Wouter Laroy

N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus†

We evaluated the use of blood serum N‐glycan fingerprinting as a tool for the diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis induced by hepatitis B virus (HBV). A group of 450 HBV‐infected patients with liver fibrosis or cirrhosis with or without HCC were studied. HCC was diagnosed by α‐fetoprotein (AFP) analysis, ultrasonography, and/or computed tomography and was studied histologically. N‐glycan profiles of serum proteins were determined with DNA sequencer–based carbohydrate analytical profiling technology. In this study, we found that a branch alpha(1,3)‐fucosylated triantennary glycan was more abundant in patients with HCC than in patients with cirrhosis, patients with fibrosis, and healthy blood donors, whereas a bisecting core alpha(1,6)‐fucosylated biantennary glycan was elevated in patients with cirrhosis. The concentration of these 2 glycans and the log ratio of peak 9 to peak 7 (renamed the GlycoHCCTest) were associated with the tumor stage. Moreover, for screening patients with HCC from patients with cirrhosis, the overall sensitivity and specificity of the GlycoHCCTest were very similar to those of AFP. Conclusion: This study indicates that a branch alpha(1,3)‐fucosylated glycan is associated with the development of HCC. The serum N‐glycan profile is a promising noninvasive method for detecting HCC in patients with cirrhosis and could be a valuable supplement to AFP in the diagnosis of HCC in HBV‐infected patients with liver cirrhosis. Its use for the screening, follow‐up, and management of patients with cirrhosis and HCC should be evaluated further. (HEPATOLOGY 2007.)

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-α-D-mannosidase for N-glycan engineering in Pichia pastoris

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein‐linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human‐compatible N‐glycans in this organism is the conversion of the yeast‐type high‐mannose glycans to mammalian‐type high‐mannose and/or complex glycans. In this perspective, we have co‐expressed an endoplasmic reticulum‐targeted Trichoderma reesei 1,2‐α‐D‐mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans‐sialidase. Analysis of the N‐glycans of the two purified proteins showed a >85% decrease in the number of α‐1,2‐linked mannose residues. Moreover, the human‐type high‐mannose oligosaccharide Man5GlcNAc2 was the major N‐glycan of the glyco‐engineered trans‐sialidase, indicating that N‐glycan engineering can be effectively accomplished in P. pastoris.

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.

N-Glycan profiling in the study of human aging

Most secreted proteins produced by the human body are modified by glycosylation. It is well known that the oligosaccharides (glycans) of glycoproteins are important for initiation of various cellular recognition signals that are essential for the maintenance of the ordered social life of each cell within a multi-cellular organism. The sugar chains can be altered by the physiological or pathophysiological condition of the cell. We describe a detailed protocol for the analysis of N-linked glycans in blood via DNA sequencing equipment-Fluorophore Assisted Carbohydrate Electrophoresis (DSA-FACE). The key features of this technique are its robustness, high throughput, high sensitivity and reliable quantification. Based on DSA-FACE technology, we previously reported that N-glycan profiling of the human serum shows substantial changes with increasing age in three major N-glycan structures. We proposed that measurement of the N-glycan level changes could provide a surrogate marker for general health or for age-related disease progression, and for monitoring the improvement of health after therapy.

Reversible changes in serum immunoglobulin galactosylation during the immune response and treatment of inflammatory autoimmune arthritis

Objectives: Improved DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology was used to monitor the changes in the galactosylation status of serum immunoglobulins during the immune response and therapy of autoimmune arthritis. Methods: Collagen-induced arthritis (CIA) was induced in susceptible DBA/1 mice and the undergalactosylation status (UGS) of serum immunoglobulins was determined using the improved DSA-FACE technology. Prophylactic intravenous tolerisation with type II collagen as well as semitherapeutic treatment with dexamethasone (DEX) were performed and UGS was analysed. Next, the serum immunoglobulin glycosylation profiles of patients with rheumatoid arthritis (RA) and spondyloarthropathy (SpA) were studied and changes in the UGS scores during anti-tumour necrosis factor (TNF)α therapy followed. Results: In the longitudinal CIA study, the undergalactosylation state of immunoglobulins was found to be significantly correlated with the clinical arthritis scores. Upon collagen-specific tolerisation as well as glucocorticoid semitherapeutic treatment, improvement of the clinical arthritis scores correlated with decreased levels of UGS. It was also demonstrated that withdrawal of DEX was associated with an increased UGS score. Interestingly, reversibility in the UGS was also shown during treatment of patients with RA and SpA with anti-TNFα. Conclusions: These findings demonstrate that the UGS of serum immunoglobulins changes during the disease course of CIA and that this UGS is inhibited by antigen-specific and antigen-independent treatment procedures. The observation that Ig galactosylation is a reversible process is also documented during treatment of patients with RA and SpA with anti-TNFα.

Secretion of functional human enzymes by Tetrahymena thermophila

BackgroundThe non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen.ResultsWe have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A1 precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern.ConclusionThe ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines.

Clinical laboratory testing in human medicine based on the detection of glycoconjugates.

The purpose of this review is to provide a concise overview of developments over the last 15 years in the field of laboratory tests in human medicine that are based on the detection of alterations in the glycan part of glycoconjugates. We show how glycosylation-based diagnostic testing is widespread in the current clinical practice, in different formats. To provide the necessary focus in this extremely broad field, we have only included assays that are either in actual clinical use or that are under active development towards clinical use, with some bias towards assays that were recently developed. The fields included are: cancer, infectious disease, genetic defects of glycoconjugate biosynthesis and catabolism, auto-immunity, drug abuse and liver disease. To conclude this review, we provide a viewpoint on the future of the glyco-diagnostics field in terms of novel technologies, especially with regard to the discovery and clinical implementation of biomarkers that are based on pathologically altered endogenous glycotopes.

Consequences of Soluble ICAM-1 N-Glycan Alterations on Receptor Binding and Signaling Kinetics in Mouse Astrocytes

Soluble intercellular adhesion molecule-1 (sICAM-1) is elevated in the cerebrospinal fluid of patients with se- vere brain trauma and mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes. The production of MIP-2 is greatly enhanced when sICAM-1 contains sialylated complex-type N-glycans (sICAM-1-CT) as produced by Chinese hamster ovary (CHO) cells. By contrast, sICAM-1 from the Lec1 mutant of CHO cells (sICAM-1-HM), containing only high mannose-type N-glycans, is relatively inactive. Here we show that the N- glycans of sICAM-1-CT are mostly  2,3-sialylated bi-, tri-, and tetraantennary complex-type structures with varying amounts of core fucosylation. Unexpectedly, sICAM-1-CT and sICAM-1-HM bound equivalently to mouse astrocytes. Enhanced MIP-2 induction by sICAM-1-CT was associated with a more rapid, higher level, and prolonged MIP-2 re- sponse as well as sICAM-1-CT accumulation at the plasma membranes of mouse astrocytes. These results show that gly- cosylation of sICAM-1 contributes to its signaling properties at the astrocyte cell surface, and suggest that altered glyco- sylation which might arise as a result of inflammation could regulate the bioactivity of sICAM-1.