RESUMEN
Glycosylphosphatidylinositol (GPI) anchoring of proteins is a ubiquitous posttranslational modification in eukaryotic cells. GPI-anchored proteins (GPI-APs) play critical roles in enzymatic, signaling, regulatory, and adhesion processes. Over 20 enzymes are involved in GPI synthesis, attachment to client proteins, and remodeling after attachment. The GPI transamidase (GPI-T), a large complex located in the endoplasmic reticulum membrane, catalyzes the attachment step by replacing a C-terminal signal peptide of proproteins with GPI. In the last three decades, extensive research has been conducted on the mechanism of the transamidation reaction, the components of the GPI-T complex, the role of each subunit, and the substrate specificity. Two recent studies have reported the three-dimensional architecture of GPI-T, which represent the first structures of the pathway. The structures provide detailed mechanisms for assembly that rationalizes previous biochemical results and subunit-dependent stability data. While the structural data confirm the catalytic role of PIGK, which likely uses a caspase-like mechanism to cleave the proproteins, they suggest that unlike previously proposed, GPAA1 is not a catalytic subunit. The structures also reveal a shared cavity for GPI binding. Somewhat unexpectedly, PIGT, a single-pass membrane protein, plays a crucial role in GPI recognition. Consistent with the assembly mechanisms and the active site architecture, most of the disease mutations occur near the active site or the subunit interfaces. Finally, the catalytic dyad is located ~22 Å away from the membrane interface of the GPI-binding site, and this architecture may confer substrate specificity through topological matching between the substrates and the elongated active site. The research conducted thus far sheds light on the intricate processes involved in GPI anchoring and paves the way for further mechanistic studies of GPI-T.
Asunto(s)
Glicosilfosfatidilinositoles , Humanos , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/química , Animales , Especificidad por Sustrato , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Retículo Endoplásmico/metabolismo , Relación Estructura-Actividad , AciltransferasasRESUMEN
GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation of live cells. This new, enhanced GPI-AP detection method has been utilized to successfully explore GPI-AP engineering, analyze GPI-APs, and profile GPI-AP expression in different cells.
Asunto(s)
ADN , Hibridación de Ácido Nucleico , Humanos , ADN/química , Proteínas Ligadas a GPI/metabolismo , Animales , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/química , Colorantes Fluorescentes/química , Azidas/químicaRESUMEN
In malaria parasites, although post-translational modification of proteins with N-. O-, and C-glycosidic bond-linked glycans is limited, it is confined to relatively fewer proteins in which the glycans are present at significant levels and may have important functions. Furthermore, several proteins are modified with glycosylphosphatidylinositols (GPIs) which represent the predominant glycan synthesized by parasites. Modification of proteins with GPIs is obligatory for parasite survival as GPI-anchored proteins (GPI-APs) play essential roles in all life cycle stages of the parasites, including development, egress, gametogenesis, motility, and host cell adhesion and invasion. Here, we discuss the current knowledge on the structures and potential functions of the glycan moieties of parasite proteins. The knowledge has important implications for the development of drugs and vaccines for malaria.
Asunto(s)
Parásitos , Animales , Glicosilación , Parásitos/metabolismo , Plasmodium falciparum , Procesamiento Proteico-Postraduccional , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Polisacáridos/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates the inositol in nascent GPI-APs. Impairment of PGAP1 activity leads to developmental diseases in humans and fatality and infertility in animals. Here, we present three PGAP1 structures (2.66-2.84 Å), revealing its 10-transmembrane architecture and product-enzyme interaction details. PGAP1 holds GPI-AP acyl chains in an optimally organized, guitar-shaped cavity with apparent energetic penalties from hydrophobic-hydrophilic mismatches. However, abundant glycan-mediated interactions in the lumen counterbalance these repulsions, likely conferring substrate fidelity and preventing off-target hydrolysis of bulk membrane lipids. Structural and biochemical analyses uncover a serine hydrolase-type catalysis with atypical features and imply mechanisms for substrate entrance and product release involving a drawing compass movement of GPI-APs. Our findings advance the mechanistic understanding of GPI-AP remodeling.
Asunto(s)
Inositol , Proteínas de la Membrana , Animales , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Inositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Hidrolasas , Control de Calidad , Glicosilfosfatidilinositoles/químicaRESUMEN
Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag. It was disclosed that this probe was more selective than our previously reported probe containing only a part structure of the GPI core for cell membrane incorporation and an improved probe for studying GPI-cell membrane interaction. Eighty-eight unique membrane proteins, many of which are related to GPIs/GPI-anchored proteins, were identified utilizing this probe. The proteomics dataset is a valuable resource for further analyses and data mining to find new GPI-related proteins and signalling pathways. A comparison of these results with those of our previous probe provided direct evidence for the profound impact of GPI glycan structure on its interaction with the cell membrane.
Asunto(s)
Glicosilfosfatidilinositoles , Polisacáridos , Glicosilfosfatidilinositoles/química , Membrana Celular/metabolismo , Polisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de SeñalRESUMEN
Many precursors of plant arabinogalactan proteins (AGPs) contain a C-terminal glycosylphosphatidylinositol (GPI)-anchoring signal. Using NtAGP1, a classical tobacco AGP, as a model, and green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags, we analyzed the localization and modification of AGP and its mutant without GPI-anchoring signal (AGPΔC) in tobacco BY-2 cells. The NtAGP1 fusion proteins migrated as large smear on SDS-polyacrylamide gel, and these proteins also localized preferentially to the plasma membrane. In contrast, fusions of AGPΔC with GFP and SPO yielded several forms: The largest were secreted, whereas others were recovered in the endomembrane organelles, including vacuoles. Comparison of the glycan structures of the microsomal SPO-AGP and the secreted SPO-AGPΔC using antibodies against the glycan epitopes of AGP indicated that the glycan structures of these proteins are different. These observations indicate that GPI-anchoring is required for the proper transport and glycosylation of the AGP precursor.
Asunto(s)
Glicosilfosfatidilinositoles , Nicotiana , Nicotiana/genética , Nicotiana/metabolismo , Glicosilación , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Secuencia de Aminoácidos , Proteínas de Plantas/metabolismoRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are regarded as the main (patho)physiological roles exerted by GPI-APs. The intercellular transfer of GPI-APs relies on the complete GPI anchor and is mediated by extracellular vesicles such as microvesicles and exosomes and lipid-free homo- or heteromeric aggregates, and lipoprotein-like particles such as prostasomes and surfactant-like particles, or lipid-containing micelle-like complexes. In mammalian organisms, non-vesicular transfer is controlled by the distance between donor and acceptor cells/tissues; intrinsic conditions such as age, metabolic state, and stress; extrinsic factors such as GPI-binding proteins; hormones such as insulin; and drugs such as anti-diabetic sulfonylureas. It proceeds either "directly" upon close neighborhood or contact of donor and acceptor cells or "indirectly" as a consequence of the induced lipolytic release of GPI-APs from PMs. Those displace from the serum GPI-binding proteins GPI-APs, which have retained the complete anchor, and become assembled in aggregates or micelle-like complexes. Importantly, intercellular transfer of GPI-APs has been shown to induce specific phenotypes such as stimulation of lipid and glycogen synthesis, in cultured human adipocytes, blood cells, and induced pluripotent stem cells. As a consequence, intercellular transfer of GPI-APs should be regarded as non-genetic inheritance of (acquired) features between somatic cells which is based on the biogenesis and transmission of matter such as GPI-APs and "membrane landscapes", rather than the replication and transmission of information such as DNA. Its operation in mammalian organisms remains to be clarified.
Asunto(s)
Glicosilfosfatidilinositoles , Micelas , Animales , Humanos , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Lipólisis , Membrana Celular/metabolismo , Glucolípidos/metabolismo , Mamíferos/metabolismoRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).
Asunto(s)
Glicosilfosfatidilinositoles , Proteínas de la Membrana , Animales , Glicosilfosfatidilinositoles/análisis , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Glucolípidos/metabolismo , Proteolisis , Mamíferos/metabolismoRESUMEN
GPI anchoring is an essential post-translational modification in eukaryotes that links proteins to the plasma membrane. In this issue, Liu et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208159) suggest, for the first time, a regulation on demand of the GPI glycolipid precursor biosynthesis.
Asunto(s)
Glicosilfosfatidilinositoles , Procesamiento Proteico-Postraduccional , Membrana Celular , Glucolípidos/biosíntesis , Glucolípidos/química , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/químicaRESUMEN
Glycosylphosphatidylinositol (GPI) anchorage of cell surface proteins to the membrane is biologically important and ubiquitous in eukaryotes. However, GPIs do not contain long enough lipids to span the entire membrane bilayer. To transduce binding signals, GPIs must interact with other membrane components, but such interactions are difficult to define. Here, a new method was developed to explore GPI-interacting membrane proteins in live cell with a bifunctional analogue of the glucosaminylphosphatidylinositol motif conserved in all GPIs as a probe. This probe contained a diazirine functionality in the lipid and an alkynyl group on the glucosamine residue to respectively facilitate the cross-linkage of GPI-binding membrane proteins with the probe upon photoactivation and then the installation of biotin to the cross-linked proteins via a click reaction for affinity-based protein isolation and analysis. Profiling the proteins pulled down from the Hela cells revealed 94 unique and 18 overrepresented proteins compared to the control, and most of them are membrane proteins and many are GPI-related. The results have proved not only the concept of using the new bifunctional GPI probe to investigate GPI-binding membrane proteins but also the important role of inositol in the biological functions of GPI anchors and GPI-anchored proteins.
Asunto(s)
Glicosilfosfatidilinositoles , Proteínas de la Membrana , Humanos , Glicosilfosfatidilinositoles/análisis , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Células HeLa , Membrana Celular/química , Proteínas de la Membrana/metabolismoRESUMEN
A new, bifunctional glycosylphosphatidylinositol (GPI) derivative containing the highly conserved core structure of all natural GPI anchors with a photoactivable diazirine in the lipid chain and clickable alkynes in the glycan was synthesized by a convergent [3+2] glycosylation strategy with late stage protecting group manipulation and regioselective phosphorylation. The challenges of this synthesis were due to the presence of several distinctive functional groups in the synthetic target, which complicated the protection tactics, in addition to the inherent difficulties associated with GPI synthesis. This bifunctional GPI derivative can cross-react with molecules in proximity upon photoactivation and be subsequently labeled with other molecular tags via click reaction. Therefore, it should be a valuable probe for biological studies of GPIs, such as analysis of GPI-interacting membrane proteins, and gaining insights into their functional mechanisms.
Asunto(s)
Glicosilfosfatidilinositoles , Proteínas de la Membrana , Glicosilfosfatidilinositoles/química , Proteínas de la Membrana/metabolismo , Glicosilación , Fosforilación , BiologíaRESUMEN
Carbohydrates have quite complicated micro heterogenic structure which may undergo different structural transitions. Due to their extreme flexibility it is very difficult to investigate such structural changes experimentally. In these studies we want to predict what structural and conformational changes are possible in the carbohydrate glycosylphosphatidylinositol anchor chain (GPI): the tetrasaccharide with the unique sequence Man-α(1â¯ââ¯2)-Man-α(1â¯ââ¯6)-Man-α(1â¯ââ¯4)-GlcN-α. This is a very important biomolecule associated with the processes of transmitting various types of signals in the cells of living organisms. In order to investigate conformational and structural changes in GPI we use in these studies the theoretical Enforced Geometry Optimization (EGO) method. In this method a molecule is exposed to a mechanical stress caused by external forces applied to selected atoms. It turned out that under external stretching forces the mannopyranose unit can change its 4C1 chair conformation into three different forms: 1S3, oS2 and B2,5. The initial 4C1 glucosamine ring can transit into the twisted boat 1S3 and the boat Bo,3 conformations. The obtained results confirm the high flexibility of the GPI anchor sugar chain.
Asunto(s)
Carbohidratos , Glicosilfosfatidilinositoles , Humanos , Glicosilfosfatidilinositoles/química , Secuencia de Carbohidratos , Estrés Mecánico , Manosa/química , Conformación de CarbohidratosRESUMEN
Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors. These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated. GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others. All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers. Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent. This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses. Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer. This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases.
Asunto(s)
Glicosilfosfatidilinositoles , Simulación de Dinámica Molecular , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Glucolípidos , Polisacáridos , Proteínas Ligadas a GPIRESUMEN
Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.
Asunto(s)
Retículo Endoplásmico , Proteínas Ligadas a GPI , Glicosilfosfatidilinositoles , Señales de Clasificación de Proteína , Humanos , Retículo Endoplásmico/metabolismo , Glicosilación , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Priones/química , Priones/metabolismo , Transporte de ProteínasRESUMEN
A bifunctional derivative of the core structure of glycosylphosphatidylinositol (GPI) anchors having a clickable alkynyl group and a photoreactive diazirine group attached to the GPI glucosamine and lipid moieties, respectively, was synthesized from myo-inositol, d-glucosamine, and (R)-1,2-O-acetonized glycerol. The target molecule should be useful for the investigation of GPI-interacting components in the cell membrane that play a key role in the signal transduction and other biological functions of GPI-anchored proteins.
Asunto(s)
Glicosilfosfatidilinositoles , Proteínas , Glucosamina , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Inositol/química , Proteínas/químicaRESUMEN
Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. The removal of a hydrophobic signal peptide and covalent attachment of GPI at the new carboxyl terminus are catalyzed by an endoplasmic reticulum membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GPI-T at a global 2.53-Å resolution, revealing an equimolar heteropentameric assembly. Structure-based mutagenesis suggests a legumain-like mechanism for the recognition and cleavage of proprotein substrates, and an endogenous GPI in the structure defines a composite cavity for the lipid substrate. This elongated active site, stemming from the membrane and spanning an additional ~22-Å space toward the catalytic dyad, is structurally suited for both substrates which feature an amphipathic pattern that matches this geometry. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.
Asunto(s)
Glicosilfosfatidilinositoles , Proteínas , Microscopía por Crioelectrón , Retículo Endoplásmico/metabolismo , Glicosilfosfatidilinositoles/química , Humanos , Señales de Clasificación de ProteínaRESUMEN
Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)-galactose-sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.
Asunto(s)
Acetilgalactosamina , Glicosilfosfatidilinositoles , Enfermedades por Prión , Priones , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animales , Encéfalo/metabolismo , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Ratones , Osteogénesis , Enfermedades por Prión/metabolismo , Priones/metabolismo , Relación Estructura-ActividadRESUMEN
The attachment of proteins to the cell membrane using a glycosylphosphatidylinositol (GPI) anchor is a ubiquitous process in eukaryotic cells. Deficiencies in the biosynthesis of GPIs and the concomitant production of GPI-anchored proteins lead to a series of rare and complicated disorders associated with inherited GPI deficiencies (IGDs) in humans. Currently, there is no treatment for patients suffering from IGDs. Here, we report the design, synthesis, and use of GPI fragments to rescue the biosynthesis of GPI-anchored proteins (GPI-APs) caused by mutation in genes involved in the assembly of GPI-glycolipids in cells. We demonstrated that the synthetic fragments GlcNAc-PI (1), Man-GlcN-PI (5), and GlcN-PI with two (3) and three lipid chains (4) rescue the deletion of the GPI biosynthesis in cells devoid of the PIGA, PIGL, and PIGW genes in vitro. The compounds allowed for concentration-dependent recovery of GPI biosynthesis and were highly active on the cytoplasmic face of the endoplasmic reticulum membrane. These synthetic molecules are leads for the development of treatments for IGDs and tools to study GPI-AP biosynthesis.
Asunto(s)
Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Biosíntesis de Proteínas , Conformación de Carbohidratos , Eliminación de Gen , Células HEK293 , Humanos , PiridoxinaRESUMEN
In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.