RESUMO
Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell-pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected ß1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel ß1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal ß1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.
Assuntos
Polissacarídeos , Xanthomonas , alfa-Manosidase , beta-Galactosidase , Galactose/metabolismo , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Manose , Manosídeos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Xanthomonas/enzimologia , alfa-Manosidase/metabolismo , beta-Galactosidase/metabolismoRESUMO
Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that plays a crucial anaplerotic role in central plant metabolism. Bacterial-type PEPC (BTPC) of developing castor oil seeds (COS) is highly expressed as a catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex. Ricinus communis Ca2+-dependent protein kinase-1 (RcCDPK1) catalyzes in vivo inhibitory phosphorylation of COS BTPC at Ser451. Autokinase activity of recombinant RcCDPK1 was detected and 42 autophosphorylated Ser, Thr or Tyr residues were mapped via liquid chromatography-tandem mass spectrometry. Prior autophosphorylation markedly attenuated the ability of RcCDPK1 to transphosphorylate its BTPC substrate at Ser451. However, fully dephosphorylated RcCDPK1 rapidly autophosphorylated during the initial stages of a BTPC transphosphorylation assay. This suggests that Ca2+-dependent binding of dephospho-RcCDPK1 to BTPC may trigger a structural change that leads to rapid autophosphorylation and subsequent substrate transphosphorylation. Tyr30 was identified as an autophosphorylation site via LC-MS/MS and immunoblotting with a phosphosite-specific antibody. Tyr30 occurs at the junction of RcCDPK1's N-terminal variable (NTVD) and catalytic domains and is widely conserved in plant and protist CDPKs. Interestingly, a reduced rate and extent of BTPC transphosphorylation occurred with a RcCDPK1Y30F mutant. Prior research demonstrated that RcCDPK1's NTVD is essential for its Ca2+-dependent autophosphorylation or BTPC transphosphorylation activities but plays no role in target recognition. We propose that Tyr30 autophosphorylation facilitates a Ca2+-dependent interaction between the NTVD and Ca2+-activation domain that primes RcCDPK1 for transphosphorylating BTPC at Ser451. Our results provide insights into links between the post-translational control of COS anaplerosis, Ca2+-dependent signaling and the biological significance of RcCDPK1 autophosphorylation.
Assuntos
Fosfoenolpiruvato Carboxilase , Ricinus communis , Bactérias/metabolismo , Cálcio/metabolismo , Ricinus communis/metabolismo , Óleo de Rícino/metabolismo , Cromatografia Líquida , Fosfoenolpiruvato Carboxilase/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Ricinus/metabolismo , Sementes/metabolismo , Espectrometria de Massas em TandemRESUMO
Characterization of the structural diversity of glycans by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains an analytical challenge in large-scale glycomics applications because of the presence of heterogeneous composition, ubiquitous isomers, lability of post-translational glycan modifications, and complexity of data interpretation. High-resolution separation of glycan isomers differentiating from positional, linkage, branching, and anomeric structures is often a prerequisite to ensure the comprehensive glycan identification. Here, we developed a straightforward method using self-packed capillary porous graphitic carbon (PGC) columns for nanoflow LC-MS/MS analyses of native glycans released from glycoproteins. The technique enables highly resolved chromatographic separation of over 20 high-mannose glycan isomers in ribonuclease B and a diverse range of hybrid and complex-type sialoglycoforms of fetuin. The distinct structures of anomeric glycans and linkage sialoglycan isomers, α2,3 and α2,6, were identified by the characteristic MS/MS fragment ions. A glycan sequencing strategy utilizing diagnostic ions and complementary fragments specific to branching residues was established to simplify the MS/MS data interpretation of closely related isomeric structures. To promote the PGC-LC-MS/MS-based method for glycome-wide applications, we extended analyses to native sulfoglycans from the egg-propagated and cell culture-derived influenza vaccines and demonstrate the high-resolution separation and structural characterization of underivatized neutral and anionic glycoforms including oligomannosidic glycan anomers, sialoglycan linkage isomers, and regioisomers of afucosylated and fucosylated sulfoglycans containing sulfated-6-GlcNAc and sulfated-4-GalNAc residues.
Assuntos
Glicoproteínas/química , Grafite/química , Polissacarídeos/análise , Cromatografia Líquida , Glicômica , Glicosilação , Isomerismo , Estrutura Molecular , Porosidade , Espectrometria de Massas em TandemRESUMO
Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions.
Assuntos
Regulação da Expressão Gênica de Plantas , Magnaporthe , Oryza , Peptídeos , Transcriptoma , Magnaporthe/fisiologia , Oryza/genética , Oryza/imunologia , Oryza/microbiologia , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , ProteômicaRESUMO
Sulfated N-glycans are biologically important structures derived from enzymatically post-glycosylational modifications of glycoproteins in many therapeutic biologics. The high-throughput analysis of sulfated N-glycomes remains a daunting technical challenge, because of negatively charged heterogeneous composition, large molecular structures, lability of sulfate attachments, and a lack of highly selective enrichment methods. Using liquid chromatography-mass spectrometry, we have analyzed the N-glycans of influenza viral hemagglutinin and neuraminidase from several subtypes of influenza vaccines, and utilized the existing resource to establish an N-glycan library consisting of 927 N-glycan structures and 387 sulfated N-glycan compositions. With the aid of database for data mining, 1380 unique N-glycopeptides were identified and manually validated by de novo glycopeptide sequencing, of which 514 were sulfated at the site-specific locations. We report here a mass spectrometric method that is able to identify and distinguish the isobaric structures of complex and hybrid N-glycans flanked by a terminal sulfation sequon on Gal-GlcNAc and GalNAc-GlcNAc of sulfated-3-Gal, sulfated-6-GlcNAc, and sulfated-4-GalNAc. The database-aided glycoproteomic analyses enable rapid determination of new sulfated-N-glycan structures in large sets of influenza vaccines, including those highly branched nonsialyl sulfo-N-glycans bearing lactosaminic extensions in both complex and hybrid N-glycans that especially interact with sulfotransferases. The novel findings highlight the tremendous structural diversity of sulfated N-glycans and strongly suggest potential functional importance of N-glycan sulfation of influenza glycoproteins.
Assuntos
Vacinas contra Influenza/química , Polissacarídeos/química , Sulfatos/química , Sequência de CarboidratosRESUMO
The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn365 and Asn422 were modified in AtPAP26-S2 to form high-mannose glycans. A 55-kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr38 and Thr39 and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating posttranscriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.
Assuntos
Fosfatase Ácida/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Galactoquinase/metabolismo , Fosfatos/deficiência , Fosfatase Ácida/isolamento & purificação , Proteínas de Arabidopsis/isolamento & purificação , Células Cultivadas , Cromatografia em Gel , Dissacarídeos , Galactoquinase/isolamento & purificação , Glucuronatos , Fosfatos/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Regulação para CimaRESUMO
MAIN CONCLUSION: Hydrogen peroxide-responsive pathways in roots of alkaligrass analyzed by proteomic studies and PutGLP enhance the plant tolerance to saline-, alkali- and cadmium-induced oxidative stresses. Oxidative stress adaptation is critical for plants in response to various stress environments. The halophyte alkaligrass (Puccinellia tenuiflora) is an outstanding pasture with strong tolerance to salt and alkali stresses. In this study, iTRAQ- and 2DE-based proteomics approaches, as well as qRT-PCR and molecular genetics, were employed to investigate H2O2-responsive mechanisms in alkaligrass roots. The evaluation of membrane integrity and reactive oxygen species (ROS)-scavenging systems, as well as abundance patterns of H2O2-responsive proteins/genes indicated that Ca2+-mediated kinase signaling pathways, ROS homeostasis, osmotic modulation, and transcriptional regulation were pivotal for oxidative adaptation in alkaligrass roots. Overexpressing a P. tenuiflora germin-like protein (PutGLP) gene in Arabidopsis seedlings revealed that the apoplastic PutGLP with activities of oxalate oxidase and superoxide dismutase was predominantly expressed in roots and played important roles in ROS scavenging in response to salinity-, alkali-, and CdCl2-induced oxidative stresses. The results provide insights into the fine-tuned redox-responsive networks in halophyte roots.
Assuntos
Genes de Plantas/genética , Glicoproteínas/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Poaceae/metabolismo , Plantas Tolerantes a Sal/metabolismo , Arabidopsis/genética , Betaína/metabolismo , Glicoproteínas/genética , Malondialdeído/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Poaceae/genética , Prolina/metabolismo , Mapas de Interação de Proteínas , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Plantas Tolerantes a Sal/genéticaRESUMO
Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 µM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKß are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.
Assuntos
Óleo de Rícino/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas Quinases/metabolismo , Ricinus/enzimologia , Sementes/metabolismo , Sequência de Aminoácidos , Formação de Anticorpos , Sítios de Ligação , Biocatálise , Fenômenos Biofísicos , Cálcio/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/metabolismo , Mitocôndrias/metabolismo , Fosforilação , Fosfosserina/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/química , Ricinus/embriologia , Ricinus/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Phosphoenolpyruvate carboxylase (PEPC) is an important regulatory enzyme situated at a key branch point of central plant metabolism. Plant genomes encode several plant-type PEPC (PTPC) isozymes, along with a distantly related bacterial-type PEPC (BTPC). BTPC is expressed at high levels in developing castor oil seeds where it tightly interacts with co-expressed PTPC polypeptides to form unusual hetero-octameric Class-2 PEPC complexes that are desensitized to allosteric inhibition by L-malate. Analysis of RNA-Seq and microarray transcriptome datasets revealed two distinct patterns of tissue-specific BTPC expression in vascular plants. Species such as Arabidopsis thaliana, strawberry, rice, maize, and poplar mainly exhibited pollen- or floral-specific BTPC expression. By contrast, BTPC transcripts were relatively abundant in developing castor, cotton, and soybean seeds, cassava tubers, as well as immature tomato, cucumber, grape, and avocado fruit. Immunoreactive 118 kDa BTPC polypeptides were detected on immunoblots of cucumber and tomato fruit extracts. Co-immunoprecipitation established that as in castor, BTPCs physically interact with endogenous PTPCs to form Class-2 PEPC complexes in tomato and cucumber fruit. We hypothesize that Class-2 PEPCs simultaneously maintain rapid anaplerotic PEP carboxylation and respiratory CO2 refixation in diverse, biosynthetically active sinks that accumulate high malate levels.
Assuntos
Magnoliopsida/genética , Malatos/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Plantas/genética , Transcriptoma/genética , Perfilação da Expressão Gênica , Magnoliopsida/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca2+-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca2+-dependent Ser11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca2+ ions [K0.5(Ca2+) < 0.4â µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that â¼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser451, RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca2+-signaling and the control of photosynthate partitioning during COS development.
Assuntos
Óleo de Rícino/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Sementes/enzimologia , Sementes/metabolismo , Concentração de Íons de Hidrogênio , Microssomos/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , FosforilaçãoRESUMO
Hydrogen peroxide (H2O2) is one of the most abundant reactive oxygen species (ROS), which plays dual roles as a toxic byproduct of cell metabolism and a regulatory signal molecule in plant development and stress response. Populus simonii × Populus nigra is an important cultivated forest species with resistance to cold, drought, insect and disease, and also a key model plant for forest genetic engineering. In this study, H2O2 response in P. simonii × P. nigra leaves was investigated using physiological and proteomics approaches. The seedlings of 50-day-old P. simonii × P. nigra under H2O2 stress exhibited stressful phenotypes, such as increase of in vivo H2O2 content, decrease of photosynthetic rate, elevated osmolytes, antioxidant accumulation, as well as increased activities of several ROS scavenging enzymes. Besides, 81 H2O2-responsive proteins were identified in the poplar leaves. The diverse abundant patterns of these proteins highlight the H2O2-responsive pathways in leaves, including 14-3-3 protein and nucleoside diphosphate kinase (NDPK)-mediated signaling, modulation of thylakoid membrane structure, enhancement of various ROS scavenging pathways, decrease of photosynthesis, dynamics of proteins conformation, and changes in carbohydrate and other metabolisms. This study provides valuable information for understanding H2O2-responsive mechanisms in leaves of P. simonii × P. nigra.
Assuntos
Peróxido de Hidrogênio/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Populus/fisiologia , Proteínas 14-3-3/metabolismo , Metabolismo dos Carboidratos , Núcleosídeo-Difosfato Quinase/metabolismo , Fotossíntese , Proteômica , Transdução de Sinais , Estresse FisiológicoRESUMO
BACKGROUND: Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS: On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS: Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 µg or 1.465 × 10(7) cells) level and close correlation (r(2) > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS: MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.
Assuntos
Antígenos de Bactérias/análise , Antígenos de Bactérias/química , Escherichia coli/química , Flagelos/química , Espectrometria de Massas/métodos , Sorotipagem/métodos , Sorotipagem/normas , Antígenos de Bactérias/imunologia , Canadá , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Flagelos/imunologia , Sensibilidade e EspecificidadeRESUMO
N-glycosylation has a great impact on glycoprotein structure, conformation, stability, solubility, immunogenicity and enzyme activity. Structural characterization of N-glycoproteome has been challenging but can provide insights into the extent of protein folding and surface topology. We describe a highly sensitive proteomics method for large-scale identification and quantification of glycoproteins in Arabidopsis through (15) N-metabolic labeling, selective enrichment of glycopeptides, data-dependent MS/MS analysis and automated database searching. In-house databases of Arabidopsis glycoproteins and glycopeptides containing Asn-X-Ser/Thr/Cys motifs were constructed by reducing 20% and 90% of the public database size, respectively, to enable a rapid analysis of large datasets for comprehensive identification and quantification of glycoproteins and heterogeneous N-glycans in a complex mixture. Proteome-wide analysis identified c. 100 stress-related N-glycoproteins, of which the endoplasmic reticulum (ER) resident proteins were examined to be up-regulated. Quantitative measurements provided a molecular signature specific to glycoproteins for determining the degree of plant stress at low temperature. Structural N-glycoproteomics following time-course cold treatments revealed the stress-responsive degradation of high-mannose type N-glycans in ER in response to chilling stress, which may aid in elucidating the cellular mechanisms of protein relocation, transport, trafficking, misfolding and degradation under stress conditions.
Assuntos
Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Congelamento , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Proteólise , Estresse Fisiológico , Regulação para Cima , Sequência de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Temperatura Baixa , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Ensaios de Triagem em Larga Escala , Polissacarídeos/química , Reprodutibilidade dos TestesRESUMO
Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.
Assuntos
Fosfoenolpiruvato Carboxilase/genética , Proteínas de Plantas/genética , Sorghum/genética , Germinação , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fosforilação , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sorghum/enzimologia , Sorghum/crescimento & desenvolvimento , Espectrometria de Massas em Tandem , UbiquitinaçãoRESUMO
The Jk(a-b-) phenotype, referred to as Jknull, is rare in most populations. This blood type is characterized by the absence of Kidd glycoprotein on the surface of red blood cells (RBCs) and moderately reduced ability to concentrate urine. The molecular basis for Jknull phenotype includes splice-site mutations, missense mutations, and a partial gene deletion in the JK(SLC14A1) gene that encodes the human urea transporter protein. In this study, we have analyzed 10 Chinese Jknull samples to determine their molecular bases. In addition to the well known Polynesian Jknull allele, three Jknull alleles were detected including one novel Jknull allele: JKA (130A, 220G).
Assuntos
Alelos , Genes Recessivos , Sistema do Grupo Sanguíneo Kidd/genética , Proteínas de Membrana Transportadoras/genética , Povo Asiático , China , Feminino , Humanos , Masculino , Transportadores de UreiaRESUMO
Sucrose synthase (SUS) catalyzes the UDP-dependent cleavage of sucrose into UDP-glucose and fructose and has become an important target for improving seed crops via metabolic engineering. A UDP-specific SUS homotetramer composed of 93-kDa subunits was purified to homogeneity from the triacylglyceride-rich endosperm of developing castor oil seeds (COS) and identified as RcSUS1 by mass spectrometry. RcSUS1 transcripts peaked during early development, whereas levels of SUS activity and immunoreactive 93-kDa SUS polypeptides maximized during mid-development, becoming undetectable in fully mature COS. The cytosolic location of the enzyme was established following transient expression of RcSUS1-enhanced YFP in tobacco suspension cells and fluorescence microscopy. Immunological studies using anti-phosphosite-specific antibodies revealed dynamic and high stoichiometric in vivo phosphorylation of RcSUS1 at its conserved Ser-11 residue during COS development. Incorporation of (32)P(i) from [γ-(32)P]ATP into a RcSUS1 peptide substrate, alongside a phosphosite-specific ELISA assay, established the presence of calcium-dependent RcSUS1 (Ser-11) kinase activity. Approximately 10% of RcSUS1 was associated with COS microsomal membranes and was hypophosphorylated relative to the remainder of RcSUS1 that partitioned into the soluble, cytosolic fraction. Elimination of sucrose supply caused by excision of intact pods of developing COS abolished RcSUS1 transcription while triggering the progressive dephosphorylation of RcSUS1 in planta. This did not influence the proportion of RcSUS1 associated with microsomal membranes but instead correlated with a subsequent marked decline in SUS activity and immunoreactive RcSUS1 polypeptides. Phosphorylation at Ser-11 appears to protect RcSUS1 from proteolysis, rather than influence its kinetic properties or partitioning between the soluble cytosol and microsomal membranes.
Assuntos
Glucosiltransferases/metabolismo , Membranas Intracelulares/enzimologia , Microssomos/enzimologia , Proteínas de Plantas/metabolismo , Ricinus communis/enzimologia , Sementes/enzimologia , Fosforilação/fisiologia , ProteóliseRESUMO
Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up-regulated in the cell walls of phosphate (Pi)-starved (-Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of -Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil-localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under -Pi conditions. Coupled with potent mixed-type inhibition of AtPAP25 by Pi (I50 = 50 µm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter-GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of -Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT-PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non-specific scavenger of Pi from extracellular P-monoesters.
Assuntos
Fosfatase Ácida/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fósforo/metabolismo , Aclimatação , Fosfatase Ácida/genética , Adaptação Fisiológica , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Glicoproteínas/metabolismo , Glicosilação , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras GenéticasRESUMO
WWP2 (WW domain-containing protein 2) is an E3 ubiquitin ligase belonging to the NEDD4-like protein family involved in various cell regulations, such as carcinogenesis, transcription control and cellular transport. Compared with homologues, WWP2 is difficult to express and no practical protocols have been developed for WWP2 preparation in large scale. Recently, domain structures of homologues of WWP2 have been determined by crystallography and NMR, but none for WWP2 has been attained. In this work, through a combination of extensive screening of â¼100 constructs, expression strategies and host systems, we have found a soluble HECT domain truncation (WHP2) of WWP2 which is amendable for preparation scale expression in Escherichia coli. We have also established a relatively simple purification process to achieve highly pure WHP2 protein by employing immobilized metal-affinity chromatography followed by salting out, ion exchange chromatography and finally, size exclusion chromatography. We are able to obtain about 60mg/L of the soluble WHP2. The identity and structure of the expressed WHP2 have been analyzed by mass spectrometry and circular dichroism. The native ability of WHP2 to bind different partners has been revealed by pull-down assay.
Assuntos
Corpos de Inclusão/química , Plasmídeos/química , Proteínas Recombinantes de Fusão/química , Ubiquitina-Proteína Ligases/química , Sequência de Aminoácidos , Sulfato de Amônio/química , Cromatografia/métodos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , PTEN Fosfo-Hidrolase/química , Plasmídeos/metabolismo , Ligação Proteica , Redobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificaçãoRESUMO
Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110 kDa and 107 kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460 kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme's feedback inhibition by L-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell's immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon-nitrogen interactions.
Assuntos
Germinação , Lisina/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Sorghum/enzimologia , Ubiquitinação , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Cinética , Espectrometria de Massas , Peso Molecular , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , Sorghum/crescimento & desenvolvimentoRESUMO
BACKGROUND: Serratia marcescens phage η is a temperate unclassified member of the Siphoviridae which had been reported as containing hypermodified guanine residues. METHODS: The DNA was characterized by enzymatic digestion followed by HPLC analysis of the nucleoside composition, and by DNA sequencing and proteomic analysis. Its ability to form stable lysogens and integrate was also investigated. RESULTS: Enzymatic digestion and HPLC analysis revealed phage η DNA did not contain modified bases. The genome sequence of this virus, determined using pyrosequencing, is 42,724 nucleotides in length with a mol% GC of 49.9 and is circularly permuted. Sixty-nine putative CDSs were identified of which 19 encode novel proteins. While seven close genetic relatives were identified, they shared sequence similarity with only genes 40 to 69 of the phage η genome, while gp1 to gp39 shared no conserved relationship. The structural proteome, determined by SDS-PAGE and mass spectrometry, revealed seven unique proteins. This phage forms very unstable lysogens with its host S. marcescens.