Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles.

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation.

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut-microbiota interface.

Longitudinal changes of serum protein N-Glycan levels for earlier detection of pancreatic cancer in high-risk individuals.

BACKGROUND: Surveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities is challenging. We aimed to identify changes in serum glycosylation levels over time to earlier detect PDAC in high-risk individuals. METHODS: Individuals with a hereditary predisposition to develop PDAC were followed in two surveillance programs. Those, of which at least two consecutive serum samples were available, were included. Mass spectrometry analysis was performed to determine the total N-glycome for each consecutive sample. Potentially discriminating N-glycans were selected based on our previous cross-sectional analysis and relative abundances were calculated for each glycosylation feature. RESULTS: 165 individuals ("FPC-cohort" N = 119; Leiden cohort N = 46) were included. In total, 97 (59%) individuals had a genetic predisposition (77 CDKN2A, 15 BRCA1/2, 5 STK11) and 68 (41%) a family history of PDAC without a known genetic predisposition (>10-fold increased risk of developing PDAC). From each individual, a median number of 3 serum samples (IQR 3) was collected. Ten individuals (6%) developed PDAC during 35 months of follow-up; nine (90%) of these patients carried a CDKN2A germline mutation. In PDAC cases, compared to all controls, glycosylation characteristics were increased (fucosylation, tri- and tetra-antennary structures, specific sialic linkage types), others decreased (complex-type diantennary and bisected glycans). The largest change over time was observed for tri-antennary fucosylated glycans, which were able to differentiate cases from controls with a specificity of 92%, sensitivity of 49% and accuracy of 90%. CONCLUSION: Serum N-glycan monitoring may support early detection in a pancreas surveillance program.

Mass spectrometry in clinical glycomics: The path from biomarker identification to clinical implementation.

Over the past decades, the genome and proteome have been widely explored for biomarker discovery and personalized medicine. However, there is still a large need for improved diagnostics and stratification strategies for a wide range of diseases. Post-translational modification of proteins by glycosylation affects protein structure and function, and glycosylation has been implicated in many prevalent human diseases. Numerous proteins for which the plasma levels are nowadays evaluated in clinical practice are glycoproteins. While the glycosylation of these proteins often changes with disease, their glycosylation status is largely ignored in the clinical setting. Hence, the implementation of glycomic markers in the clinic is still in its infancy. This is for a large part caused by the high complexity of protein glycosylation itself and of the analytical techniques required for their robust quantification. Mass spectrometry-based workflows are particularly suitable for the quantification of glycans and glycoproteins, but still require advances for their transformation from a biomedical research setting to a clinical laboratory. In this review, we describe why and how glycomics is expected to find its role in clinical tests and the status of current mass spectrometry-based methods for clinical glycomics.

Amino acid analysis using chromatography-mass spectrometry: An inter platform comparison study.

The analysis of amino acids has become a central task in many aspects. While amino acid analysis has traditionally mainly been carried out using either gas chromatography (GC) in combination with flame ionization detection or liquid chromatography (LC) with either post-column derivatization using ninhydrin or pre-column derivatization using o-phthalaldehyde, many of today's analysis platforms are based on chromatography in combination with mass spectrometry (MS). While derivatization is mandatory for the GC-based analysis of amino acids, several LC platforms have emerged, particularly in the dawn of targeted metabolite profiling using hydrophilic interaction liquid chromatography (HILIC) coupled to MS, allowing the analysis of underivatized amino acids. Among the numerous analytical platforms available for amino acid analysis today, we here compare three prominent approaches, being GC-MS and LC-MS after amino acid derivatization using chloroformate and HILIC-MS of underivatized amino acids. We compare and discuss practical issues as well as performance characteristics, e.g., the use of (13)C-labeled internal standards, of the different platforms and present data on their practical implementation in our laboratory. Finally, we compare the real-life applicability of all three platforms for a complex biological sample. While all three platforms are very-well suited for the analysis of complex biological samples they all show advantages and disadvantages for some analytes as discussed in detail in this manuscript.

Total plasma N-glycome changes during pregnancy.

During pregnancy, the mother faces a major immunological challenge. Most of the major plasma proteins have important immunological functions, and altered levels of these major proteins have been reported during pregnancy, potentially providing immunosuppression. A large number of the high abundance plasma proteins are post-translationally modified by N-glycans, and while it is now understood that these glycans may also affect the immunological functions, their pattern has not been studied in relation to pregnancy. Here, the N-glycosylation profile of 32 pregnant women was determined over the course of their pregnancy using a multiplexed CGE-LIF method. Moreover, for 6 women, the glycosylation profiles of the proteins IgG, IgA, and alpha1-antitrypsin were monitored. For total plasma, 16 glycan signals showed differential expression during pregnancy. In general the levels of largely sialylated bi-, tri-, and tetra-antennary glycans were increased during pregnancy, while biantennary glycans with no more than one sialic acid were decreased. Similarly altered glycosylation profiles were observed for the individual proteins IgG, with a decrease of digalactosylated biantennary glycans after delivery, and alpha1-antitrypsin, on which increased levels of triantennary glycans were observed during pregnancy. Overall, these results show altered glycosylation profiles both for total plasma glycoproteins and on individual proteins during pregnancy, which may contribute to immunosuppression and have other biological functions.

Glycan labeling strategies and their use in identification and quantification.

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.

Relevance and use of capillary coatings in capillary electrophoresis-mass spectrometry.

Over the last two decades, coupled capillary electrophoresis (CE)-mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool.

Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

Serum antibody screening by surface plasmon resonance using a natural glycan microarray.

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.

Antibody responses to Ascaris-derived proteins and glycolipids: the role of phosphorylcholine.

In addition to proteins, glycolipids can be targets of antibody responses and contribute to host-pathogen interaction. Following the structural analysis of Ascaris lumbricoides-derived glycolipids, the antibody responses of a group of children with no, light and heavy infections were analysed. The role of the phosphorylcholine moiety, present on Ascaris glycoproteins and glycolipids, in antibody reactivity of these infected individuals was determined. Children carrying heavy infections showed highest IgG reactivity to glycolipids compared to lightly or non-infected children. Substantial IgG antibody reactivity to both (glyco)proteins and glycolipids was found to be directed to the phosphorylcholine moiety as determined by either removal of this group or a competition assay. This was most pronounced for glycolipids, where removal of the phosphorylcholine moieties by hydrofluoric acid treatment abrogated IgG antibody reactivity. Measurement of IgG4 and IgE isotypes showed no IgG4 reactivity to Ascaris glycolipids, but raised IgE responses were detected in subjects with light or no Ascaris infections, suggesting that IgE responses to glycolipids may play a role in controlling parasite burden. Differences found in antibody profiles to glycolipids and (glyco)proteins, indicate that these different classes of compounds may have distinct roles in shaping of and interacting with humoral immune responses.

Mapping fucosylated epitopes on glycoproteins and glycolipids of Schistosoma mansoni cercariae, adult worms and eggs.

The developmental expression of the antigenic fucosylated glycan motifs Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X), and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) in Schistosoma mansoni cercariae, adult worms and eggs, was surveyed using previously defined anti-carbohydrate monoclonal antibodies (mAbs). Lewis X was found both on glycolipids and glycoproteins, yet with completely different expression patterns during the life-cycle: on glycolipids, Lewis X was mainly found in the cercarial stage, while protein-conjugated Lewis X was mainly present in the egg stage. Also protein-conjugated LDN-F and LDN-DF were most highly expressed in the egg-stage. On glycolipids LDN-DF was found in all three examined stages, whereas LDN-F containing glycolipids were restricted to adult worms and eggs. The motifs F-LDN and F-LDN-F were found both on glycoproteins and glycolipids of the cercarial and egg stage, while in the adult stage, they appeared to occur predominantly on glycolipids. Immunofluorescence assays (IFA) showed that these F-LDN and F-LDN-F containing glycolipids were localized in a yet undefined duct or excretory system of adult worms. Murine infection serum showed major reactivity with this adult worm duct-system, which could be fully inhibited by pre-incubation with keyhole limpet haemocyanin (KLH). Clearly, the use of defined mAbs provides a quick and convenient way to map expression profiles of carbohydrate epitopes.

Bovine lymphocytes express functional receptors for Escherichia coli Shiga toxin 1.

Interactions of Shiga toxins (Stxs) and immune cells contribute to the pathogenesis of diseases due to Stx-producing Escherichia coli (STEC) infections in humans and facilitate the persistence of infection in asymptomatically infected cattle. Our recent findings that bovine B and T lymphocytes express Gb(3)/CD77, the human Stx-receptor, prompted us to determine whether the bovine homologue also mediates binding and internalization of Stx1. In fact, Stx1 holotoxin and recombinant B subunit (rStxB1) bound to stimulated bovine peripheral blood mononuclear cells, especially to those subpopulations (B cells, BoCD8(+) T cells) that are highly sensitive to Stx1. Competition and HPTLC-binding studies confirmed that Stx1 binds to bovine Gb(3), but different receptor isoforms with varying affinities for rStxB1 were expressed during the course of lymphocyte activation. At least one of these isoforms mediated toxin uptake. An anti-StxB1 mouse monoclonal antibody, used as a model for bovine serum antibodies specific for Stx1, modulated rather than generally prevented rStxB1 binding to and internalization by the receptors. The presence of functional Stx1-receptors on bovine lymphocytes explains the immunomodulatory effect of Stx1 observed in cattle at a molecular level. Furthermore, expression of such receptors by bovine but not human T cells enlightens the background for the differential outcome of STEC infections in cattle and man, i.e., persistent infection and development of disease, respectively.

Globotriaosylceramide (Gb(3)/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro.

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb(3) syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb(3)/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD7+ cells was highest for BoCD8+ cells, followed by B-cells and BoCD4+ cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(alpha1-4)Gal(1-4)Glc(1-1)ceramide (Gb(3)). Biochemically, Gb(3) was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb(3) in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb(3) by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb(3)/CD77 predominantly by quantitative changes in the Gb(3) metabolism. This report presents Gb(3)/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.

Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.

Local variability of the phosphoglycerate kinase-triosephosphate isomerase fusion protein from Thermotoga maritima MSB 8.

The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.

The liver flukes Fasciola gigantica and Fasciola hepatica express the leucocyte cluster of differentiation marker CD77 (globotriaosylceramide) in their tegument.

Glycosphingolipids from the parasitic liver flukes Fasciola gigantica and Fasciola hepatica were isolated and their carbohydrate moieties were structurally analysed by methylation analysis, exoglycosidase treatment, on-target exoglycosidase cleavage and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively. From F. gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal beta4-Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal alpha4Gal beta4-Glc1-ceramide, which is designated as either globotriaosylceramide, Pk-blood group antigen or CD77 leucocyte cluster of differentiation antigen. To our knowledge, this is the first report on the expression of globo-series glycosphingolipids in non-mammalian species. Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2-hydroxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines. By the use of an anti-CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F. hepatica cryosections. The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.

Stage-associated expression of ceramide structures in glycosphingolipids from the human trematode parasite Schistosoma mansoni.

Glycosphingolipids of Schistosoma mansoni adults, cercariae and eggs comprise ceramide monohexosides (CMH) with glucose or galactose and ceramide dihexosides (CDH) with the schistosome-specific structure GalNAc(beta1-4)Glc(1-1)ceramide. Ceramide analysis revealed C18- and C20-phytosphingosines in egg CMH, C18-sphinganine as well as C18-, C19- and C20-phytosphingosines in cercarial CMH, and C18- and C20-phytosphingosines as well as C18-sphingosine and C18-sphinganine in adult CMH. For all three life cycle stages, the predominant fatty acid was C16h:0. As a characteristic feature, a range of saturated, unsaturated and hydroxylated long-chain fatty acids with 24-28 carbon atoms were additionally found in minor cercarial CMH species. The corresponding ceramides represented major constituents in cercarial CDH, while adult and egg CDH were dominated by ceramides with short fatty acid chains. The resultant ceramide patterns could be correlated with the differential expression of carbohydrate antigens on schistosomal glycolipids at various stages. A possible impact of ceramide structure on the biosynthesis of the carbohydrate moieties is discussed.

A fucose-containing epitope is shared by keyhole limpet haemocyanin and Schistosoma mansoni glycosphingolipids.

The glycolipids of Schistosoma mansoni adult worms, cercariae and eggs are recognised by schistosome infection serum and the monoclonal antibody M2D3H. The haemocyanin of the keyhole limpet, Megathura crenulata, is known to be immunoreactive to schistosomal infection sera and is, therefore, under investigation for the diagnosis of and vaccination against schistosomiasis. By dot-blot, inhibition-ELISA and inhibition-HPTLC immunostaining we have demonstrated that the M2D3H epitope is shared by both S. mansoni glycolipids and keyhole limpet haemocyanin (KLH). Analogously to the established epitopic importance of fucose to the immunorecognition of S. mansoni glycolipids, we have similarly defined the significance of the fucose residue(s) for the immunoreactivity between KLH and schistosomal infection serum and the monoclonal antibody M2D3H. Fucose was specifically removed from KLH by partial hydrolysis, monitored by ultrafiltration and carbohydrate component analysis. On removal of the fucose residue(s) the serological and immunological reactivity of KLH was greatly diminished, which implied that the fucose-containing M2D3H antigenic determinant was common to both S. mansoni glycolipids and KLH.