Sortase A-mediated chemoenzymatic synthesis of complex glycosylphosphatidylinositol-anchored protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Wu Z
PROVIDER: S-EPMC3876033 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
Publications
Sortase A-mediated chemoenzymatic synthesis of complex glycosylphosphatidylinositol-anchored protein.
Wu Zhimeng Z Guo Xueqing X Gao Jian J Guo Zhongwu Z
Chemical communications (Cambridge, England) 20131201 99
Green fluorescent protein and a glycosylphosphatidylinositol (GPI) anchor containing the common core structure and a lipid chain were synthesized and then coupled together in the promotion of bacterial sortase A (SrtA), which was the first example for the synthesis of a full-size GPI-anchored protein by SrtA, demonstrating that this can be a generally useful method for GPI-anchored protein synthesis. ...[more]
Similar Datasets
Project description:MUC1 glycopeptide was efficiently coupled to glycosylphosphatidylinositol (GPI) derivatives by sortase A (SrtA), verifying that SrtA can accept sterically hindered glycopeptide as substrate for ligation with GPIs. This work has established a practical method for the chemoenzymatic synthesis of GPI-linked glycopeptides.
| S-EPMC3166627 | biostudies-literature
Project description: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.
| S-EPMC8609528 | biostudies-literature
Project description:Glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive posttranslational modifications and remodeling, including the addition and subsequent removal of phosphoethanolamine (EtNP) from mannose 1 (Man1) and mannose 2 (Man2) of the glycan moiety. Removal of EtNP from Man1 is catalyzed by Cdc1p, an event that has previously been considered to occur in the endoplasmic reticulum (ER). We establish that Cdc1p is in fact a cis/medial Golgi membrane protein that relies on the COPI coatomer for its retention in this organelle. We also determine that Cdc1p does not cycle between the Golgi and the ER, and consistent with this finding, when expressed at endogenous levels ER-localized Cdc1p-HDEL is unable to support the growth of cdc1? cells. Our cdc1 temperature-sensitive alleles are defective in the transport of a prototypical GPI-AP-Gas1p to the cell surface, a finding we posit reveals a novel Golgi-localized quality control warrant. Thus, yeast cells scrutinize GPI-APs in the ER and also in the Golgi, where removal of EtNP from Man2 (via Ted1p in the ER) and from Man1 (by Cdc1p in the Golgi) functions as a quality assurance signal.
| S-EPMC7927193 | biostudies-literature
Project description:Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity.In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha.PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.
| S-EPMC2628761 | biostudies-literature
Project description:Cold acclimation results in changes of the plasma membrane (PM) composition. The PM is considered to contain specific lipid/protein-enriched microdomains which can be extracted as detergent-resistant plasma membrane (DRM). Previous studies in animal cells have demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be targeted to microdomains and/or the apoplast. However, the functional significance of GPI-APs during cold acclimation in plants is not yet fully understood. In this study, we aimed to investigate the responsiveness of GPI-APs to cold acclimation treatment in Arabidopsis We isolated the PM, DRM, and apoplast fractions separately and, in addition, GPI-AP-enriched fractions were prepared from the PM preparation. Label-free quantitative shotgun proteomics identified a number of GPI-APs (163 proteins). Among them, some GPI-APs such as fasciclin-like arabinogalactan proteins and glycerophosphoryldiester phosphodiesterase-like proteins predominantly increased in PM- and GPI-AP-enriched fractions while the changes of GPI-APs in the DRM and apoplast fractions during cold acclimation were considerably different from those of other fractions. These proteins are thought to be associated with cell wall structure and properties. Therefore, this study demonstrated that each GPI-AP responded to cold acclimation in a different manner, suggesting that these changes during cold acclimation are involved in rearrangement of the extracellular matrix including the cell wall towards acquisition of freezing tolerance.
| S-EPMC5014161 | biostudies-literature
Project description:To define the ability of GPIHBP1 to bind other lipase family members and other apolipoproteins (apos) and lipoproteins.GPIHBP1, a GPI-anchored lymphocyte antigen (Ly)6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1, but other members of the lipase family do not. We also examined the binding of apoAV-phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1's amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind high-density lipoprotein. Chylomicrons bind avidly to GPIHBP1-transfected Chinese hamster ovary cells, but this binding is dependent on GPIHBP1's ability to bind LPL within the cell culture medium.GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but does not bind apoAI or high-density lipoprotein. The ability of GPIHBP1-transfected Chinese hamster ovary cells to bind chylomicrons is mediated by LPL; chylomicron binding does not occur unless GPIHBP1 first captures LPL from the cell culture medium.
| S-EPMC3004026 | biostudies-literature
Project description:Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (?)thioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an (?)thioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.
| S-EPMC3465687 | biostudies-literature
Project description:Glycosylphosphatidylinositol-anchored proteins (GPI-APs), which are anchored at the outer leaflet of plasma membranes (PM) only by a carboxy-terminal GPI glycolipid, are known to fulfill multiple enzymic and receptor functions at the cell surface. Previous studies revealed that full-length GPI-APs with the complete GPI anchor attached can be released from and inserted into PMs in vitro. Moreover, full-length GPI-APs were recovered from serum, dependent on the age and metabolic state of rats and humans. Here, the possibility of intercellular control of metabolism by the intercellular transfer of GPI-APs was studied. Mutant K562 erythroleukemia (EL) cells, mannosamine-treated human adipocytes and methyl-ß-cyclodextrin-treated rat adipocytes as acceptor cells for GPI-APs, based on their impaired PM expression of GPI-APs, were incubated with full-length GPI-APs, prepared from rat adipocytes and embedded in micelle-like complexes, or with EL cells and human adipocytes with normal expression of GPI-APs as donor cells in transwell co-cultures. Increases in the amounts of full-length GPI-APs at the PM of acceptor cells as a measure of their transfer was assayed by chip-based sensing. Both experimental setups supported both the transfer and upregulation of glycogen (EL cells) and lipid (adipocytes) synthesis. These were all diminished by serum, serum GPI-specific phospholipase D, albumin, active bacterial PI-specific phospholipase C or depletion of total GPI-APs from the culture medium. Serum inhibition of both transfer and glycogen/lipid synthesis was counteracted by synthetic phosphoinositolglycans (PIGs), which closely resemble the structure of the GPI glycan core and caused dissociation of GPI-APs from serum proteins. Finally, large, heavily lipid-loaded donor and small, slightly lipid-loaded acceptor adipocytes were most effective in stimulating transfer and lipid synthesis. In conclusion, full-length GPI-APs can be transferred between adipocytes or between blood cells as well as between these cell types. Transfer and the resulting stimulation of lipid and glycogen synthesis, respectively, are downregulated by serum proteins and upregulated by PIGs. These findings argue for the (patho)physiological relevance of the intercellular transfer of GPI-APs in general and its role in the paracrine vs. endocrine (dys)regulation of metabolism, in particular. Moreover, they raise the possibility of the use of full-length GPI-APs as therapeutics for metabolic diseases.
| S-EPMC9267055 | biostudies-literature
Project description:Phosphorylation of S403 or S407 of the autophagic receptor protein p62 has recently been discovered to enhance the binding of p62 with ubiquitinated protein substrates to upregulate selective autophagy. To elucidate the molecular mechanism of how phosphorylation regulates the recruitment of ubiquitinated proteins, we report the first chemical synthesis of homogeneously phosphorylated p62, which enables the setting up of accurate in vitro systems for biochemical studies. Our synthesis employs the technology of sortase A-mediated protein hydrazide ligation, which successfully affords three types of phosphorylated p62 at the multi-milligram scale. Quantitative biochemical measurements show that the binding affinity of S403/S407-bisphosphorylated p62 to K63 diubiquitin is significantly higher than that of mono-phosphorylated p62. This finding suggests that phosphorylated S403 and S407 sites should bind to different epitopes on the ubiquitin chain. Furthermore, glutamate mutation is found to give a significantly impaired binding affinity, implying the necessity of using chemically synthesized phosphorylated p62 for the biochemical study of selective autophagy.
| S-EPMC5636944 | biostudies-literature
Project description:Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site-specific fashion. This reaction is applicable to proteins in solution and on the living cell surface. The method described in this unit only requires incubation of the target protein, which has been engineered to contain a sortase recognition site either at the C terminus or within solvent-accessible loops, with a purified sortase enzyme and a suitably functionalized oligoglycine peptide.
| S-EPMC5551486 | biostudies-other