Autophosphorylation Inhibits RcCDPK1, a Dual-Specificity Kinase that Phosphorylates Bacterial-Type Phosphoenolpyruvate Carboxylase in Castor Oil Seeds.

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.

Remarkable Structural Diversity of N-Glycan Sulfation on Influenza Vaccines.

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.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

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.

The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

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+) < 0.4 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 ∼20% 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.

The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

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.

Surface modifications of influenza proteins upon virus inactivation by ß-propiolactone.

Inactivation of intact influenza viruses using formaldehyde or ß-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.

Regulatory monoubiquitination of phosphoenolpyruvate carboxylase in germinating castor oil seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme situated at the core of plant C-metabolism. Although its anaplerotic role and control by allosteric effectors, reversible phosphorylation, and oligomerization have been well documented in the endosperm of developing castor oil seeds (COS), relatively little is known about PEPC in germinating COS. The initial phase of COS germination was accompanied by elevated PEPC activity and accumulation of comparable amounts of pre-existing 107-kDa and inducible 110-kDa immunoreactive PEPC polypeptides (p107 and p110, respectively). A 440-kDa PEPC heterotetramer composed of an equivalent ratio of non-phosphorylated p110 and p107 subunits was purified from germinated COS. N-terminal microsequencing, mass spectrometry, and immunoblotting revealed that both subunits arose from the same gene (RcPpc3) that encodes the p107 subunit of a phosphorylated 410-kDa PEPC homotetramer in developing COS but that p110 is a monoubiquitinated form of p107. Tandem mass spectrometry sequencing of a diglycinated tryptic peptide identified Lys-628 as p110's monoubiquitination site. This residue is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a human deubiquitinating enzyme (USP-2 core) converted the p110:p107 PEPC heterotetramer into a p107 homotetramer while significantly reducing the enzyme's K(m)(PEP) and sensitivity to allosteric activators (hexose-Ps, glycerol-3-P) and inhibitors (malate, aspartate). Monoubiquitination is a non-destructive and reversible post-translational modification involved in the control of diverse processes such as transcription, endocytosis, and signal transduction. The current study demonstrates that tissue-specific monoubiquitination of a metabolic enzyme can also occur and that this modification influences its kinetic and regulatory properties.

A selenoprotein in the plant kingdom. Mass spectrometry confirms that an opal codon (UGA) encodes selenocysteine in Chlamydomonas reinhardtii gluththione peroxidase.

Selenoproteins that contain the rare amino acid selenocysteine in their primary structure have been identified in diverse organisms such as viruses, bacteria, archea, and mammals, but so far not in yeast or plants. Among the most thoroughly investigated families of selenoenzymes are the animal glutathione peroxidases (GPXs). In the last few years, genes encoding GPX-like homologues from Chlamydomonas and higher plants have been isolated, but, unlike the animal ones, all of them have cysteine (rather than selenocysteine) residues in their catalytic site. In all organisms investigated that contain selenoproteins, selenocysteine is encoded by a UGA opal codon, which is usually a stop codon. We report here that, in Chlamydomonas reinhardtii, the cDNA-cloned sequence of a GPX homologue contains an internal TGA codon in frame to the ATG. Specific mRNA expression, protein production, and enzyme activity are selenium-dependent. Sequence analysis of the peptides produced by proteolytic digestion, performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirmed the presence of a selenocysteine residue at the predicted site and suggest its location in the mitochondria. Thus, our data present the first direct proof that a UGA opal codon is decoded in the plant kingdom to incorporate selenocysteine.