FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy.

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and ß-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.

Relative quantitation of multi-antennary N-glycan classes: combining PGC-LC-ESI-MS with exoglycosidase digestion.

In the search for N-glycan disease biomarkers current glycoanalytical methods may not be revealing a complete picture of precious samples, and we may be missing valuable structural information that fall outside analysis windows. We report a targeted strategy combining PGC-LC-ESI-MS with exoglycosidases to improve the relative quantitation of tri and tetra-antennary glycan classes.

Acute exercise increases hippocampal TNF-α, Caspase-3 and Caspase-7 expression in healthy young and older mice.

AIM: Regular exercise may protect against cognitive decline by preventing central inflammation. The effect of an acute exercise bout on central cytokine and apoptotic protein expression is not known. The brain may be protected from transient oxidative stress such as that induced by acute-exercise. The purpose of this exploratory study was to determine the effect of a single bout of intense exercise on hippocampal expression of inflammatory mediators (TNF-α) and apoptotic proteins (caspase-3, caspase-7), and to evaluate any potential age-related differences. METHODS: Using a C57BL/6 mouse model (N.=98), we evaluated the effect of an acute exercise bout (90 minutes of treadmill running: 10 min warm-up, 30 min at 22 m.min⁻¹, 30 min at 25 m.min⁻¹, and 30 min at 28 m.min⁻¹, 2° slope) on hippocampal inflammation in young (3-4 months), middle-aged (13-14 months) and older (16-17 months) C57BL/6 mice. RESULTS: Our results show post-exercise increases in hippocampal TNF-α and caspase-3/7 in each age group (main effect of acute exercise, P<0.05). Older mice displayed higher TNF-α (main effect of age, P<0.05) expression compared with younger animals at baseline. Young mice demonstrated greater increases in caspase-7 following acute exercise, compared to older mice (interaction effect for caspase-7, P<0.05). CONCLUSION: Given the relationship between aging, inflammation and neurodegenerative disease, and the protective effects of exercise, we cautiously propose that acute-exercise induced inflammation may be a normal physiologic response that elicits a favorable (anti-inflammatory) hippocampal environment.

The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration.

BACKGROUND: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. METHODS: An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. RESULTS: Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. CONCLUSIONS: This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Apoptotic and inflammatory cytokine protein expression in intestinal lymphocytes after acute treadmill exercise in young and old mice.

AIM: Gastrointestinal disturbances are common in athletes following intense exercise. Variations in apoptotic protein expression and cell death may contribute to acute exercise-induced intestinal inflammation. The effect of age on apoptotic protein response in the intestinal compartment in response to exercise is not known. Using a mouse model, we examined the effects of a single bout of treadmill running in young and old mice on intestinal lymphocyte (IL) expression of the apoptosis-inducing cytokine TNF-α, the pro-apoptotic proteins caspase-3 and 7, the anti-apoptotic protein Bcl-2, and IL apoptotic status (% AnnexinV+). METHODS: Young (3-4 months, N.=44) and old (13-14 months, N.=45) female C57Bl/6 mice were randomized to treadmill exercise (10 min warm-up, 20 min at 22 m min-1, 30 min at 25 m min-1, 30 min at 28 m min-1, 2º slope) with sacrifice immediately (IMM) or 2hr after (2Hr), or to a non-exercised control (SED). IL were removed and prepared for analysis of % apoptosis (flow cytometry) and determination of apoptotic protein and cytokine expression (Western blotting). Plasma corticosterone and 8-iso-PGF2α were measured by EIA. RESULTS: Exercise was associated with a higher IL expression of caspase-3 in IMM and 2Hr groups vs. SED (P<0.001), a higher expression of TNF-α in the IMM group vs. SED (P<0.001), and a lower Bcl-2 expression in the IMM and 2Hr groups vs. SED (P<0.01). There was a trend (P=0.07) for increased caspase-7 expression after exercise. IL caspase-3 and 7 and TNF-α expression did not differ by age whereas Bcl-2 expression was lower (P<0.001) and % Annexin V+ IL was higher (P<0.05) in old vs. young mice. Plasma corticosterone and 8-iso-PGF2α were higher (P<0.001 and P<0.05) in IMM vs. SED mice but did not differ by age. CONCLUSION: The expression of the pro-apoptotic proteins, caspase-3 and caspase-7, and the apoptosis-inducing cytokine, TNF-α, in IL did not differ by age in this animal model in response to a single intense exercise challenge. However, Old mice had lower expression of the 'protective' anti-apoptotic protein Bcl-2 and a higher percentage of early apoptotic IL. Whether repeated exercise results in less IL resiliency in elderly individuals remains to be determined.

Does exercise protect from cognitive decline by altering brain cytokine and apoptotic protein levels? A systematic review of the literature.

Regular exercise is thought to provide protection against age-related cognitive decline and possibly reduce risk of dementias. The mechanisms for the exercise protective effects are not known although changes in inflammatory cytokine levels may be involved. We conducted a systematic review of the literature to assess (1) the effects of exercise on cytokines in the brain, (2) the methodological rigour of studies which have examined these exercise effects and (3) the potential role of regular exercise in reducing the pro-inflammatory cytokine milieu that may contribute to dementia. We also reviewed the effects of exercise on concurrent pro and anti-apoptotic protein expression in the brain as related to cytokine changes. Five databases were searched until January 2010 with an initial 630 articles identified; 61 articles were retrieved of which 10 met study inclusion criteria. Investigations of both acute and chronic (training) exercise were assessed for methodological quality using a modified PEDro scale. Two studies were carried out with human participants and eight with mouse or rat models; studies differed markedly in design and methodological rigour; the types, intensities and durations of exercise, the cytokine and apoptotic proteins measured, and the regions of the brain (or proxy compartments) sampled. Despite variations in design, specific cytokine outcomes, and exercise type, the 10 studies provide limited evidence that acute strenuous exercise increases and exercise training decreases pro-inflammatory cytokines centrally. Two animal studies relate training associated decreases in pro-inflammatory cytokines with improved cognitive function using behavioural assessments such as the Morris maze. Recommendations for the design of future research on exercise, central cytokines, and cognition are offered.

Freewheel training decreases pro- and increases anti-inflammatory cytokine expression in mouse intestinal lymphocytes.

Intestinal inflammation and inflammatory bowel diseases (IBD) may occur due to imbalances in pro- and anti-inflammatory cytokines. Long-term exercise reduces the risk for IBD. The purpose of this study was to determine the effect of long-term wheel running in healthy mice on intestinal lymphocyte (IL) expression of pro- and anti-inflammatory cytokine proteins. In addition, pro- and anti-apoptotic proteins and the percentage of early apoptotic, late apoptotic, and dead IL were measured with wheel running and following acute aerobic exercise. Female C57BL/6 mice were given 16 weeks of wheel running (WR) or a control condition (No WR) and at the end of training were assigned to a single acute treadmill exercise session with sacrifice immediately, 2h after, or 24h after completion of exercise, or were not run (sedentary) with respect to the acute treadmill exercise. Intestinal lymphocytes were assessed for pro-(TNF-α, IL-17) and anti-(IL-10) inflammatory, and pleiotropic (IL-6) cytokines, and pro-(caspase 3 and 7, AIF) and anti-(Bcl-2) apoptotic protein expression. The percent of early (Annexin(+)) and late (Annexin(+)PI(+)) apoptotic, and dead (PI(+)) IL was determined. WR mice had lower TNF-α and caspase 7, and higher IL-10 and IL-6 expression in IL than No WR mice. A single exposure to intense aerobic treadmill exercise increased pro-(TNF-α) and anti-(IL-10) inflammatory cytokine and pro-apoptotic protein (caspase 3) expression in IL. The percent of early and late apoptotic, and dead IL were higher after acute exercise. Although long-term voluntary wheel running did not protect against acute exercise-induced changes in IL cytokine expression or apoptosis, there was an overall 'anti-inflammatory' effect observed as a result of wheel running in healthy mice.

Does physical activity affect quality of life, disease symptoms and immune measures in patients with inflammatory bowel disease? A systematic review.

Inflammatory bowel diseases (IBD) are a group of chronic, episodic inflammatory conditions of the large and small intestines. Individuals with IBD have been reported to use physical activity (PA) as a complementary therapy although the effectiveness of PA for reducing disease burden in patients with IBD is not known. The review objective is to evaluate published studies on physical activity and IBD focusing on quality of life, disease burden markers and immunological outcomes. A literature search was carried out using MEDLINE, WEB OF SCIENCE, CINHAL, and SCOPUS (to December 2008). Studies were included if they 1) were provided in English; 2) dealt with IBD in humans; 3) focused on the outcome measures of health related quality of life, clinical disease indicators or immune function; and 4) included PA as a primary intervention for IBD cases. In total, 7 studies were included in this systematic review: 5 were on PA and quality of life measures and inflammatory disease markers, and 2 on PA and immune measures. Four studies showed that PA significantly increased quality of life for IBD patients as assessed by various questionnaires. PA was also associated with decreased disease activity. There was no evidence that PA affected immune outcomes in patients with IBD. The role of PA as an adjunctive therapy for patients with IBD has not been well characterized in the literature. However, there is some evidence that PA may improve quality of life and reduce disease activity in patients with IBD.

Proteomic analysis of the genetic premature aging disease Hutchinson Gilford progeria syndrome reveals differential protein expression and glycosylation.

Proteomics has revealed differential protein expression and glycosylation in membrane proteins from premature aging Hutchinson-Gilford progeria syndrome fibroblasts (progeria). Progeria is a rare autosomal dominant genetic disorder of premature aging characterized by marked growth retardation and specific, progressive, premature senescent changes of the skin and other tissues. Affected children live to an average age of 13 years. The 1q20-24 region of chromosome 1 which codes for one of these proteins, lamin A/C, has previously been implicated by Brown et al. (1990) who described identical twins with progeria, where cytogenetic analysis showed an inverted insertion in the long arm of the chromosome in 70% of cells. Luengo et al. (2002) similarly reported an interstitial deletion of chromosome 1q23, in a 9-year-old patient with a classic clinical picture of progeria.

Data standardisation in GlycoSuiteDB.

GlycoSuiteDB, a database of glycan structures, has been constructed with an emphasis on quality, consistency and data integrity. Importance has been placed on making the database a reliable and useful resource for all researchers. This database can help researchers to identify what glycan structures are known to be attached to certain glycoproteins, as well as more generally identifying what types of glycan structures are associated with different states, for example, different species, tissues and diseases. To achieve this, a major effort has gone into data standardisation. Many rules and standards have been adopted, especially for representing glycan structure and biological source information. This paper describes some of the challenges faced during the continuous development of GlycoSuiteDB.

GlycoMod--a software tool for determining glycosylation compositions from mass spectrometric data.

GlycoMod (http://www.expasy.ch/tools/glycomod/) is a software tool designed to find all possible compositions of a glycan structure from its experimentally determined mass. The program can be used to predict the composition of any glycoprotein-derived oligosaccharide comprised of either underivatised, methylated or acetylated monosaccharides, or with a derivatised reducing terminus. The composition of a glycan attached to a peptide can be computed if the sequence or mass of the peptide is known. In addition, if the protein is known and is contained in the SWISS-PROT or TrEMBL databases, the program will match the experimentally determined masses against all the predicted protease-produced peptides (including any post-translational modifications annotated in these databases) which have the potential to be glycosylated with either N- or O-linked oligosaccharides. Since many possible glycan compositions can be generated from the same mass, the program can apply compositional constraints to the output if the user supplies either known or suspected monosaccharide constituents. Furthermore, known oligosaccharide structural constraints on monosaccharide composition are also incorporated into the program to limit the output.

GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources.

GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco-protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O:-linked oligosaccharides from the last 50 years and most N:-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono-isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N:- or O:-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at http://www.glycosuite.com.

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.

Multiple O-glycoforms on the spore coat protein SP96 in Dictyostelium discoideum. Fuc(alpha1-3)GlcNAc-alpha-1-P-Ser is the major modification.

A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of the spore coat. Here the post-translational modifications of a major spore coat protein, SP96, are studied in a wild type strain (X22) and a fucosylation-defective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc(alpha1-3)GlcNAc-alpha-1-P-Ser(,) was identified by electrospray ionization mass spectrometry and NMR. It was shown using monosaccharide and gas chromatography mass spectrometry analysis that SP96 in the mutant HU2470 contained approximately 20% of wild type levels of fucose, as a result of a missing terminal fucose on the novel glycan structure. The results support previous predictions, based on inhibition studies on different fucose-deficient strains, about the nature of monoclonal antibody epitopes identified by monoclonal antibodies MUD62 and MUD166, which are known to identify O-linked glycans (Champion, A., Griffiths, K., Gooley, A. A., Gonzalez, B. Y., Gritzali, M., West, C. M., and Williams, K. L. (1995) Microbiology 141, 785-797). Quantitative studies on wild type SP96 indicated that there were approximately 60 sites with phosphodiester-linked N-acetylglucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites as phosphoserine. Threonine and tyrosine residues were not found to be modified.

Proteomics: capacity versus utility.

Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High-throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.

BOLD--a biological O-linked glycan database.

Glycans can be O-linked to proteins via the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline. Sometimes the glycan is O-linked to the hydroxyl group via a phosphodiester bond. The core monosaccharide residue may be N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, fucose, mannose, xylose or arabinose. These O-linked glycans can remain as a monosaccharide, but often a complex structure is built up by stepwise addition of monosaccharides. Monosaccharides known to be added include galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid, N-glycolylneuraminic acid and 2-keto-3-deoxynonulosonic acid. O-linked glycans can also contain sulfate and phosphate residues. This leads to the possibility of the existence of numerous O-glycan structures. The biological O-linked database (BOLD) is a relational database that contains information on O-linked glycan structures, their biological sources (with a link to the SWISS-PROT protein database), the references in which the glycan was described (with a link to MEDLINE), and the methods used to determine the glycan structure. The database provides a valuable resource for glycobiology researchers interested in O-linked oligosaccharide structures that have been previously described on proteins from different species and tissues.

Studies on the "insoluble" glycoprotein complex from human colon. Identification of reduction-insensitive MUC2 oligomers and C-terminal cleavage.

The "insoluble" glycoprotein complex was isolated from human colonic tissue and mucin subunits were prepared following reduction. Antibodies raised against peptide sequences within MUC2 revealed that virtually all of this mucin occurs in the insoluble glycoprotein complex. In addition, reduction released a 120-kDa C-terminal MUC2 fragment, showing that proteolytic cleavage in this domain may occur and leave the fragment attached to the complex via disulfide bonds. The variable number tandem repeat region and the irregular repeat domain were isolated after trypsin digestion and shown to have molecular weights of 930,000 and 180,000, respectively, suggesting a molecular weight for the entire MUC2 monomer of approximately 1.5 million. Gel chromatography and agarose gel electrophoresis revealed several populations of MUC2 subunits, and analytical ultracentrifugation showed that these have molecular weights on the order of 2 million, 4 million, and 5 million, corresponding to monomers, dimers, and trimers, respectively. Agarose gel electrophoresis of subunits from individuals expressing both a "long" and a "short" MUC2 allele revealed a larger number of populations, consistent with the presence of short and long monomers and oligomers arising from permutations of the two types of monomers. In addition to disulfide bonds, MUC2 monomers are apparently joined by a "novel," reduction-insensitive bond.