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Correction for 'The biology of environmental stress: molecular biomarkers in Sydney rock oysters (Saccostrea glomerata)' by D. A. Raftos et al., Environ. Sci.: Processes Impacts, 2016, 18, 1129-1139.

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This review describes our recent work on environmental stress in Sydney rock oysters, focusing on the identification of molecular biomarkers for ecotoxicological analysis. We begin by describing the environmental pressures facing coastal estuaries in Australia, with particular reference to Sydney Harbour. After providing that context, we summarise our transcriptional and proteomic analyses of Sydney rock oysters responding to chemical contamination and other forms of environmental stress. This work has shown that the intracellular processes of oysters are highly responsive to environmental threats. Our data agree with the broader literature, which suggests that there is a highly conserved intracellular stress response in oysters involving a limited number of biological processes. We conclude that many effective molecular markers for environmental biomonitoring are likely to lie within these biological pathways.

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Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.

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Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.

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The use of artificial control sediments in toxicity tests offers several advantages over field-collected control sediments, especially for laboratory-cultured organisms such as Neanthes arenaceodentata. Ten side-by-side trials were conducted using a field-collected sediment from West Beach, Whidbey Island, Washington State, USA and an artificial sediment composed of a silica quartz sand commonly used for masonry applications. After a 20-d exposure to sediment and clean overlying seawater, a similar biological response (as measured by the final average individual dry weight of Neanthes) was observed. The average individual dry weight was 14.77 +/- 3.83 and 14.70 +/- 3.57 mg/worm for the artificial and field-collected negative controls, respectively.

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Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine alpha-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.

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In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.

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We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low-cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS-PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC-electrospray mass spectrometry.

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In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.

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Mammalian cell invasion by the intracellular protozoan parasite Trypanosoma cruzi is mediated by recruitment and fusion of host cell lysosomes, an unusual process that has been proposed to be dependent on the ability of parasites to trigger intracellular free calcium concentration ([Ca2+]i) transients in host cells. Previous work implicated the T.cruzi serine hydrolase oligopeptidase B in the generation of Ca2+-signaling activity in parasite extracts. Here we show that deletion of the gene encoding oligopeptidase B results in a marked defect in host cell invasion and in the establishment of infections in mice. The invasion defect is associated with the inability of oligopeptidase B null mutant trypomastigotes to mobilize Ca2+ from thapsigargin-sensitive stores in mammalian cells. Exogenous recombinant oligopeptidase B reconstitutes the oligopeptidase B-dependent Ca2+ signaling activity in null mutant parasite extracts, demonstrating that this enzyme is responsible for the generation of a signaling agonist for mammalian cells.

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We have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The efficiency of peptide recovery from trypsin-digested gel bands or electroblotted membrane slices was examined using 35S-labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC-ESI-MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit. We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow-split microspray HPLC-MS interface system. In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng. In general, protein identification was slightly better in samples prepared from gels rather than membranes. A dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydrase and 100 ng of BSA.

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There are a number of different glycoproteins that have been identified relatively recently which contain oligosaccharides linked to serine or threonine in a peptide backbone via phosphodiesters. It is possible that these glycoproteins may form an alternative structural class of glycosylation. This modification has been referred to as phosphoglycosylation (Mehta et al., 1996; J. Biol. Chem., 271, 10897-10903), and has been reported in slime molds and several unicellular parasites. In this review, examples of phosphoglycosylation from different biological sources are discussed. Those which are well characterized have been found to be highly variable with respect to the glycan moiety, while sharing some common features. An experimental approach detailing how to determine whether a protein is phosphoglycosylated is also presented.

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We have investigated the subcellular location of the Trypanosoma cruzi surface glycoprotein, Gp72, by introducing epitope-tagged copies of gp72 null-mutant cells. A tagged Gp72, containing three tandemly repeated copies of a human influenza hemagglutinin nonapeptide (HA) adjacent to the mature Gp72 amino terminus, was able to complement the null mutant phenotype, as well as being recognized in Western blots by both anti-HA antibody and the carbohydrate-specific monoclonal antibody WIC29.26. Integration of this epitope-tagged gp72 into the chromosomal gp72 locus produced a clonal cell line, 72HAN3.1G7, which was used for studies of the subcellular location of the epitope-tagged Gp72. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 epimastigotes showed that GP72 was evenly distributed over the cell body and somewhat concentrated in the proximal region of the flagellum. No fluorescence could be detected in the distal tip of the flagellum. Immunoelectron microscopy of fixed 72HAN3.1G7 epimastigotes revealed that Gp72 was predominantly membrane-associated and located on the cell surface. Indirect immunofluorescence microscopy of live 72HAN3.1G7 epimastigote cells showed a similar pattern of fluorescence on the flagellum, but no fluorescence was detected on the cell body, which was attributed to masking by other cell-surface components. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 amastigotes revealed that Gp72, which has long been considered to be expressed only in epimastigotes and metacyclic trypomastigotes, can be expressed in amastigotes, but it no longer contains the WIC29.26 carbohydrate epitope.

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Affinity-purified glycopeptides were prepared from Trypanosoma cruzi using the carbohydrate-specific monoclonal antibody WIC29.26. These glycopeptides contain rhamnose, fucose, xylose, and galactose, in the ratio 1:1:2:3. A series of oligosaccharides was released from the glycopeptides by mild acid hydrolysis, while, in contrast, no oligosaccharides were released by either peptide N-glycosidase F or conventional base-catalyzed beta-elimination and reduction. This suggested the presence of a phosphodiester linkage between the carbohydrate and peptide, which was further supported by the detection of phosphothreonine in the glycopeptides. The mild acid liberated (MAL) fraction was resolved into two major acidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutral oligosaccharides (MAL-P1b and MAL-P2b) and a neutral fraction (MAL-N1), consisting of Gal and Xyl monosaccharides. The MAL-P1 and MAL-P2 oligosaccharides proved to be hexa- and hepta-saccharides that shared a common xylose reducing terminus, but differed by one galactofuranose residue, and their negative charge was shown to be due to the presence of cyclic-phosphate attached to nonreducing terminal galactofuranose residues. The MAL-P1b and MAL-P2b oligosaccharides appeared to be nonphosphorylated versions of MAL-P1 and MAL-P2. Partial structures of MAL-P1 and MAL-P2 are suggested, based on compositional analyses, electrospray mass spectrometry, and tandem mass spectrometry before and after permethylation. The origin and significance of these unique trypanosomatid glycoconjugates is discussed.

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An immunodominant 72-kDa surface glycoprotein (Gp72) of Trypanosoma cruzi is involved in adhesion of the flagellum to the cell body (Cooper, R, Ribeiro de Jesus, A and Cross, G.A.M (1993) J. Cell Biol. 122, 149-156). We have characterized a gene, flagellum-adhesion glycoprotein genel (fla1), from Trypanosoma brucei that encodes a 546 amino-acid protein (Fla1) with high similarity to Gp72. Their sequence similarity and cellular localization suggest that Fla1 and Gp72 have similar functions. We could disrupt individual fla1 alleles but not both, suggesting that fla1 is essential in T. brucei, in contrast to the situation for gp72 in T. cruzi. Using affinity-purified polyclonal antibody, raised against part of the amino-terminal domain of Fla1 expressed in Escherichia coli, we showed that Fla1 is concentrated along the flagellum and in the flagellar pocket in both bloodstream-form and procyclic trypanosomes. Fla1 from both life-cycle stages is N-glycosylated. Fla1 from bloodstream-form T. brucei contains additional glycans, which can be liberated by treatment with mild acid, suggestive of phosphodiester linkages.

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The present study was undertaken to examine the extent to which the lgt locus varies among strains of gonococci. This locus encodes five glycosyl transferases involved in the synthesis of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae. We examined seven gonococcal strains and found that the structure of the lgt locus is conserved among six of these strains. The locus is strikingly altered in strain 15253. This is one of the few strains where extensive structural analysis of its LOS is available, and therefore, we defined the altered lgt locus and focused on the reactivity of mAB 2C7. We found that strain 15253 contains only two lgt genes, lgtA and lgtE. As in F62, lgtA encodes a GlcNAc transferase and is subject to phase variation. In addition, by analysis of deletion mutants, we found that lgtE, which encodes a galactosyl transferase that is required for elongating the alpha-chain, is also necessary for completing the beta chain.

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We previously reconstituted the ATP-dependent inactivation of asialoglycoprotein receptors (ASGPRs) in digitonin-permeabilized hepatocytes (Medh, J. D., and Weigel, P. H. (1991) J. Biol. Chem. 266, 8771-8778). Here we report that rat hepatic lectin 1 (RHL1) is the only ASGPR subunit that becomes radiolabeled when permeabilized washed hepatocytes are incubated at 4 degrees C in the presence of [gamma-32P]ATP; RHL2 and RHL3 are not radiolabeled. Phosphorylation of RHL1 was rapid (t1/2 appoximately 4 min) and complete within 30 min. Inclusion of 20 mM EDTA inhibited phosphorylation of RHL1 completely. Phosphoamino acid analysis identified Tyr(P) as the predominant (> 90%) radiolabeled phosphoamino acid. Addition of vanadate enhanced phosphorylation of Tyr in RHL1 4-fold. Phosphorylation of RHL1 occurred to the same extent in hepatocytes permeabilized with either 0.006% (w/v) or 0.055% digitonin and in the presence or the absence of ligand (50 micrograms/ml asialo-orosomucoid; ASOR) and/or 10 mM CaCl2. Sequential purification of active ASGPRs (using ASOR-Sepharose) and inactive ASGPRs from the ASOR-Sepharose flow-through (using anti-ASGPR antibody-Sepharose) demonstrated that radiolabeled RHL1 was present almost exclusively in active ASGPR oligomers. When permeabilized hepatocytes radiolabeled with [gamma-32P]ATP at 4 degrees C were warmed to 37 degrees C, a temperature at which ATP-dependent ASGPR inactivation occurs, RHL1 was dephosphorylated rapidly (t1/2 approximately 4 min) and completely within approximately 30 min. Western blot analysis using a monoclonal anti-Tyr(P) antibody showed that the steady-state level of endogenous Tyr(P) in RHL1 doubled as a result of ATP treatment at 4 degrees C and then decreased to undetectable levels upon warming to 37 degrees C. The protein-tyrosine kinase inhibitor tyrphostin 51 inhibited phosphorylation of RHL1 at 4 degrees C and also prevented ATP-dependent ASGPR inactivation at 37 degrees C. We conclude that phosphorylation of Tyr in RHL1 of active ASGPRs is a prerequisite for ATP-dependent ASGPR inactivation.

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The rat asialoglycoprotein receptor (ASGPR) is an integral transmembrane glycoprotein composed of three polypeptide subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Each subunit contains one or more Ser and Thr residues in its cytoplasmic domain that are potential sites of phosphorylation; in addition, RHL1 also contains one cytoplasmic Tyr. Based on [32P]PO4 metabolic radiolabeling experiments, Takahashi et al. (Takahashi, T., Nakada, H., Okumura, T., Sawamura, T., and Tashiro, Y. (1985) Biochem. Biophys. Res. Commun. 126, 1054-1060) concluded that RHL2 and RHL3 are phosphoproteins but that RHL1 is not. We report here that RHL1 in active ASGPR is, in fact, a phosphoprotein. Western blot analysis using anti-Tyr(P) antibody identified Tyr(P) in RHL1 of affinity-purified ASGPRs. RHL2 and RHL3, which do not contain Tyr in their cytoplasmic domains, did not react with this antibody. When isolated hepatocytes were radiolabeled metabolically with [32P]PO4, RHL1, RHL2, and RHL3 became radiolabeled. Each ASGPR subunit was radiolabeled to a similar extent in the presence or absence of the ligand asialo-orosomucoid, indicating that functioning of the ASGPR does not change its steady-state 23P-radiolabeling. Phosphoamino acid analysis of radiolabeled ASGPR subunits identified Ser(P) as the predominant (approximately 95%) and Thr(P) as a minor (approximately 5%) phosphoamino acid in each polypeptide and confirmed the presence of Tyr(P) (approximately 1%) in RHL1. Furthermore, treatment of hepatocytes with 3 mM vandate at 37 degrees C for 30 min doubled the steady-state level of Tyr(P) in RHL1.

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