later, it was shown that the GPI tetrasaccharide backbone forms a rather stiff rod with a residual bending elasticity and mechanical compressibility [51]. Moreover, GPIs are not only membrane-anchoring devices but also prevent transient interactions between attached proteins and the underlying lipid bilayer, thereby permitting their rapid diffusion in the lipid bilayer [40]. Clearly, GPIs have unique properties that may be critical for the proper functioning of GPI-anchored proteins. For example, delipidation of GPI-anchored proteins could induce a conformational change that can affect antibody affinity [52].

Studies have shown that GPI-anchored proteins prefer to localize in the ordered regions of cell membrane, such as the lipid rafts [53]. Accordingly, the lipid compositions of GPIs and membranes could affect GPI-membrane association and interaction. Results obtained with lipid bilayers disclosed that the membrane-anchoring domain of GPIs had a significant impact on the distribution of GPI-anchored proteins in lipid rafts [54]. GPIs and GPI-anchored proteins could also form cholesterol-dependent microdomains in the cell membrane [55], which is affected by both the lipid chain and head-group. GPI-anchored proteins with normally remodeled GPIs had elevated oligomerization tendency and immunoreactivity as compared to that with unremodeled GPIs [56]. Studies on a truncated GPI analog in monolayers revealed that phase-separation occurred above a threshold concentration owing to strong head-group interactions, whereas the GPI analog could mix with disordered lipid and induce order in a highly cooperative manner below this concentration [57]. The anchored proteins may also affect microdomain formation [58]. In resting cells, virtually all GPI-anchored proteins form transient homodimers via ectodomain protein interactions. This works with the general, low-affinity cholesterol-dependent raft-lipid interaction to generate greater rafts in steady-state cells and in response to extracellular stimulation. GPI and GPI-anchored protein buildup and microdomain formation in the cell membrane are critical for their biological functions. In addition to the examples mentioned above that delipidation and incorrect lipid structure could reduce GPI-anchored protein affinity [52] and immunoreactivity [56], raft-based lipid interaction was discovered to play a role in signal transduction by GPI-anchored protein [58]. GPIs are also involved in protein sorting, trafficking and targeting [49].

The lipid tails of GPIs do not completely extend through the lipid bilayer. Therefore, GPI-linked proteins are not so tightly anchored to the membrane as transmembrane proteins and can migrate from one cell to another to enable cell communication [59]. Moreover, GPIs can be selectively cleaved by phospholipases, such as phosphatidylinositol-specific phospholipases C and D, to release the anchored proteins. This may be a regulating mechanism for GPI-anchored proteins [60]. On the other hand, inositol phospholipid may participate in signal transduction pathways [61].

Despite the challenge to access and study GPIs due to their complex structure and amphiphilic property, biophysical and biological investigation of GPIs has been expanding. GPIs possess many unique features that are difficult to find in simple lipids or transmembrane proteins. This may explain why nature spends much energy to synthesize and utilize these complex molecules to anchor proteins to the cell membrane. With increasing availability of synthetic GPIs and GPI analogs, studies to gain insights into the structure, function, and structure-activity relationship of GPI anchors will prosper.

6. Conclusion

GPIs represent a distinctive class of protein anchors. Their many unique properties may be critical for the proper functioning of anchored proteins. However, GPI research has been impeded by the difficult access to homogeneous and structurally defined GPIs and GPI-anchored proteins. Fortunately, progress in GPI total synthesis has made it possible to chemically secure GPIs and related derivatives. We project that one of the flourishing topics in the future will be using synthetic GPIs, especially those with fluorescent, affinity and other molecular tags, to gain insights into GPI anchorage. In contrast, access to homogeneous GPI-anchored proteins remains a significant challenge. A potential solution is using enzymes to catalyze site-specific conjugation of synthetic GPIs and recombinant proteins. For this purpose, GPI-T should be ideal, but the complex structure of GPI-T makes its expression and application difficult. Alternatively, synthetic GPI-anchored protein analogs may be used, but whether these molecules can really represent natural GPI-anchored proteins is an open question. Thus, synthetic strategies leading to natural GPI-anchored proteins are highly desirable. Finally, practical methods and tools to study GPIs and GPI-anchored proteins in vivo or in living cells is another future direction. Reports [62] in this area have already started to emerge.

Highlights.

  • Great progress has been made in revealing GPI and GPI-anchored protein biosynthesis.

  • Progress in GPI and GPI-anchored protein synthesis has made their access practical.

  • Synthetic GPIs and GPI-anchored proteins have facilitated the study of GPI biology.

Acknowledgments

Our research program has been supported by the National Major Scientific and Technological Special Project for “Significant New Drug Development” (2012ZX09502001-005) and the State Major Basic Research Development (973) Program (2012CB822102) of China, and by the National Science Foundation (NSF, CHE-0320878, 0715275 and 1053848) and National Institutes of Health (NIH, R01 GM090270) of the United States.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Eisenhaber B, Bork P, Eisenhaber F. Post-translational GPI lipid anchor modification of proteins in kingdoms of life: Analysis of protein sequence data from complete genomes. Protein Eng. 2001;14:17–25. doi: 10.1093/protein/14.1.17. [DOI] [PubMed] [Google Scholar]
  • 2.Ferguson MAJ, Homans SW, Dwek RA, Rademacher TW. Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science. 1988;239:753–759. doi: 10.1126/science.3340856. [DOI] [PubMed] [Google Scholar]
  • 3.Ferguson MAJ. The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. J Cell Sci. 1999;112:2799–2809. doi: 10.1242/jcs.112.17.2799. [DOI] [PubMed] [Google Scholar]
  • 4.Tiede A, Bastisch I, Schubert J, Orlean P, Schmidt RE. Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes. Biol Chem. 1999;380:503–523. doi: 10.1515/BC.1999.066. [DOI] [PubMed] [Google Scholar]
  • 5.Masterson WJ, Doering TL, Hart GW, Englund PT. A novel pathway for glycan assembly: Biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989;56:793–800. doi: 10.1016/0092-8674(89)90684-3. [DOI] [PubMed] [Google Scholar]
  • 6.Güther ML, Ferguson MA. The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei. EMBO J. 1995;14:3080–3093. doi: 10.1002/j.1460-2075.1995.tb07311.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Stevens VL, Zhang H. Coenzyme A dependence of glycosylphosphatidylinositol biosynthesis in a mammalian cell-free system. J Biol Chem. 1994;269:31397–31403. [PubMed] [Google Scholar]
  • 8.Udenfriend S, Kodukula K. How glycosylphosphatidylinositol-anchored membrane protein are made. Ann Rev Biochem. 1995;64:563–591. doi: 10.1146/annurev.bi.64.070195.003023. [DOI] [PubMed] [Google Scholar]
  • 9.Eckert V, Gerold P, Schwarz RT. Biosynthesis of glycosylated phosphatidylinositol in parasitic protozoa, yeast, and higher eukaryotes. Comp Nat Prod Chem. 1999;3:295–309. [Google Scholar]
  • 10.Ikezawa H. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol Pharm Bull. 2002;25:409–417. doi: 10.1248/bpb.25.409. [DOI] [PubMed] [Google Scholar]
  • 11.Kinoshita T, Inoue N. Dissecting and manipulating the pathway for glycosylphos-phatidylinositol-anchor biosynthesis. Curr Opin Chem Biol. 2000;4:632–638. doi: 10.1016/s1367-5931(00)00151-4. [DOI] [PubMed] [Google Scholar]
  • 12.Amthauer R, Kodukula K, Gerber L, Udenfriend S. Evidence that the putative COOH-terminal signal transamidase involved in glycosylphosphatidylinositol protein synthesis is present in the endoplasmic reticulum. Proc Natl Acad Sci USA. 1993;90:3973–3977. doi: 10.1073/pnas.90.9.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • •13.Chen R, Knez JJ, Merrick WC, Medof ME. Comparative effciencies of C-terminal signals of native glycophosphatidylinositol (GPI)-anchored proproteins in conferring GPI-anchoring. J Cell Biochem. 2002;84:68–83. doi: 10.1002/jcb.1267. The strcuture of the GPI attachment signal was found to have a significant influence on the efficiency of GPI attachment to proteins catalyzed by GPI transamidase. [DOI] [PubMed] [Google Scholar]
  • •14.Fujita M, Yoko-o T, Jigami Y. Inositol deacylation by BST1P is required for the quality control of glycosylphosphatidylinositol-anchored proteins. Mol Biol Cell. 2006;17:834–850. doi: 10.1091/mbc.E05-05-0443. Inositol deacylation was shown to be important for the quality control and transportation of GPI-anchored proteins. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Field MC, Menon AK, Cross GAM. A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei: Lipid structure and biosynthesis. EMBO J. 1991;10:2731–2739. doi: 10.1002/j.1460-2075.1991.tb07821.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Roberts WL, Santikarn S, Reinhold VN, Rosenberry TL. Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry. J Biol Chem. 1988;263:18776–18784. [PubMed] [Google Scholar]
  • 17.Treumann A, Lifely MR, Schneider P, Ferguson MAJ. Primary structure of CD52. J Biol Chem. 1995;270:6088–6099. doi: 10.1074/jbc.270.11.6088. [DOI] [PubMed] [Google Scholar]
  • 18.Masterson WJ, Raper J, Doering TL, Hart GW, Englund PT. Fatty acid remodeling: A novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors. Cell. 1990;62:73–80. doi: 10.1016/0092-8674(90)90241-6. [DOI] [PubMed] [Google Scholar]
  • •19.Nagamune K, Ohishi K, Ashida H, Hong Y, Hino J, Kangawa K, Inoue N, Maeda Y, Kinoshita T. GPI transamidase of Trypanosoma brucei has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits. Proc Natl Acad Sci USA. 2003;100:10682–10687. doi: 10.1073/pnas.1833260100. The multiunit construct of GPI transamidase was discussed, and the enzymes from different species were compared. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Grimme SJ, Gao X, Martin PS, Tu K, Tcheperegine SE, Corrado K, Farewell AE, Orlean P, Bi E. Deficiencies in the endoplasmic reticulum (ER)-membrane protein GAB1P perturb transfer of glycosylphosphatidylinositol to proteins and cause perinuclear ER-associated actin bar formation. Mol Biol Cell. 2004;15:2758–2770. doi: 10.1091/mbc.E04-01-0035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Meyer U, Benghezal M, Imhof I, Conzelmann A. Active site determination of GPI8P, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins. Biochemistry. 2000;39:3461–3471. doi: 10.1021/bi992186o. [DOI] [PubMed] [Google Scholar]
  • •22.Kang X, Szallies A, Rawer M, Echner H, Duszenko M. GPI anchor transamidase of Trypanosoma brucei: In vitro assay of the recombinant protein and VSG anchor exchange. J Cell Sci. 2002;115:2529–2539. doi: 10.1242/jcs.115.12.2529. GPI8 was demonstrated to be the catalytic subunit of GPI transamidase and was found to mediate the cleavage of a synthetic peptide carrying the GPI attachment signal. [DOI] [PubMed] [Google Scholar]
  • 23.Eisenhaber B, Maurer-Stroh S, Novatchkova M, Schneider G, Eisenhaber F. Enzymes and auxiliary factors for GPI lipid anchor biosynthesis and post-translational transfer to proteins. Bioessays. 2003;25:367–385. doi: 10.1002/bies.10254. [DOI] [PubMed] [Google Scholar]
  • 24.Ohishi K, Inoue N, Kinoshita T. PIG-S, and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1, and GPI8. EMBO J. 2001;20:4088–4098. doi: 10.1093/emboj/20.15.4088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Vainauskas S, Maeda Y, Kurniawan H, Kinoshita T, Menon AK. Structural requirements for the recruitment of GAA1 into a functional glycosylphosphatidylinositol transamidase complex. J Biol Chem. 2002;277:30535–30542. doi: 10.1074/jbc.M205402200. [DOI] [PubMed] [Google Scholar]
  • 26.Murakata C, Ogawa T. A total synthesis of GPI anchor of Trypanosoma brucei. Tetrahedron Lett. 1991;32:671–674. [Google Scholar]
  • 27.Xue J, Guo Z. Convergent synthesis of a GPI containing an acylated inositol. J Am Chem Soc. 2003;125:16334–16339. doi: 10.1021/ja0382157. [DOI] [PubMed] [Google Scholar]
  • •28.Nikolaev AV, Al-Maharik N. Synthetic glycosylphosphatidylinositol (GPI) anchors: How these complex molecules have been made. Nat Prod Rep. 2011;28:970–1020. doi: 10.1039/c0np00064g. A comprehensive review about the chemical synthesis of GPI anchors (see references cited therein) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • •29.Swarts BM, Guo Z. Chemical synthesis of glycosylphosphatidylinositol anchors. Adv Carbohydr Chem Biochem. 2012;67:137–219. doi: 10.1016/B978-0-12-396527-1.00004-8. A comprehensive review about the chemical synthesis of GPI anchors (see references cited therein) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Azzouz N, Kamena F, Seeberger PH. Synthetic glycosylphosphatidylinositol as tools for glycoparasitology research. OMICS: J Integ Biol. 2010;14:445–454. doi: 10.1089/omi.2009.0138. [DOI] [PubMed] [Google Scholar]
  • 31.Almeida IC, Camargo MM, Procopio DO, Silva LS, Mehlert A, Travassos LR, Gazzinelli RT, Ferguson MAJ. Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J. 2000;19:1476–1485. doi: 10.1093/emboj/19.7.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • •32.Yashunsky DV, Borodkin VS, Ferguson MAJ, Nikolaev AV. The chemical synthesis of bioactive glycosylphosphatidylinositols from Trypanosoma cruzi containing an unsaturated fatty acid in the lipid. Angew Chem Int Ed. 2006;45:468–474. doi: 10.1002/anie.200502779. The first chemical synthesis of a GPI anchor containing unsaturated lipids. [DOI] [PubMed] [Google Scholar]
  • 33.Yashunsky Dmitry V, Borodkin Vladimir S, McGivern Philip G, Ferguson Michael AJ, Nikolaev Andrei V. Frontiers in Modern Carbohydrate Chemistry. Vol. 960. American Chemical Society; 2007. The chemical synthesis of glycosylphosphatidylinositol anchors from Trypanosoma cruzi trypomastigote mucins; pp. 285–306. [Google Scholar]
  • ••34.Swarts BM, Guo Z. Synthesis of a glycosylphosphatidylinositol anchor bearing unsaturated lipid chains. J Am Chem Soc. 2010;132:6648–6650. doi: 10.1021/ja1009037. An efficient strategy for the synthesis of GPI anchors carrying unsaturated lipids using the para-methoxybenzyl group for global hydroxyl protection was described. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Burgula S, Swarts BM, Guo Z. Total synthesis of a glycosylphosphatidylinositol anchor of the human lymphocyte CD52 antigen. Chem Eur J. 2012;18:1194–1201. doi: 10.1002/chem.201102545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • •36.Swarts BM, Guo Z. Chemical synthesis and functionalization of clickable glycosylphosphatidylinositol anchors. Chem Sci. 2011;2:2342–2352. doi: 10.1039/C1SC00440A. Synthetic GPI analogs with an azido group and an alkynyl group were synthesized and then coupled to a fluorescent tag and an affinity (biotin) tag, respectively. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Tsai Y-H, Götze S, Azzouz N, Hahm HS, Seeberger PH, Varon Silva D. A general method for synthesis of GPI anchors illustrated by the total synthesis of the low-molecular-weight antigen from Toxoplasma gondii. Angew Chem Int Ed. 2011;50:9961–9964. doi: 10.1002/anie.201103483. [DOI] [PubMed] [Google Scholar]
  • ••38.Tsai YH, Gotze S, Vilotijevic I, Grube M, Silva DV, Seeberger PH. A general and convergent synthesis of diverse glycosylphosphatidylinositol glycolipids. Chem Sci. 2013;4:468–481. A convergent and diversity-oriented strategy for the synthesis of GPI anchors was described. [Google Scholar]
  • 39.Tanaka Y, Nakahara Y, Hojo H, Nakahara Y. Studies directed toward the synthesis of protein-bound GPI anchor. Tetrahedron. 2003;59:4059–4067. [Google Scholar]
  • •40.Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR. Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers. J Am Chem Soc. 2007;129:11543–11550. doi: 10.1021/ja073271j. The first report of coupling a large protein to a GPI anchor analog by native chemical ligation. [DOI] [PubMed] [Google Scholar]
  • 41.Becker CFW, Liu X, Olschewski D, Castelli R, Seidel R, Seeberger PH. Semisynthesis of a glycosylphosphatidylinositol-anchored prion protein. Angew Chem Int Ed. 2008;47:8215–8219. doi: 10.1002/anie.200802161. [DOI] [PubMed] [Google Scholar]
  • 42.Xue J, Shao N, Guo Z. First total synthesis of a GPI-anchored peptide. J Org Chem. 2003;68:4020–4029. doi: 10.1021/jo034213t. [DOI] [PubMed] [Google Scholar]
  • 43.Shao N, Xue J, Guo Z. Chemical synthesis of a skeleton structure of sperm CD52 - a GPI-anchored glycopeptide. Angew Chem Int Ed. 2004;43:1569–1573. doi: 10.1002/anie.200353251. [DOI] [PubMed] [Google Scholar]
  • ••44.Guo X, Wang Q, Swarts BM, Guo Z. Sortase-catalyzed peptide-glycosylphosphatidylinositol analog ligation. J Am Chem Soc. 2009;131:9878–9879. doi: 10.1021/ja903231v. The first application of Sortase A to ligating peptides and GPI analogs for chemoenzymatic synthesis of GPI-anchored proteins. [DOI] [PubMed] [Google Scholar]
  • 45.Wu Z, Guo X, Wang Q, Swarts BM, Guo Z. Sortase A-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymatic synthesis of GPI-anchored proteins. J Am Chem Soc. 2010;132:1567–1571. doi: 10.1021/ja906611x. [DOI] [PubMed] [Google Scholar]
  • 46.Wu Z, Guo X, Guo Z. Chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides. Chem Comm. 2010:5773–5774. doi: 10.1039/c0cc00828a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • •47.Marraffini LA, DeDent AC, Schneewind O. Sortases and the art of anchoring proteins to the envelopes of Gram-positive bacteria. Microbiol Mol Biol Rev. 2006;70:192–221. doi: 10.1128/MMBR.70.1.192-221.2006. A comprehensive review about sortases. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Wu Z, Guo Z. Sortase-mediated transpeptidation for site-specific modification of peptides, glycopeptides, and proteins. J Carbohydr Chem. 2012;31:48–66. doi: 10.1080/07328303.2011.635251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • •49.Paulick MG, Bertozzi CR. The glycosylphosphatidylinositol anchor: A complex membrane-anchoring structure for proteins. Biochemistry. 2008;47:6991–7000. doi: 10.1021/bi8006324. A comprehensive review about the properties, functions, and other related aspects of the GPI anchor. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Chevalier F, Lopez-Prados J, Groves P, Perez S, Martín-Lomas M, Nieto PM. Structure and dynamics of the conserved protein gpi anchor core inserted into detergent micelles. Glycobiology. 2006;10:969–980. doi: 10.1093/glycob/cwl015. [DOI] [PubMed] [Google Scholar]
  • ••51.Wehle M, Vilotijevic I, Lipowsky R, Seeberger PH, Silva DV, Santer M. Mechanical compressibility of the glycosylphosphatidylinositol (GPI) anchor backbone governed by independent glycosidic linkages. J Am Chem Soc. 2012;134:18964–18972. doi: 10.1021/ja302803r. The tetrasaccharide backbone of the GPI anchor was found to form a rather stiff rod with a residual bending elasticity and mechanical compressibility. [DOI] [PubMed] [Google Scholar]
  • •52.Bradley JE, Chan JM, Hagood JS. Effect of the GPI anchor of human Thy-1 on antibody recognition and function. Lab Invest. 2013;93:365–374. doi: 10.1038/labinvest.2012.178. Delipidation of GPI-anchored proteins was found to affect protein conformation and antibody affinity. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Giocondi M-C, Besson F, Dosset P, Milhiet PE, Le Grimellec C. Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase. Langmuir. 2007;23:9358–9364. doi: 10.1021/la700892z. [DOI] [PubMed] [Google Scholar]
  • •54.Garner AE, Smith DA, Hooper NM. Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers. Mol Mem Biol. 2007;24:233–242. doi: 10.1080/09687860601127770. The nature of the membrane-anchoring domain of the GPI anchor was shown to have a significant impact on the association and distribution of GPI-anchored proteins with lipid rafts. [DOI] [PubMed] [Google Scholar]
  • 55.Varma R, Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998;394:798–801. doi: 10.1038/29563. [DOI] [PubMed] [Google Scholar]
  • ••56.Seong J, Wang Y, Kinoshita T, Maeda Y. Implications of lipid moiety in oligomerization and immunoreactivities of GPI-anchored proteins. J Lipid Res. 2013;54:1077–1091. doi: 10.1194/jlr.M034421. The lipid structure of the GPI anchor was found to have an impact on the oligomerization and function of GPI-anchored proteins. GPI-anchored proteins with normally remodeled GPIs had enhanced oligomerization and immunoreactivity as compared to that with unremodeled GPIs. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • ••57.Stefaniu C, Vilotijevic I, Santer M, Silva DV, Brezesinski G, Seeberger PH. Subgel phase structure in monolayers of glycosylphosphatidylinositol glycolipids. Angew Chem Int Ed. 2012;51:12874–12878. doi: 10.1002/anie.201205825. It was demonstrated that a truncated GPI analog could mix with disordered lipid and induce order in a highly cooperative manner below a certain threshold concentration. [DOI] [PubMed] [Google Scholar]
  • ••58.Suzuki KGN, Kasai RS, Hirosawa KM, Nemoto YL, Ishibashi M, Miwa Y, Fujiwara TK, Kusumi A. Transient GPI-anchored protein homodimers are units for raft organization and function. Nat Chem Biol. 2012;8:774–783. doi: 10.1038/nchembio.1028. The interactions between the anchored proteins were shown to play a role in the microdomain formation of GPI-anchored proteins on the cell surface. [DOI] [PubMed] [Google Scholar]
  • 59.Kooyman DL, Byrne GW, McClellan S, Nielsen D, Tone M, Waldmann H, Coffman TM, McCurry KR, Platt JL, Logan JS. In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium. Science. 1995;269:89–92. doi: 10.1126/science.7541557. [DOI] [PubMed] [Google Scholar]
  • 60.Low MG, Stiernberg J, Waneck GL, Flavell RA, Kincade PW. Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C. J Immunol Methods. 1988;113:101–111. doi: 10.1016/0022-1759(88)90386-9. [DOI] [PubMed] [Google Scholar]
  • 61.Corda D, Zizza P, Varone A, Bruzik KS, Mariggio S. The glycerophosphoinositols and their cellular functions. Biochem Soc Trans. 2012;40:101–107. doi: 10.1042/BST20110679. [DOI] [PubMed] [Google Scholar]
  • •62.Paulick MG, Forstner MB, Groves JT, Bertozzi CR. A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor. Proc Natl Acad Sci USA. 2007;104:20332–20337. doi: 10.1073/pnas.0710139104. A new strategy was developed for the study of the functions of GPI anchors in living cells by using synthetic GPI anchor and GPI-anchored protein analogs that carry specific molecular tags. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES