Screening for abnormal glycosylation in a cohort of adult liver disease patients

Congenital Disorders of Glycosylation (CDG) are a rapidly expanding group of rare genetic defects in glycosylation. In a novel CDG subgroup of Vacuolar-ATPase assembly defects various degrees of hepatic injury have been described, including end stage liver disease. However, the CDG diagnostic workflow can be complex as liver disease per se may be associated with abnormal glycosylation. Therefore, we collected serum samples of patients with a wide range of liver pathology to study the performance and yield of two CDG screening methods. Our aim was to identify glycosylation patterns that could help to differentiate between primary and secondary glycosylation defects in liver disease. To this end, we analyzed serum samples of 1042 adult liver disease patients. This cohort consisted of 567 liver transplant candidates and 475 chronic liver disease patients. Our workflow consisted of screening for abnormal glycosylation by transferrin isoelectric focusing (tIEF), followed by in-depth analysis of the abnormal samples with quadruple time-of-flight mass spectrometry (QTOF-MS). Screening with tIEF resulted in identification of 247 (26%) abnormal samples. QTOF-MS analysis of 110 of those did not reveal glycosylation abnormalities comparable with those seen in V-ATPase assembly factor deficiencies. However, two patients presented with isolated sialylation deficiency. Fucosylation was significantly increased in liver transplant candidates compared to healthy controls and patients with chronic liver disease. In conclusion, a significant percentage of patients with liver disease presented with abnormal CDG screening results, however, not indicative for a V-ATPase assembly factor defect. Advanced glycoanalytical techniques assist in the dissection of secondary and primary glycosylation defects. This article is protected by copyright. All rights reserved

Examining the distribution and impact of single nucleotide polymorphisms in the capsular locus of Streptococcus pneumoniae serotype 19A

Streptococcus pneumoniae serotype 19A prevalence has increased after implementation of PCV7 and PCV10 vaccines. In this study, we have provided, with high accuracy, the genetic diversity of the 19A serotype in a cohort of Dutch invasive pneumococcal disease patients and asymptomatic carriers obtained in the period 2004-2016. Whole genomes of the 338 pneumococcal isolates in this cohort were sequenced and their capsule (cps) loci compared to examine the diversity and determine the impact on the production of CPS sugar precursors and CPS shedding. We discovered 79 types with a unique CPS locus sequence. Most variation was observed in the rmlB and rmlD genes of the TDP-Rha synthesis pathway, and in the wzg gene, of unknown function. Interestingly, gene variation in the cps locus was conserved in multiple alleles. Using RmlB and RmlD protein models, we predict that enzymatic function is not affected by the single nucleotide polymorphisms as identified. To determine if RmlB and RmlD function was affected, we analyzed nucleotide sugar levels using UHPLC-MS. CPS precursors differed between 19A cps locus subtypes, including TDP-Rha, but no clear correlation was observed. Also, a significant difference in multiple nucleotide sugar levels was observed between phylogenetically branched groups. Because of indications of a role for Wzg in capsule shedding, we analyzed if this was affected. No clear indication of a direct role in shedding was found. We thus describe genotypic variety in rmlB, rmlD and wzg in serotype 19A the Netherlands, for which we have not discovered an associated phenotype

Mutations in GDP-mannose pyrophosphorylase b cause congenital and limb-girdle muscular dystrophies associated with hypoglycosylation of α-dystroglycan

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG. © 2013 The American Society of Human Genetics.Funding for UK10K was provided by the Wellcome Trust under award WT091310

Carbohydrate-based chemical probes for the proteomic profiling of glucosidases and the emerging cancer marker galectin-3

This thesis describes the synthesis of carbohydrate-based chemical probes to either profile glucosidases, or specifically label the emerging cancer marker galectin-3 in cell lysates. In general, chemical probe based methods can tag certain classes of proteins, and covalently label a protein or a protein family. This method should allow quantification within a mixture of other proteins. It could also give information about the localization of the protein in the cell, and about interactions of the target with other proteins. The method is fast, robust and easy to perform. It starts with incubation of the probe with the target protein or a cell lysate of interest, followed by photolabeling. In the next step a fluorescent reporter molecule is introduced to allow visualization of the captured proteins in a gel. An alkyne moiety on the probe allows for “click chemistry” to introduce the fluorescent reporter molecule. Instead of the fluorescent reporter, a biotin-tag or a 6xhistidine-tag was used in attempts to enrich the labeled proteins from the proteome. Competition experiments with a known ligand confirmed selective binding of the probe. One part of the project focuses on profiling glucosidases within a proteome. A glucose-based probe and iminosugar-based probes were synthesized, as well as a probe based on an acyclic mimic of the iminosugar. One iminosugar-based probe showed very high affinity towards non-lysosomal glucoceramidase, and was able to specifically label lysosomal glucocerebrosidase in the presence of a human cell lysate. This enzyme is defective in Gaucher patients. The other part of the project, which is the main part, focuses on the synthesis and evaluation of chemical probes to label galectin-3. This human protein is considered an emerging cancer marker in the literature. Because techniques used to obtain information about its function (mostly antibody based) often give conflicting results, chemical probes were designed and synthesized to selectively label galectin-3. A divalent lactose probe was synthesized, which increases the affinity for the protein over its monovalent derivative, due to a multivalency effect. Peptidic photoprobes were also synthesized, which were able to label galectin-3. The best probes in labeling galectin-3 are based on the high-affinity thiodigalactoside ligand. The probes were synthesized with benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. Besides labeling the protein, these aromatic photolabels also greatly enhance the affinity of the probes towards galectin-3, due to the interaction of the photolabel with two arginine guanidinium groups of the protein. The linkage between the sugar and the photolabel was varied as an ester, an amide, and a triazole. For the amide and triazole derivatives, a versatile synthetic route towards a symmetrical 3-azido-3-deoxy-thiodigalactoside was developed. The results described in this thesis clearly show that creating a succesful probe is a multidimensional challenge involving issues of lipophilicity, affinity and photoreactivity. Above all, one of the probes showed such a good selectivity for its target protein in the presence of a human cancer cell lysate that this compound may have potential in future cancer research or diagnostics

Clinical glycomics for the diagnosis of congenital disorders of glycosylation

Contains fulltext : 191891.pdf (publisher's version ) (Open Access

Detection of galectin-3 by novel peptidic photoprobes

Photoprobes were prepared with specificity for binding, labeling, and visualizing galectin-3 in a mixture of proteins. The probes were derived from a galectin-3 binding 15-mer peptide sequence in which a benzophenone photolabel was incorporated at the N-terminus and in another case as a phenyl alanine replacement in the middle of the sequence. Detection of galectin-3 was possible in Escherichia coli lysates that were spiked with various amounts of galectin-3