Structural characterisation of the N-glycan moiety of the barnacle settlement-inducing protein complex (SIPC).

Many barnacle species are gregarious and their cypris larvae display a remarkable ability to explore surfaces before committing to permanent attachment. The chemical cue to gregarious settlement behaviour - the settlement-inducing protein complex (SIPC) - is an α(2)-macroglobulin-like glycoprotein. This cuticular protein may also be involved in cyprid reversible adhesion if its presence is confirmed in footprints of adhesive deposited during exploratory behaviour, which increase the attractiveness of surfaces and signal other cyprids to settle. The full-length open-reading frame of the SIPC gene encodes a protein of 1547 amino acids with seven potential N-glycosylation sites. In this study on Balanus amphitrite, glycan profiling of the SIPC via hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-fluorescence) provided evidence of predominantly high mannose glycans (M2-9), with the occurrence of monofucosylated oligomannose glycans (F(6)M2-4) in lower proportions. The high mannose glycosylation found supports previous observations of an interaction with mannose-binding lectins and exogenous mannose increasing settlement in B. amphitrite cypris larvae. Transmission electron microscopy of the deglycosylated SIPC revealed a multi-lobed globular protein with a diameter of ~8 nm. Obtaining a complete structural characterisation of the SIPC remains a goal that has the potential to inspire solutions to the age-old problem of barnacle fouling.

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells.

BACKGROUND: The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. RESULTS: Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. CONCLUSIONS: The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms.

Despite the reduced incidence of gastric cancer in the developed world, a diagnosis of stomach carcinoma still carries a poor prognosis due to the asymptomatic nature of the disease in the early stages, subsequent advanced stage diagnosis, and a low 5 year survival rate. Endoscopy remains the primary standard for diagnosis of stomach carcinoma and the current marker, carbohydrate antigen 19-9 (CA19-9) lacks the levels of sensitivity and specificity required in order to make it clinically useful for diagnostic monitoring. Therefore, there is a current need for additional markers to improve the diagnostic accuracy for the early stages of stomach cancer. Together, glycomic, proteomic, and glycoproteomic analyses of serum have the potential to identify such probable markers. A discovery study is reported here using preoperative serum from 80 stomach cancer patients, 10 patients bearing benign stomach disease, and 20 matched controls. Glycomic analysis of the total and immunoaffinity depleted serum revealed statistically significant increases in the levels of sialyl Lewis X epitopes (SLe(X)) present on triantennary glycans accompanied by increased levels of core fucosylated agalactosyl biantennary glycans present on IgG (referred to as the IgG G0 glycoform) which are associated with increasing disease pathogenesis. Protein expression analysis using 2D-DiGE returned a number of differentially expressed protein candidates in the depleted serum, many of which were shown to carry triantennary SLe(X) during subsequent glycomic investigations. Biological pathway analysis of the experimental data returned complement activation and acute phase response signaling as the most significantly altered pathways in the stomach cancer patient serum. Upon the basis of these findings, it is suggested that increased expression of IgG G0 and complement activation are a host response to the presence of the stomach tumor while the increased expression of SLe(X) and acute phase response proteins is a result of pro-inflammatory cytokine signaling, including IL-6, during carcinogenesis. The approach presented herein provides an insight into the underlying mechanisms of disease and the resulting changes in the glycome and glycoproteome offer promise as potential markers for diagnosis and prognostic monitoring in stomach cancer.

Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 µm hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, haptoglobin, transferrin, and α1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and α1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease progression. The speed and selectivity offered by the sub-2 µm HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome.

Glycan characterization of PSA 2-DE subforms from serum and seminal plasma.

Prostate-specific antigen (PSA) two-dimensional electrophoresis (2-DE) subforms (F1-F5) have been described to be altered in prostate cancer (PCa) compared to benign prostatic hyperplasia (BPH). To understand their molecular differences, characterization of these subforms from PCa serum and seminal plasma, namely, at the glycan level, was performed. PSA 2-DE subforms from two serum PCa samples and seminal plasma were analyzed by N-glycan sequencing using high-performance liquid chromatography (HPLC) combined with exoglycosidase array digestions and by mass spectrometry. F1, F2, and F3 subforms showed the same N-glycan pattern, which contained higher levels of sialic acid than the F4 subform, whereas the F5 subform was unglycosylated. When comparing PSA subforms from PCa with seminal plasma, a decrease in sialylation was observed. Furthermore, the analysis of F3, the more abundant PSA subform, showed a higher proportion of alpha 2-3 sialic acid and a decrease in core fucosylated glycans in the PCa sample. These N-glycan changes in PCa PSA subforms highlight the importance of glycosylation as an indicator of PCa disease.

Control of choline oxidation in rat kidney mitochondria.

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173+/-64 microM and a Vmax of 0.4+/-0.1 nmol/min/mg mitochondrial protein (at 10 degrees C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 degrees C (125+/-6 pmol O2/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96+/-4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.

In vitro nuclear interactome of the HIV-1 Tat protein.

BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.

2D-DIGE as a strategy to identify serum markers for the progression of prostate cancer.

Prostate cancer is the most common solid organ malignancy affecting men in the United States and Western Europe. Currently, the main diagnostic tools used to look for evidence of prostate cancer include physical examination using digital rectal exam (DRE), serum concentrations of prostate specific antigen (PSA) and biopsy. However, due to the low specificity of PSA in differentiating prostate cancer from other benign conditions, many patients undergo overtreatment for their disease. There is an urgent need for additional markers to improve the diagnostic accuracy for early stages of prostate cancer. Proteomic analysis of serum has the potential to identify such markers. An initial discovery study has been completed using 12 serum samples from patients with different grades of prostate cancer (Gleason score 5 and 7) undergoing radical prostatectomy. Serum samples were subjected to immunoaffinity depletion and protein expression analysis using 2D-DIGE. Image analysis isolated 63 spots that displayed differential expression between the Gleason score 5 and 7 cohorts (p < 0.05), 13 of which were identified as statistically significant using two independent image analysis packages. Identification of differentially expressed spots was carried out using LC-MS/MS. Because of their functional relevance and potential significance with regards to prostate cancer progression, two of these proteins, pigment epithelium-derived factor (PEDF) and zinc-alpha2-glycoprotein (ZAG), have undergone extensive validation in serum and tissue samples from the original cohort and also from a larger independent cohort of patients. These results have indicated that PEDF is a more accurate predictor of early stage prostate cancer. We are confident that proteomics-based approaches have the potential to provide more insight into the underlying molecular mechanisms of the disease and also hold great promise for biomarker discovery in prostate cancer.

Variability, heritability and environmental determinants of human plasma N-glycome.

Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels.

The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS).

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2(-/-) macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2-D DIGE was applied to compare protein patterns from wild-type and Tyk2(-/-) macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty-one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N-myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS-induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS-mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

Fasting status as a consideration for human serum collection and preparation prior to depletion and analysis.

The use of comparative serum proteomic analysis has the potential to reveal protein expression changes present at different stages of disease progression. Depletion strategies allow for the enrichment of low-abundance proteins, which are more likely to be clinically significant biomarkers. We have observed that patient serum samples filtered through 0.22 microm cellulose acetate spin filters prior to depletion showed a variable level of retention of patient material on the upper part of the filter. This could potentially be related to the fasting status of the patient as a reduction in the lipid content of samples through the incorporation of a centrifugation step prior to filtration was found to reduce this effect. In order to determine if proteins were being selectively retained during filtration, a 2-D difference gel electrophoresis (2-D DIGE) experiment was performed. This demonstrated no significant selective retention of protein within crude serum samples. However, as this analysis was carried out on crude serum, it must be emphasised that protein loss could be manifest in the low-abundance proteins which would be masked in our analysis. Depletion of the retentate was not possible due to technical limitations, however based on our results a centrifugation step might act as an alternative to filtration in serum processing prior to depletion.

Integrative proteomics: structure, function, and interaction report on the 3rd joint meeting of the British Society for Proteome Research and the European Bioinformatics Institute, July 2006.

This report summarizes the highlights of the recent British Society for Proteome Research (BSPR) meeting jointly organized with the European Bioinformatics Institute (EBI) which was held at the Wellcome Trust Genome Campus, Hinxton, Cambridge, UK in July 2006. This was the third annual scientific meeting organized by the BSPR and EBI and the theme of this years meeting was Integrative Proteomics: Structure, function and interaction. A wealth of local and overseas speakers were invited to discuss both their own work and specific challenges present in modern day proteomic based experiments.

CyDye immunoblotting for proteomics: co-detection of specific immunoreactive and total protein profiles.

The development of ECL-Plex CyDye-conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre-labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2-D immunoprobing and protein confirmation following MS analysis.

2-D DIGE as a quantitative tool for investigating the HUPO Brain Proteome Project mouse series.

Brain development and aging is a complex process involving proliferation, differentiation and apoptosis. Elucidating proteome changes in these processes can help to understand the mechanisms of brain development and maintenance as well as neurodegenerative diseases. The research reported here is a contribution to the HUPO Brain Proteome Project mouse pilot study. Whole, frozen C57BL/6J mouse brain comprising three different developmental stages (embryonic day 16, postnatal day 7, and postnatal days 54-58) were processed by using 2-D DIGE. A total of 1999 spots were matched between all gels. Of these, 206 spots were differentially expressed between the different stages: 122 spots were highest in intensity in embryonic stage E16, 26 highest in the juvenile group P7 and 58 spots highest in P56, the adult stage. The results show a pattern of temporal expression. Based on the expression patterns we tentatively suggest that proteins involved in the establishment of primary structures in the brain are expressed highest in the embryonic mouse. Proteins involved in the development of the brain are expressed highest in the juvenile phase and proteins that make utilization of the brain possible by delivering energy are expressed highest in the adult mice.

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet-specific integrin alphaIIb beta3 has endogenous thiol isomerase activity associated with the CXXC motifs within the beta subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited alphaIIb beta3 activation, as detected by PAC-1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified alphaIIb beta3 with a 50% inhibitory concentration of 15.5 micromol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin-independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of alphaIIb beta3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Identification of the phosphotyrosine proteome from thrombin activated platelets.

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.