Dieter Vanderschaeghe

Multiple phenotypes in phosphoglucomutase 1 deficiency

BACKGROUND Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).

Abscisic acid deficiency causes changes in cuticle permeability and pectin composition that influence tomato resistance to Botrytis cinerea

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.

GlycoFibroTest Is a Highly Performant Liver Fibrosis Biomarker Derived from DNA Sequencer-based Serum Protein Glycomics

Liver fibrosis is currently assessed by liver biopsy, a costly and rather cumbersome procedure that is unsuitable for frequent patient monitoring, which drives research into biomarkers for this purpose. To investigate whether the serum N-glycome contains information suitable for this goal, we developed a 96-well plate-based serum N-glycomics sample preparation protocol that only involves fluid transfer steps and incubations in a PCR thermocycler yielding 8-aminopyrene-1,3,6-trisulfonic acid-labeled N-glycans. These N-glycans are then ready for analysis on the capillary electrophoresis-based DNA sequencers that are the current standard in clinical genetics laboratories worldwide. Subsequently we performed a multicenter, blinded study of 376 consecutive chronic hepatitis C virus patients for which liver biopsies and extensive serum biochemistry data were available. Among patients, the METAVIR fibrosis stage distribution was as follows: 10.6% F0, 44.4% F1, 20.5% F2, 18.4% F3, and 6.1% F4. We found that the ratio of two N-glycans, here called GlycoFibroTest, correlates with the histological fibrosis stage equally well as FibroTest (ρ = 0.4–0.5 in F1–F4), which is used in the clinic today. Finally using affinity chromatography we depleted sera of immunoglobulin G, and this resulted in a complete removal of the undergalactosylated biantennary glycans from the N-glycome, which are partially determining GlycoFibroTest.

High-Throughput Profiling of the Serum N-Glycome on Capillary Electrophoresis Microfluidics Systems: Toward Clinical Implementation of GlycoHepatoTest

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use.

Mutations in GMPPA Cause a Glycosylation Disorder Characterized by Intellectual Disability and Autonomic Dysfunction

In guanosine diphosphate (GDP)-mannose pyrophosphorylase A (GMPPA), we identified a homozygous nonsense mutation that segregated with achalasia and alacrima, delayed developmental milestones, and gait abnormalities in a consanguineous Pakistani pedigree. Mutations in GMPPA were subsequently found in ten additional individuals from eight independent families affected by the combination of achalasia, alacrima, and neurological deficits. This autosomal-recessive disorder shows many similarities with triple A syndrome, which is characterized by achalasia, alacrima, and variable neurological deficits in combination with adrenal insufficiency. GMPPA is a largely uncharacterized homolog of GMPPB. GMPPB catalyzes the formation of GDP-mannose, which is an essential precursor of glycan moieties of glycoproteins and glycolipids and is associated with congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-dystroglycan. Surprisingly, GDP-mannose pyrophosphorylase activity was unchanged and GDP-mannose levels were strongly increased in lymphoblasts of individuals with GMPPA mutations. This suggests that GMPPA might serve as a GMPPB regulatory subunit mediating feedback inhibition of GMPPB instead of displaying catalytic enzyme activity itself. Thus, a triple-A-like syndrome can be added to the growing list of congenital disorders of glycosylation, in which dysregulation rather than mere enzyme deficiency is the basal pathophysiological mechanism.

Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis

Mycobacterium bovis bacille Calmette‐Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α‐1,2‐oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer‐term survival. Persistence of the SapM‐mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC‐IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.

N-glycan based biomarker distinguishing non-alcoholic steatohepatitis from steatosis independently of fibrosis

BACKGROUND Non-alcoholic fatty liver disease is a spectrum of disorders ranging from steatosis to non-alcoholic steatohepatitis (NASH). Steatosis of the liver is benign, whereas NASH can progress to cirrhosis or even hepatocellular carcinoma. Currently, a liver biopsy is the only validated method to distinct NASH from steatosis. AIM The objective of this study was to identify a biomarker specific for NASH based on the N-glycosylation of serum proteins. METHODS N-glycosylation patterns were assessed using DNA sequencer-assisted fluorophore-assisted capillary electrophoresis and compared with histology. RESULTS Initially, a glycomarker (log[NGA2F]/[NA2]) was developed based on the results obtained in 51 obese non-alcoholic patients scheduled for bariatric surgery. Multivariate analysis showed that our glycomarker had the lowest P-value of all biomarkers in distinguishing NASH from steatosis (P=0.069). The glycomarker was validated in a cohort of 224 non-alcoholic fatty liver disease patients. In both pilot and validation study, glycomarker score increased in ascending amount of lobular inflammation (single-factor ANOVA, P ≤ 0.001 and P=0.012, respectively). The N-glycan profile of immunoglobulin G in the NASH population confirmed the significantly increased undergalactosylation present in these patients. CONCLUSION Our glycomarker specifically recognises liver inflammation in obese individuals which is the main trigger for the development of steatohepatitis and can differentiate between steatosis and NASH.

Expression analysis of the nucleocytoplasmic lectin ‘Orysata’ from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose‐specific, jacalin‐related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin‐related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomycesα‐factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N‐glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high‐mannose structures, Man9–11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high‐mannose as well as with more complex N‐glycan structures. Orysata has potent anti‐human immunodeficiency virus and anti‐respiratory syncytial virus activity in cell culture compared with other jacalin‐related lectins.

Long-Lasting Cross-Protection Against Influenza A by Neuraminidase and M2e-based immunization strategies

There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

Urinary prostate protein glycosylation profiling as a diagnostic biomarker for prostate cancer

Serum prostate‐specific antigen (sPSA) measurement is widely used as opportunistic screening tool for prostate cancer (PCa). sPSA suffers from considerable sensitivity and specificity problems, particularly in the diagnostic gray zone (sPSA 4–10 µg/L). Furthermore, sPSA is not able to discriminate between poorly‐, moderately‐, and well‐differentiated PCa. We investigated prostatic protein glycosylation profiles as a potential PCa biomarker.