Carbohydrate Epitopes Research Articles

Glycosylation in cancer: its application as a biomarker and recent advances of analytical techniques.

Despite significant worldwide investment in research, cancer remains one of the most common cause of death in the world. Early detection and reliable diagnosis are the keys to effectively treating cancer and improving the long-term survival of cancer patients. Therefore, there is an urgent need to develop a single, non-invasive biomarker with high sensitivity and specificity. Aberrant glycosylation is well defined as a hallmark of cancer and represents a promising source of potential biomarkers. Tumor-associated carbohydrate epitopes have long been detected by using monoclonal antibodies and lectins. Recent advances in analytical technologies allow us to analyze cancer-specific glycoforms on a target protein in a variety of clinical samples. A number of glycan analysis techniques, such as lectin-based detection methods and mass spectrometry combined with various types of chromatography, have been developed to establish novel glycoform-specific cancer biomarkers. Several glycan-based cancer biomarkers are already in clinical use. The aim of this review is to outline the role of glycan as a cancer biomarker and to summarize the current status and the potential for contribution of the serum glycoproteome to cancer diagnostics, monitoring, and prognostics.

Comparative mass spectrometry analysis of N-glycans from the glycoproteins of eight allergy-inducing plants

Extensive cross-allergic reactivity exists among food-borne plants. As most allergens are glycoproteins, protein sensitization has been extensively studied. However, little attention has been given to the glycan component of glycoproteins. Here, we qualitatively and quantitatively compared the N-glycans of eight cross-reactive plant glycoproteins derived from peach pollen, soy, pine nut, cashew nut, pistachio, walnut, almond, and hazelnut. Cross-reactive carbohydrate determinants (CCDs) were widely present in the allergic glycoproteins according to the following order: pollen > soybean > pistachios > pine nuts > walnuts > cashews > hazelnuts > almonds. The N-glycan structure presented clear differences between the germplasm and vegetative tissue. Importantly, fucosylation and xylosylation levels correlated positively with extensive cross-reactivity between plants, which suggests that CCDs may be one of the causes of cross-allergy. This result provides the foundation for mechanistic studies on plant-derived CCDs as a source of food cross-allergies.

Alpha-gal as the newest member of the glycan epitopes recognized in allergen nomenclature for cross-reactive carbohydrates

The first publications on cross-reactive carbohydrate epitopes as targets of IgE antibodies to pollen protein appeared over 30 years ago, but in general they were not relevant to pollen symptoms. By contrast, the recently described IgE specific for alpha-gal which is cross reactive over many mammalian molecules, are causally related to two forms of anaphylaxis.

Sialyl-Tn antigen facilitates extracellular vesicle-mediated transfer of FAK and enhances motility of recipient cells.

Protein glycosylation plays a pivotal role in tumour development by modulating molecular interactions and cellular signals. Sialyl-Tn (sTn) antigen is a tumour-associating carbohydrate epitope whose expression correlates with metastasis and poor prognosis of various cancers; however, its pathophysiological function is poorly understood. Extracellular vesicles (EVs) derived from cancer cells act as a signal mediator amongst tumour microenvironments by transferring cargo molecules. sTn antigen has been found in the glycans of EVs, thereby the functional relevance of sTn antigen to the regulation of tumour microenvironments could be expected. In the present study, we showed that sTn antigen induced TP53 and tumour suppressor-activated pathway 6 (TSAP6) and consequently enhanced EV production. Besides, the genetic attenuation of TSAP6 resulted in the reduction of the EV production in the sTn antigen expressing cells. The enhanced EV production in the sTn antigen-expressing cells consequently augmented the delivery of EVs to recipient cells. The produced EVs selectively and abundantly encased focal adhesion kinase and transferred it to EV-recipient cells, and thus, their cellular motility was enhanced. These findings would contribute to facilitate the elucidation of the pathophysiological significance of the sTn antigen in the tumour microenvironments and tumour development.

Developing an Effective Glycan-Based Vaccine for Streptococcus Pyogenes.

Streptococcus pyogenes is a primary infective agent that causes approximately 700 million human infections each year, resulting in more than 500 000 deaths. Carbohydrate-based vaccines are proven to be one of the most promising subunit vaccine candidates, as the bacterial glycan pattern(s) are different from mammalian cells and show increased pathogen serotype conservancy than the protein components. In this Review we highlight reverse vaccinology for use in the development of subunit vaccines against S. pyogenes, and report reproducible methods of carbohydrate antigen production, in addition to the structure-immunogenicity correlation between group A carbohydrate epitopes and alternative vaccine antigen carrier systems. We also report recent advances used to overcome hurdles in carbohydrate-based vaccine development.

Developing an Effective Glycan‐Based Vaccine for Streptococcus Pyogenes

AbstractStreptococcus pyogenes is a primary infective agent that causes approximately 700 million human infections each year, resulting in more than 500 000 deaths. Carbohydrate‐based vaccines are proven to be one of the most promising subunit vaccine candidates, as the bacterial glycan pattern(s) are different from mammalian cells and show increased pathogen serotype conservancy than the protein components. In this Review we highlight reverse vaccinology for use in the development of subunit vaccines against S. pyogenes, and report reproducible methods of carbohydrate antigen production, in addition to the structure–immunogenicity correlation between group A carbohydrate epitopes and alternative vaccine antigen carrier systems. We also report recent advances used to overcome hurdles in carbohydrate‐based vaccine development.

Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions.

Pollen Allergy Screening with Allergen-Specific and Total Immunoglobulin E Titers.

BackgroundAllergic rhinitis is a typical type I hypersensitivity reaction, commonly caused by inhalant allergens. Accurate identification of the causative antigen is important for rapid diagnosis and treatment initiation.ObjectiveThis study examined the efficiency of serum-based allergen-specific immunoglobulin E and total immunoglobulin E antibody titers in screening for pollen allergy. We also examined the effect of cross-reactive carbohydrate determinants on specific immunoglobulin E titers in screening for pollen allergy, one of the causes of false positivity in specific immunoglobulin E measurements.MethodsA questionnaire was used to evaluate the symptoms of pollinosis among participants who underwent a medical examination. One hundred and thirty-two participants reported pollen allergy symptoms and 127 reported an absence of symptoms. Specific immunoglobulin E levels were measured using the AlaSTAT 3g Allergy method. Seventeen components, including four types of cross-reactive carbohydrate determinant-specific immunoglobulin E antibodies, were measured and evaluated comparatively.ResultsThe sensitivity and specificity of the tests in predicting the presence or absence of pollen allergy were analyzed. The values of the areas under the curves for immunoglobulin E antibody levels against cedar, cypress, orchard grass, and ragweed pollen were 0.87, 0.82, 0.63, and 0.56, respectively. A cross-reactive carbohydrate determinant-related false-positive effect on the pollen specific immunoglobulin E titer was noted in pollen screening.ConclusionCedar pollen-specific immunoglobulin E titers showed sufficient accuracy for use in pollen allergy screening. The study of cross-reactive carbohydrate determinants suggested that subjects who tested positive for pollen often had false-positive results due to the impact of cross-reactive carbohydrate determinants.

Serum IgE profiles in Chinese pollinosis patients with grass pollen sensitisation

PurposePollen from trees, grasses, and weeds is a common allergen source. The characteristics of pollen allergy in China are obviously different from Europe. Most studies have focused on tree and weed pollen, but there is a paucity of data on grass pollen sensitisation in China. Therefore, we used component-resolved diagnostics to investigate the serum-specific immunoglobulin E (sIgE) to grass pollen in Chinese patients with pollinosis. MethodsWe retrospectively analysed 547 patients with pollen allegy from an outpatient Allergy Department in Beijing, China. All the patients answered questionnaires about their clinical allergy histories. Total immunoglobulin E (IgE) and sIgE levels to grass pollen (Bermuda, Timothy grass) were quantified by ImmunoCAP using 0.35 kUA/L as a threshold for positivity. ResultsOf the 547 pollinosis patients, 389 (71.1%) showed a positive sIgE reaction to either grass pollen, or both. The prevalence of food allergy was significantly lower in patients with grass pollen sensitisation. Among the 389 patients with grass pollen sensitisation, the prevalence of sIgE to allergen extracts of bermuda, mugwort, ragweed, plane, hop, ash, birch, and timothy grass was 97%, 96%, 94%, 88%, 88%, 84%, 78%, and 78%, respectively. However, only 134/389 (34%) were positive for Cyn d 1, 29/389 (7%) for Phl p 1, and 8/389 (2%) for Phl p 5b. For pollinosis patients, 62/547 (11%) were sIgE-positive for cross-reactive carbohydrate determinants (CCDs), and their grass pollen-sIgE was also positive. ConclusionsThe prevalence of in vitro IgE sensitisation to grass pollen extract is high in Chinese patients with pollinosis. But mostly spurious and characterized by IgE sensitisation to profilins and CCD, induced by other pollen. Component-resolved diagnostics is an extremely useful tool precise diagnostics of pollen allergy in China.

Targeting P-selectin ligands: ushering in a new treatment paradigm for Sickle Cell Disease

Sickle cell disease (SCD) is a debilitating systemic disease with limited treatment options for disease-related complications. Microvascular vaso-occlusion is the hallmark of the disease with profound morbidity and increased mortality. Vaso-occlusion underlies intricate pattern of complications related to hyper-adhesiveness of circulating blood cells and endothelium. Over-expression of P-selectin is central to initiation of the vaso-occlusive cascade by downstream engagement of multiple adhesion receptor/ligand pairs. Blocking P-selectin has been shown to effectively prevent vaso-occlusion by blocking these adhesive interactions. However, the model of the E-selectin inhibitor uproleselan in acute myeloid leukemia (AML) should instigate the development of similar agents that target selectin-ligand adhesive interactions in order to attenuate the clinical effects of vaso-occlusion. Interestingly, the sialylated and fucosylated tetrasaccharide sialyl Lewis(X) (sLeX) was found to be the glycan (carbohydrate) determinant for selectin binding. Therefore, novel synthetic lectins that target glycan determinants on P-selectin-ligands could prove more precise and efficient drugs, not only for prevention, but also for treating patients with vaso-occlusion crises (VOCs).

The α-Gal Syndrome and Potential Mechanisms.

The α-Gal syndrome is a complex allergic disease characterized by the development of specific IgE antibodies against the carbohydrate galactose-α-1,3-galactose (α-Gal), an oligosaccharide present in cells and tissues of non-primate mammals. Individuals with IgE antibodies to α-Gal suffer from a delayed form of anaphylaxis following red meat consumption. There are several features that make the α-Gal syndrome such a unique allergic disease and distinguish it from other food allergies: (1) symptoms causing IgE antibodies are directed against a carbohydrate moiety, (2) the unusual delay between the consumption of the food and the onset of the symptoms, and (3) the fact that primary sensitization to α-Gal occurs via tick bites. This review takes a closer look at the immune response against α-Gal, in healthy and in α-Gal allergic individuals. Furthermore, the similarities and differences between immune response against α-Gal and against the other important glycan moieties associated with allergies, namely cross-reactive carbohydrate determinants (CCDs), are discussed. Then different mechanisms are discussed that could contribute to the delayed onset of symptoms after consumption of mammalian meat. Moreover, our current knowledge on the role of tick bites in the sensitization process is summarized. The tick saliva has been shown to contain proteins carrying α-Gal, but also bioactive molecules, such as prostaglandin E2, which is capable of stimulating an increased expression of anti-inflammatory cytokines while promoting a decrease in the production of proinflammatory mediators. Together these components might promote Th2-related immunity and trigger a class switch to IgE antibodies directed against the oligosaccharide α-Gal. The review also points to open research questions that remain to be answered and proposes future research directions, which will help to get a better understanding and lead to a better management of the disease.

Simple and Label-Free Electrochemical Immuno Determination of the Gastric Cancer Biomarker Carbohydrate Antigen 72-4 with a Carbon Nanotube-Graphene Oxide Hybrid as the Sensing Platform and Ferrocyanide/Ferricyanide as the Probe

Developing simple and effective method for carbohydrate antigen 72-4 (CA72-4) detection is important to achieve the early diagnosis and treat of gastric cancer. In this work, multiwalled carbon nanotubes (MWCNT) were treated with a simple wet-chemistry method to form MWCNT/graphene oxide (MWCNT/GO) core/shell nanohybrids that provide high electron transfer, high dispersibility, and a large surface area to immobilize CA72-4 antibodies (Ab) due to the carboxyl groups on the GO shell. Using ferrocyanide/ferricyanide ([Fe(CN)6]3−/4−) as the signaling probe, a simple, label-free and effective electrochemical immunosensing platform was constructed for CA72-4. Upon optimizing the experimental conditions, the simple MWCNT/GO hybrids-based immunosensor showed desirable sensing including a wide linear range and low detection limit. Furthermore, the reproducibility, selectivity and practical applications demonstrated potential value to determine CA72-4 in clinical applications for diagnosing gastric cancer.

Idiopathic anaphylaxis: Diagnosis and management.

Introduction: Idiopathic anaphylaxis (IA) is a diagnosis of exclusion and is based on the inability to identify a causal relationship between a trigger and an anaphylactic event, despite a detailed patient history and careful diagnostic assessment. The prevalence of IA among the subset of people who experienced anaphylaxis is challenging to estimate and varies widely, from 10 to 60%; most commonly noted is ∼20% in the adult anaphylactic population. Comorbid atopic conditions, such as food allergy, allergic rhinitis, and asthma, are present in up to 48% of patients with IA. Improved diagnostic technologies and an increased understanding of conditions that manifest with symptoms associated with anaphylaxis have improved the ability to determine a more accurate diagnosis for patients who may have been initially diagnosed with IA. Methods: Literature search was conducted on PubMed, Google Scholar and Embase. Results: Galactose-α-1,3-galactose (α-gal) allergy, mast cell disorders, and hereditary a-tryptasemia are a few differential diagnoses that should be considered in patients with IA. Unlike food allergy, when anaphylaxis occurs within minutes to 2 hours after allergen consumption, α-gal allergy is a 3-6-hour delayed immunoglobulin E-mediated anaphylactic reaction to a carbohydrate epitope found in red meat (e.g., beef, lamb, pork). The more recently described hereditary α-tryptasemia is an inherited autosomal dominant genetic trait caused by increased germline copies of tryptase human gene alpha-beta 1 (TPSAB1), which encodes α tryptase and is associated with elevated baseline serum tryptase. Acute management of IA consists of carrying an epinephrine autoinjector to be administered immediately at the first signs of anaphylaxis. Long-term management for IA with antihistamines and other agents aims to potentially reduce the frequency and severity of the anaphylactic reactions, although the evidence is limited. Biologics are potentially steroid-sparing for patients with IA; however, more research on IA therapies is needed. Conclusion: The lack of diagnostic criteria, finite treatment options, and intricacies of making a differential diagnosis make IA challenging for patients and clinicians to manage.

Analysis of Peanut Allergen Components Sensitization and Cross Reaction with Pollen Allergen in Chinese Southerners with Allergic Rhinitis and/or Asthma.

ObjectivePeanut is one of the most frequently reported allergens causing severe allergies in western countries. In China, however, there have been few reports of severe allergies caused by peanuts. We investigated the peanut allergen components sensitization and cross-reaction with pollen allergen in Chinese Southerners with allergic rhinitis and/or asthma.MethodsTotal IgE (tIgE) and specific IgE (sIgE) antibodies against Ara h 1, Ara h 8, Juglans pollen, Platanus pollen, birch pollen, Bet v 1, Bet v 4, and cross-reactive carbohydrate determinant (CCD) of 58 allergic patients, of whom 33 were peanut-sIgE positive and 25 were negative, were detected by the ImmunoCAP system. The relationships between peanut allergen and pollen allergens were analyzed.ResultsA 9.1% (3/33) of the patients with peanut sensitization were sensitized to Ara h 8, while 21.2% (7/33) were sensitized to Ara h 1. The peanut-sensitized group had significantly higher positive rates for sIgE antibodies against CCD (69.7% vs 4.0%), Juglans pollen (87.9% vs 12.0%), Platanus pollen (90.9% vs 16.0%), and birch pollen (60.6% vs 4.0%) than the peanut tolerance group (all P < 0.05). Spearman correlation showed that peanut-sIgE were significantly correlated with sIgE to CCD (rs=0.859), Juglans pollen (rs=0.772), Platanus pollen (rs=0.838) and birch pollen (rs=0.816).ConclusionThe majority of patients sensitized to peanut allergen in Southern China tested positive for multiple pollen allergens. Peanut sensitization was highly correlated with Platanus, Juglans, and birch pollen sensitization, which suggested there may be cross-reactions between peanut and pollen allergens. Clinicians should pay attention to distinguish diagnosis in clinical peanut allergy diagnosis and treatment.

Impact of Specific N-Glycan Modifications on the Use of Plant-Produced SARS-CoV-2 Antigens in Serological Assays.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N-glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N-glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying β1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.

From Allergen Molecules to Molecular Immunotherapy of Nut Allergy: A Hard Nut to Crack

Peanuts and tree nuts are two of the most common elicitors of immunoglobulin E (IgE)-mediated food allergy. Nut allergy is frequently associated with systemic reactions and can lead to potentially life-threatening respiratory and circulatory symptoms. Furthermore, nut allergy usually persists throughout life. Whether sensitized patients exhibit severe and life-threatening reactions (e.g., anaphylaxis), mild and/or local reactions (e.g., pollen-food allergy syndrome) or no relevant symptoms depends much on IgE recognition of digestion-resistant class I food allergens, IgE cross-reactivity of class II food allergens with respiratory allergens and clinically not relevant plant-derived carbohydrate epitopes, respectively. Accordingly, molecular allergy diagnosis based on the measurement of allergen-specific IgE levels to allergen molecules provides important information in addition to provocation testing in the diagnosis of food allergy. Molecular allergy diagnosis helps identifying the genuinely sensitizing nuts, it determines IgE sensitization to class I and II food allergen molecules and hence provides a basis for personalized forms of treatment such as precise prescription of diet and allergen-specific immunotherapy (AIT). Currently available forms of nut-specific AIT are based only on allergen extracts, have been mainly developed for peanut but not for other nuts and, unlike AIT for respiratory allergies which utilize often subcutaneous administration, are given preferentially by the oral route. Here we review prevalence of allergy to peanut and tree nuts in different populations of the world, summarize knowledge regarding the involved nut allergen molecules and current AIT approaches for nut allergy. We argue that nut-specific AIT may benefit from molecular subcutaneous AIT (SCIT) approaches but identify also possible hurdles for such an approach and explain why molecular SCIT may be a hard nut to crack.

A qualitative and quantitative comparison of IgE antibody profiles with two multiplex platforms for component-resolved diagnostics in allergic patients.

Clinically complex phenotypes require more and more sophisticated and comprehensive diagnostic approaches, able to discriminate genuine sensitizations from cross-reactivity. Interpretative complexity of multiplex diagnostic arrays has somewhat limited their diffusion. This study compares two currently available methods, namely ISAC® test and ALEX2® test. In total, 140 allergic individuals, with a history of atopic dermatitis, adverse food reactions, allergic rhinitis and/or bronchial asthma were studied by Allergy Explorer-ALEX2® macroarray and ImmunoCAP ISAC112® . Lin's concordance correlation coefficient, intraclass correlation coefficient and Bland-Altman plots were used to verify the agreement between continuous values. Cohen's kappa coefficient (k) was assessed for the molecules available in both tests. The degree of relationship was analysed using Spearman's correlation (quantitative variables) and Pearson's χ2 or Fisher's exact test (categorical variables). A substantial agreement (κ=0.795) was observed between the two methods with 94,3% concordant results when results were dichotomized as negative or positive, but if double-negative results were discarded, the agreement dropped to 71%. Conversely, little or no concordance was observed comparing raw data. Considering the 102molecules shared by both systems, 28/102 (27%) showed an almost perfect agreement (k>0.81), and concordance was good (k>0.61) in a further 32 (31%) cases. A perfect to substantial agreement was observed by comparing species-specific aeroallergens. Heterogeneous results emerged comparing panallergens (co-recognition ranging from 30% for tropomyosin/serum albumins to 70% for PR-10/profilin). The correlation among LTP, profilin and PR-10 assayed with ISAC® was better than ALEX2® , but the latter identified more positive cases due to the wider number of molecules available. The CCD blocker provided by ALEX® test abolishes the carbohydrate determinants signal in 60% of the 33 cases reactive to MUXF3 on the ISAC® test. Despite the excellent concordance of the species-specific markers, the analysis of the panallergens provided in both methods suggests a better performance of the ISAC® test on those components, while the ALEX2® test, which includes a larger number of allergens, allowing a broader molecular detection.

Dynamic changes in primexine during the tetrad stage of pollen development.

Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.

B4GALNT2 Controls Sda and SLex Antigen Biosynthesis in Healthy and Cancer Human Colon.

The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down‐regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens’ expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex. In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.

A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.