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Zwischen Protein und Membran
Abstract
Spezielle Glykolipide verankern eukaryotische Proteine an der äußeren Zellmembran. Bislang ist jedoch wenig über die Funktionen dieser Glykosylphosphatidylinositole bekannt. Erkenntnisse über ihre biologischen Eigenschaften lassen sich mit synthetischen Molekülen gewinnen.
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Modularer Aufbau: Eine neue, allgemeine Synthesestrategie, die die Herstellung verzweigter Glycosylphosphatidylinositole (GPI) ermöglicht, lieferte das Glycolipid 1 des Parasiten Toxoplasma gondii. Die Struktur von 1 wurde durch Affinitätsexperimente mit monoklonalen Antikörpern verifiziert.
Glycosylphosphatidylinositol (GPI) glycolipids anchor a large number of proteins in the cell membrane of eukaryotic cells. Their conserved pseudopentasaccharide core carries additional phosphoethanolamine, saccharide and lipid substituents. These structural variations are characteristic for a species or a tissue but their functional significance remains largely unknown. Studies that would link a specific function to a structurally unique GPI rely on availability of homogeneous samples of these glycolipids. To address this need we have developed a general synthetic route to GPI glycolipids. Our convergent synthesis starts from common building blocks and relies on a fully orthogonal set of protecting groups that enables the regioselective introduction of phosphodiesters and efficient assembly of the GPI glycans. Here, we report on the development of this synthetic strategy, evaluation of the set of protecting groups with respect to phosphorylation methods, evaluation of the assembly plan for the GPI glycan, optimization of the glycosylation reactions, and the application of this strategy to the total syntheses of four structurally diverse branched GPI glycolipids.
Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 x 10(-3) s-1 to 2.39 x 10(-3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.
Protein engineering of cell surfaces is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer. This technology exploits the fact that proteins that are anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, incorporate into their surface membranes and are fully functional. By substituting 3'-mRNA end sequence of naturally GPI-anchored proteins (i.e., a sequence that contains the signals that direct GPI anchoring) for endogenous 3'-mRNA end sequence, virtually any protein of interest can be expressed as a GPI-anchored derivative. The GPI-anchored product then can be purified from transfectants and the purified protein used to "paint" any target cell. Such protein engineering or "painting" of the cell surface offers several advantages over conventional gene transfer. Among these advantages are that 1) GPI-anchored proteins can be painted onto cells that are difficult to transfect, 2) cells can be altered immediately without previous culturing, 3) the amount of protein added to the surface can be precisely controlled, and 4) multiple GPI-anchored proteins can be sequentially or concurrently inserted into the same cells. Emerging applications for the technology include its use for the analysis of complex cell-surface interactions, the engineering of antigen presenting cells, the development of cancer vaccines, and possibly the protection against graft rejection.
Cells that are sensitive to the channel-forming toxin aerolysin contain surface glycoproteins that bind the toxin with high affinity. Here we show that a common feature of aerolysin receptors is the presence of a glycosylphosphatidylinositol anchor, and we present evidence that the anchor itself is an essential part of the toxin binding determinant. The glycosylphosphatidylinositol (GPI)-anchored T-lymphocyte protein Thy-1 is an example of a protein that acts as an aerolysin receptor. This protein retained its ability to bind aerolysin when it was expressed in Chinese hamster ovary cells, but could not bind the toxin when expressed in Escherichia coli, where the GPI anchor is absent. An unrelated GPI-anchored protein, the variant surface glycoprotein of trypanosomes, was shown to bind aerolysin with similar affinity to Thy-1, and this binding ability was significantly reduced when the anchor was removed chemically. Cathepsin D, a protein with no affinity for aerolysin, was converted to an aerolysin binding form when it was expressed as a GPI-anchored hybrid in COS cells. Not all GPI-anchored proteins bind aerolysin. In some cases this may be due to differences in the structure of the anchor itself. Thus the GPI-anchored proteins procyclin of Trypanosoma congolense and gp63 of Leishmania major did not bind aerolysin, but when gp63 was expressed with a mammalian GPI anchor in Chinese hamster ovary cells, it bound the toxin.
Glycosylphosphatidylinositol (GPI) anchors are commonly found in all eukaryotic cells. However, compared to mammalian cells, protozoan parasites express about one hundred times more GPI glycolipids per cell. GPIs are commonly employed by the parasites to anchor surface antigens on the extracellular membrane, although not protein-linked or free GPIs can also be found. Parasitic GPIs are believed to regulate the immune response of the host by protozoa. However, a detailed structure function relationship of GPIs has not been established due to the difficulties in obtaining sufficient quantities of homogeneous material. This review summarizes the structures of characterized parasitic GPIs and their roles in triggering host immune responses. We focus on the recent progress in the chemical synthesis of GPI anchors and application of synthetic materials for development of vaccines and glycan arrays.
Köpfe und Ketten: GPI‐Glycolipid‐Monoschichten an der Grenzfläche Luft/Flüssigkeit sind durch eine Ordnung ganzer Moleküle, wie mit GIXD beobachtet, charakterisiert. Starke Wasserstoffbrücken vermitteln die Ordnung der Kopfgruppen, die zur Bildung eines Übergitters führt. Diese Kräfte sind sehr wahrscheinlich der Schlüssel für die Wechselwirkungen von GPI‐verankerten Proteinen und/oder GPIs in Zellmembranen.
Komplexe posttranslationale Modifikation: Die chemische Synthese eines Cystein-modifizierten Glycosylphosphatidylinositol(GPI)-Ankers eröffnet den Zugang zu homogenem GPI-verankertem Prionprotein mithilfe der Ligation exprimierter Proteine (Expressed Protein Ligation; siehe Schema). Mit diesem Verfahren sollte es möglich sein, den Einfluss dieser komplexen posttranslationalen Modifikation auf die Proteinstruktur und -funktion zu untersuchen.
Die Vielfalt an kovalenten Proteinvarianten (das Proteom) übersteigt bei weitem die Zahl an Proteinen, die sich aus der Codierkapazität der DNA ableitet. Ursache für diese Diversität sind gerichtete posttranslationale Modifikationen. Unter den Enzymen, die solche Modifikationen ausführen, finden sich etwa 500 humane Proteinkinasen, 150 Proteinphosphatasen und 500 Proteasen. Die Hauptarten der kovalenten Proteinmodifikationen – Phosphorylierung, Acetylierung, Glycosylierung, Methylierung und Ubiquitinierung – können nach der modifizierten Aminosäureseitenkette, dem modifizierenden Enzym und dem Grad an Reversibilität klassifiziert werden. Auch chemische Ereignisse wie das Proteinspleißen, die Reifung des grünen Fluoreszenzproteins und die Selbstaktivierung des Proteasoms zählen zu den posttranslationalen Modifikationen. Ein gründliches Verständnis von Umfang und Muster der posttranslationalen Modifikationen vermittelt Einblicke in die Funktion und Dynamik eukaryotischer Proteomzusammensetzungen.
More than 100 mammalian proteins are post-translationally modified by glycosylphosphatidylinositol (GPI) at their C-termini and are anchored to the cell surface membrane via the lipid portion. GPI-anchored proteins (GPI-APs) have various functions, such as hydrolytic enzymes, receptors, adhesion molecules, complement regulatory proteins and other immunologically important proteins. GPI-anchored proteins are mainly associated with membrane microdomains or membrane rafts enriched in sphingolipids and cholesterol. It is thought that association with membrane rafts is important for GPI-APs in signal transduction and other functions. Here, we review recent progress in studies on biosynthesis, remodelling and functions of mammalian GPI-APs.
Glycosylphosphatidylinositols (GPIs) are a class of natural glycosylphospholipids that anchor proteins, glycoproteins and lipophosphoglycans to the membrane of eukaryotic cells. GPI anchors are widely present in parasitic protozoa, where GPI-anchored mucins and phosphoglycans are abundant and form a dense protective layer (glycocalyx) on the surface of the parasites. This type of anchor appears to be present in these organisms with a much higher frequency than in higher eukaryotes. Since the first full assignment of a GPI structure in 1988, more than 50 glycosylphosphatidylinositols have been structurally characterised. The functions of GPI anchors (in addition to the clear one of linking the above biopolymers to membranes) have been extensively discussed. The high lateral mobility of GPIs and GPI-anchored polymers seems to actively facilitate the selective release of molecules from the cell surface and the exchange of membrane proteins between cells. There is also evidence that GPIs and/ or their metabolites can act as secondary messengers, modulating biological events including insulin production, insulin-mediated signal transduction, cellular proliferation and cell-cell recognition. Their discovered role as mediators of regulatory processes makes the chemical preparation of these compounds and their analogues of great interest. This comprehensive review highlights the progress in the chemical synthesis of GPI anchors and related glycoconjugate structures from protozoan parasites, yeast and mammals in the last two decades. The synthesis of a structurally related prokaryotic glycoconjugate of Mycobacterium tuberculosis is also discussed.
Several peptides/small proteins and glycosylphosphatidylinositol (GPI) derivatives were synthesized and compared as substrates of sortase A (SrtA), a bacterial transpeptidase, for enzymatic coupling. It was observed that peptides containing the LPKTGGS and LPKTGGRS sequences as sorting signals at the peptide C-terminus were effectively coupled to GPI derivatives having one or two glycine residues attached to the phosphoethanolamine group at the nonreducing end. This reaction was employed to prepare several analogues of the human CD52 and CD24 antigens, which are naturally GPI-anchored glycopeptides/glycoproteins. It was further observed that the trisaccharide GPI analogues 5 and 6 were better SrtA substrates than monosaccharide GPI analogue 4, suggesting that steric hindrance of the GPI analogues does not affect their peptidation reaction mediated by SrtA. Therefore, this synthetic strategy may be useful for the preparation of more complex GPI-anchored peptides, glycopeptides, proteins, and glycoproteins.
A class of membrane molecules has been identified whose primary translation product includes a COOH-terminal protein sequence that signals attachment of a glycosyl-phosphatidylinositol anchor at a COOH-terminal residue that is newly formed by cleavage of the signaling sequence. This class includes a wide diversity of protein types from eukaryotes at many stages of evolution. The structures of the glycosyl-phosphatidylinositol anchors are being resolved, but their functions aside from membrane attachment and dynamics remain to be determined.
Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification.