SapM mutation to improve the BCG vaccine: Genomic, transcriptomic and preclinical safety characterization.

The Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFNγ producing Th1 and Tc1 cells after sapM::Tn BCG vaccination. These findings corroborate that a live attenuated vaccine that affords improved long-term survival upon TB infection can be obtained by a mutation that further attenuates BCG. These findings suggest that an analysis of the effectiveness of innate immune control of the vaccine bacteria could be instructive also for other live attenuated TB vaccines that are currently under development, and encourage further studies of SapM mutation as a strategy in developing a more protective live attenuated TB vaccine.

Endoglycosidase S Enables a Highly Simplified Clinical Chemistry Procedure for Direct Assessment of Serum IgG Undergalactosylation in Chronic Inflammatory Disease.

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn's and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry because of a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput. We addressed this issue by using endo-ß-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker toward clinical implementation.

Glycome Patterns of Perfusate in Livers Before Transplantation Associate With Primary Nonfunction.

BACKGROUND & AIMS: Primary nonfunction (PNF) is a rare complication after liver transplantation that requires urgent retransplantation. PNF is associated with livers from extended criteria donors. Clinical and biochemical factors have not been identified that reliably associate with graft function after liver transplantation. Serum patterns of N-glycans associate with changes in the liver. We analyzed perfusate from grafted liver to identify protein glycosylation patterns associated with PNF. METHODS: We performed a prospective study of consecutive patients who underwent liver transplantation (66 patients, from 1 center, in the derivation set, and 56 patients, from 2 centers, in the validation set) in Belgium, from October 1, 2011, through April 30, 2017. All donor grafts were transported using cold static storage, and perfusate samples were collected from the livers by flushing of hepatic veins before transplantation. Protein-linked N-glycans were isolated from perfusate samples and analyzed with a multicapillary electrophoresis-based ABI3130 sequencer. We compared glycan patterns between patients with vs without PNF of transplanted livers. PNF was defined as the need for urgent retransplantation when a graft had no evidence of function, after exclusion of other causes, such as hepatic artery thrombosis or acute cellular rejection. RESULTS: The relative abundance of a single glycan, agalacto core-alpha-1,6-fucosylated biantennary glycan (NGA2F) was significantly increased in perfusate of livers given to 4 patients who developed PNF after liver transplantation compared with livers given to patients who did not develop PNF. Level of NGA2F identified patients with PNF with 100% accuracy. This glycomarker was the only factor associated with PNF in multivariate analysis in the derivation and the validation sets (P < .0001). CONCLUSIONS: In an analysis of patients who underwent liver transplantation, we associated graft perfusate level of glycan NGA2F present on perfusate proteins with development of PNF with 100% accuracy, and validated this finding in a separate cohort of patients. This biomarker might be used to assess grafts before transplantation, especially when high-risk organs are under consideration.

A Glycomics-Based Test Predicts the Development of Hepatocellular Carcinoma in Cirrhosis.

Purpose: Cirrhosis is a major risk factor for the development of hepatocellular carcinoma but remains underdiagnosed in the compensated stage. Fibrosis progression and cirrhosis are associated with changes in blood serum glycomic profiles. Previously, the serum glycomics-based GlycoCirrhoTest was shown to identify 50% to 70% of compensated cirrhosis cases in chronic liver disease cohorts, at >90% specificity. This study assessed GlycoCirrhoTest for the risk of hepatocellular carcinoma development in compensated cirrhosis.Experimental Design: Serum glycomics were analyzed in sera of 133 patients, with compensated cirrhosis collected between 1995 and 2005 in a surveillance protocol for hepatocellular carcinoma using an optimized glycomic technology on a DNA sequencer.Results: Baseline GlycoCirrhoTest values were significantly increased in patients who developed hepatocellular carcinoma after a median follow-up of 6.4 years as compared with patients who did not. For patients with a baseline GlycoCirrhoTest exceeding 0.2, the HR for hepatocellular carcinoma development over the entire study (Cox regression) was 5.1 [95% confidence interval (CI), 2.2-11.7; P < 0.001], and the HR for hepatocellular carcinoma development within 7 years was 12.1 (95% CI, 2.8-51.6; P = 0.01) based on the cut-off value optimized in the same cohort. An absolute increase in GlycoCirrhoTest of 0.2 was associated with an HR of 10.29 (95% CI, 3.37-31.43; P < 0.001) for developing hepatocellular carcinoma. In comparison, the HR for the development of hepatocellular carcinoma within 7 years for AFP levels above the optimal cutoff in this study (5.75 ng/mL) was 4.65 (95% CI, 1.59-13.61).Conclusions: This prognostic study suggests that GlycoCirrhoTest is a serum biomarker that identifies compensated cirrhotic patients at risk for developing hepatocellular carcinoma. Screening strategies could be guided by a positive test on GlycoCirrhoTest. Clin Cancer Res; 23(11); 2750-8. ©2016 AACR.

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.

BACKGROUND: 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. METHODS: Differences in total urine N-glycosylation profile of prostatic proteins were determined between healthy volunteers (n = 54), patients with benign prostate hyperplasia (BPH; n = 93) and newly diagnosed PCa patients (n = 74). Variations in N-glycosylation profile and prostate volume were combined into one urinary glycoprofile marker (UGM). Additionally, differences in N-glycosylation were identified between Gleason <7, =7, and >7. RESULTS: The UGM was able to discriminate BPH from PCa, overall and in the diagnostic gray zone (P < 0.001). The UGM showed comparable diagnostic accuracy to sPSA, but gave an additive diagnostic value to sPSA (P < 0.001). In the diagnostic gray zone the UGM performed significantly better than sPSA (P < 0.001). A significant difference was found in core-fucosylation of biantennary structures and overall core-fucosylation of multiantennary structures between Gleason < 7 and Gleason > 7 (P = 0.010 and P = 0.020, respectively) and between Gleason = 7 and Gleason > 7 (P = 0.011 and P = 0.025, respectively). CONCLUSIONS: The UGM shows high potential as PCa biomarker, particularly in the diagnostic gray zone. Further research is needed to validate these findings.

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.).

Characterization of a type D1A EUL-related lectin from rice expressed in Pichia pastoris.

OrysaEULD1A is one of the five EUL genes in rice (Oryza sativa) encoding a putative carbohydrate-binding protein belonging to the family of Euonymus related lectins (EUL). The OrysaEULD1A sequence comprises two highly similar EUL domains (91% sequence similarity and 72% sequence identity) separated by a 23 amino acid linker sequence and preceded by a 19 amino acid N-terminal sequence. In the present study, the full-length protein OrysaEULD1A as well as its individual domains OrysaEULD1A domain 1 and 2 were expressed in Pichia pastoris. After purification of the recombinant proteins, their carbohydrate-binding specificity was analyzed and compared. Interestingly, all recombinant lectins showed clear specificity towards galactosylated structures. Furthermore, all recombinant proteins agglutinated red blood cells, indicating that the full-length protein OrysaEULD1A and its domains are true lectins. These results taken together with data previously reported for single-domain EUL proteins indicate that although the amino acids--responsible for the formation of the carbohydrate-binding site--are identical for all EUL proteins in rice, these lectins show different carbohydrate specificities. This promiscuity of the carbohydrate-binding site can be attributed to gene divergence.

Capillary electrophoresis of urinary prostate glycoproteins assists in the diagnosis of prostate cancer.

Prostate marker assays are widely used for detection of prostate cancer (PCa) but are associated with considerable sensitivity and specificity problems. Therefore, we investigated prostatic protein glycosylation profiles as a potential biomarker. We determined the urinary asparagine-linked glycan (N-glycan) profile of prostatic proteins of healthy volunteers (n = 25), patients with benign prostate hyperplasia (BPH; n = 62) and newly diagnosed PCa patients (n = 42) using DNA-sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Through squeezing of the prostate, a sufficient amount of prostatic proteins was obtained for direct structural analyses of N-glycan structures. N-glycans of PCa compared to BPH were characterized by a significant decrease in triantennary structures (p = 0.047) and overall fucosylation (p = 0.026). Prostate-specific antigen (PSA) and the urinary glycoprofile marker showed comparable overall receiver operating characteristic curve analysis as well as in the diagnostic gray zone with serum PSA values between 4 and 10 µg/L. However, when combining PSA and the urinary glycoprofile marker, the latter gave an additive diagnostic value to serum PSA (p ≤ 0.001). In conclusion, N-glycosylation profiling demonstrated differences between BPH and PCa. These changes could lead to the discovery of a new biomarker for PCa.

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.

High-throughput profiling of the serum N-glycome on capillary electrophoresis microfluidics systems.

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward.

CE analysis of γ-globulin mobility and potential clinical utility.

Serum protein electrophoresis is widely used in clinical laboratories to measure the relative abundance of each obtained fraction. Moreover, we found that the migration time of the γ-globulin fraction can be reproducibly determined (CV = 1.1%). Immunoglobulins were purified from serum using protein L-agarose and their N-glycosylation was studied using CE on a DNA sequencer. Liver fibrosis patients showed a lower level of sialylation and this moderately correlates with the migration time of the γ-globulins (r = 0.2-0.4). This allowed us to differentiate healthy individuals from these patients with an acceptable diagnostic accuracy (area under the curve = 0.75). This glycomics approach could become a significant added value to a daily, routine clinical test.

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.

A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes.

We manufactured a novel type of lipid-coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high-resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N-glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.

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 Saccharomyces α-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.

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-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.

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.

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.

Diagnostic value of the hemopexin N-glycan profile in hepatocellular carcinoma patients.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common and rapidly fatal cancer. Current diagnostic methods for HCC have poor sensitivity and specificity, are invasive, and carry risk for complications. Newer markers are needed to overcome these problems and allow diagnosis of HCC at an earlier stage. In view of known associations between glycosylation changes and liver disease, we focused on the serum glycoprotein hemopexin and the specific characteristics of this liver-synthesized glycoprotein. METHODS: We studied 49 healthy volunteers and 81 patients divided into the categories of fibrosis, cirrhosis, and HCC with cirrhosis. Hemopexin was purified from study participants' serum by use of heme agarose beads. The hemopexin N-glycan profile was determined by use of the DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis technique. RESULTS: We found that branching alpha-1,3-fucosylated multiantennary glycans on hemopexin were increased in the HCC group compared with the cirrhosis without HCC, fibrosis, and healthy volunteer groups, whereas nonmodified biantennary glycans decreased progressively across groups from fibrosis to the cirrhosis and HCC groups. Summarization of this information in a new marker, called the hemopexin glycan marker, enabled distinction of patients with HCC and cirrhosis from healthy volunteers and patients with fibrosis or cirrhosis with a sensitivity and specificity of 79% and 93%, respectively. CONCLUSIONS: This study demonstrated hemopexin to be a model protein for studying liver-specific N-glycosylation. The hemopexin glycan marker could be a valuable complementary test to alpha-fetoprotein measurements for detection of HCC in patients with cirrhosis. Additional study of its utility for diagnosis and follow-up is recommended.

Glycome profiling using modern glycomics technology: technical aspects and applications.

Glycomics research has become indispensable in many research fields such as immunity, signal transduction and development. Moreover, changes in the glycosylation of proteins and lipids have been reported in several diseases including cancer. The analysis of a complex post-translational modification such as glycosylation depends on the availability or development of appropriate analytical technologies. The research goal determines the sensitivity, resolution and throughput requirements and guides the choice of a particular technology. This review highlights the evolution of glycan profiling tools in the past 5 years. We focus on capillary electrophoresis, liquid chromatography, mass spectrometry and lectin microarrays.