O-Linked glycosylation in Acanthamoeba polyphaga mimivirus

Acanthamoeba polyphaga mimivirus is a member of the giant nucleocytoplasmic large DNA viruses, infecting various Acanthamoeba spp. The genomes of giant viruses encode components previously thought to be exclusive to cellular life, such as proteins involved in nucleic acid and protein synthesis. Recent work on enzymes involved in carbohydrate biosynthesis and metabolism show that instead of utilizing host cell resources, Mimivirus produces its own glycosylation machinery. To obtain a more detailed view of glycosylation in Mimivirus, we developed a periodate oxidation-based method to selectively enrich Mimivirus surface glycoproteins. O-Glycosylation in Mimivirus glycoproteins was identified by permethylation and matrix-assisted laser desorption/ionization-mass spectrometry analyses of beta-eliminated glycans. We sequenced 26 previously undescribed O-glycans, most of which contain glucose as their reducing end saccharide. These data will facilitate future studies on the functional significance of glycosylation in Mimiviru

N-glycosylation site occupancy in serum glycoproteins using multiple reaction monitoring liquid chromatography-mass spectrometry

Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and alpha(1)-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in alpha(1)-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glycoproteomics survey of N-glycosylation site occupancies in biological samples

An Echinococcus multilocularis coproantigen is a surface glycoprotein with unique O-gycosylation

A major surface constituent of Echinococcus multilocularis adult worms, referred to as EmA9 antigen, was immunoaffinity purified and identified as a high molecular weight glycoconjugate. Labelling studies using the monoclonal antibody MAbEmA9 indicated that this antigen undergoes a regulated expression during the development from the larval to the adult parasite. Chemical modification of carbohydrate by periodate oxidation resulted in a reduced reactivity with antigen specific antibodies. Non-reductive beta-elimination of the purified molecule indicated the presence of O-linked glycans attached to threonine residues. Carbohydrate compositional analyses indicated the presence of N- and O-glycans with the ratio of carbohydrate to protein being 1.5:1 (w/w). N- and O-linked glycans were released by hydrazinolysis and analysed as 2-aminobenzamide derivatised glycans by mass spectrometry together with HPLC and enzymatic sequencing. Novel linear O-linked saccharides with multiple beta-HexNAc extensions of reducing end Gal were identified. N-linked glycans were also detected with oligomannose and mono-, bi-, tri- and tetra-antennary type structures, most of which were found to be core-fucosylated. Taken together, the results indicate that the EmA9 antigen is a glycoprotein located at the outer surface of the adult E. multilocularis. The observation that the EmA9 antigen expression is developmentally regulated suggests an involvement of this glycoprotein in the establishment of the parasite in its canine hos

Exposure to Mimivirus collagen promotes arthritis

Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by the ORF L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intra-dermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from human healthy subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens

Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik

Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 β1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-I

Occult hypoperfusion and changes of systemic lipid levels after severe trauma: an analysis in a standardized porcine polytrauma model

BACKGROUND: Occult hypoperfusion describes the absence of sufficient microcirculation despite normal vital signs. It is known to be associated with prolonged elevation of serum lactate and later complications in severely injured patients. We hypothesized that changes in circulating lipids are related to responsiveness to resuscitation. The purpose of this study is investigating the relation between responsiveness to resuscitation and lipidomic course after poly trauma. METHODS: Twenty-five male pigs were exposed a combined injury of blunt chest trauma, liver laceration, controlled haemorrhagic shock, and femoral shaft fracture. After 1 h, animals received resuscitation and fracture stabilization. Venous blood was taken regularly and 233 specific lipids were analysed. Animals were divided into two groups based on serum lactate level at the end point as an indicator of responsiveness to resuscitation (<2 mmol/L: responder group (R group), ≧2 mmol/L: occult hypoperfusion group (OH group)). RESULTS: Eighteen animals met criteria for the R group, four animals for the OH group, and three animals died. Acylcarnitines showed a significant increase at 1 h compared to baseline in both groups. Six lipid subgroups showed a significant increase only in R group at 2 h. There was no significant change at other time points. CONCLUSIONS: Six lipid groups increased significantly only in the R group at 2 h, which may support the idea that they could serve as potential biomarkers to help us to detect the presence of occult hypoperfusion and insufficient resuscitation. We feel that further study is required to confirm the role and mechanism of lipid changes after trauma

Altered CSF Albumin Quotient Links Peripheral Inflammation and Brain Damage in MS

OBJECTIVE CNS damage can increase the susceptibility of the blood-brain barrier (BBB) to changes induced by systemic inflammation. The aim of this study is to better understand BBB permeability in patients with MS and to examine whether compromised BBB integrity in some of these patients is associated with CNS damage and systemic inflammation. METHODS Routine CSF measurements of 121 patients with MS were analyzed including number and type of infiltrating cells, total protein, lactate, and oligoclonal bands, as well as intrathecal production of immunoglobulins and CSF/serum quotients for albumin, immunoglobulins, and glucose. In addition, in a subcohort of these patients, we performed ex vivo immunophenotyping of CSF-infiltrating and paired circulating lymphocytes using a panel of 13 monoclonal antibodies, we quantified intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1), and we performed intrathecal lipidomic analysis. RESULTS Patients with MS with abnormal high levels of albumin in the CSF showed a distinct CSF cell infiltrate and markers of CNS damage such as increased intrathecal levels of NF-L and CHI3L1 as well as a distinct CSF lipidomic profile. In addition, these patients showed higher numbers of circulating proinflammatory Th1 and Th1* cells compatible with systemic inflammation. Of interest, the abnormally high levels of albumin in the CSF of those patients were preserved over time. CONCLUSIONS Our results support the hypothesis that CNS damage may increase BBB vulnerability to systemic inflammation in a subset of patients and thus contribute to disease heterogeneity

Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins

Asparagine-linked glycosylation is a common post translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site specific glycosylation analysis in many model systems and clinical applications