The mannose receptor mediates dengue virus infection of macrophages.

Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.

Detailed N-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation.

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognises both mannosylated and sulphated ligands through its C-type lectin domains (CTLDs) and cysteine-rich (CR) domain, respectively. It is widely expressed among different tissues and by certain cell types in vivo. Our previous study suggested that the glycosylation, especially terminal sialylation, regulated the functional specificities of MR. In the current investigation, the distribution of MR among various mouse tissues was studied and the N-linked glycosylation of spleen MR was analysed. Our results showed that spleen expressed the most abundant MR, consistent with its wide distribution in different cell types in this organ. Spleen MR was heterogeneously N-glycosylated. The majority of the glycans were sialylated in the alpha2 --> 6-linkage and both Neu5Ac and Neu5Gc sialic acids were detected. Most glycans were bi-antennary (74%) with approximately 22% tri-antennary and most were core fucosylated (68%). About 13% contained alpha-galactose. In the lung, MR exhibited more terminal sialic acids in the alpha2 --> 3- rather than in the alpha2 --> 6-configuration. Our study provides a profile of MR N-linked glycosylation that will facilitate our understanding of their physiological role on MR biology in vivo.

GlycoBase and autoGU: tools for HPLC-based glycan analysis.

SUMMARY: The development of robust high-performance liquid chromatography (HPLC) technologies continues to improve the detailed analysis and sequencing of glycan structures released from glycoproteins. Here, we present a database (GlycoBase) and analytical tool (autoGU) to assist the interpretation and assignment of HPLC-glycan profiles. GlycoBase is a relational database which contains the HPLC elution positions for over 350 2-AB labelled N-glycan structures together with predicted products of exoglycosidase digestions. AutoGU assigns provisional structures to each integrated HPLC peak and, when used in combination with exoglycosidase digestions, progressively assigns each structure automatically based on the footprint data. These tools are potentially very promising and facilitate basic research as well as the quantitative high-throughput analysis of low concentrations of glycans released from glycoproteins. AVAILABILITY: http://glycobase.ucd.ie

Remarkable selective glycosylation of the immunoglobulin variable region in follicular lymphoma.

Follicular lymphoma (FL) generally expresses immunoglobulin (Ig) with somatically mutated variable (V) region genes. Surprisingly, these almost always carry introduced motifs available for N-glycosylation (Asn-X-Ser/Thr). Introduced motifs are uncommon on normal B cells, but are on other germinal center (GC)-associated B-cell malignancies suggesting a site-specific role. They are not evident in mutated chronic lymphocytic leukemia (CLL) or myeloma. Recently, we found that the glycosylation sites are unusual in containing oligomannose glycans, which are apparently displayed on tumor cell surface IgM. This suggests a potential interaction with a mannose receptor in the GC. However, natural N-glycosylation sites exist in germline (GL) V region genes, particularly the V4-34 gene expressed by normal B cells and by some malignancies, including CLL, potentially undermining the selective importance for FL. To compare oligosaccharide addition at the introduced and natural sites, we expressed V region genes as single chain Fv (scFv) and analyzed the added glycans. In contrast to introduced sites, which were oligomannosylated, the natural GL motif in the V4-34 sequence had no added sugars. The remarkable selective glycosylation within the heavy chain V region gene of FL apparently permits only limited processing to oligomannose at somatically mutated motifs, creating a feature exploitable by GC lymphomas.

A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression.

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.

Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry.

Negative ion electrospray (ESI) fragmentation spectra derived from anion-adducted glycans were evaluated for structural determination of N-linked glycans and found to be among the most useful mass spectrometric techniques yet developed for this purpose. In contrast to the more commonly used positive ion spectra that contain isobaric ions formed by losses from different regions of the molecules and often lead to ambiguous deductions, the negative ion spectra contain ions that directly reflect structural features such as the branching pattern, location of fucose, and the presence of bisecting GlcNAc. These structural features are sometimes difficult to determine by traditional methods. Furthermore, the spectra give structural information from mixtures of isomers and from single compounds. The method was evaluated with well-characterized glycans from IgG and used to explore structures of N-linked glycans released from serum glycoproteins with the aim of identifying biomarkers for cancer. Quantities of glycans were measured by ESI and by matrix-assisted laser desorption ionization mass spectrometry; each technique produced virtually identical results for the neutral desialylated glycans.

HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software.

We present a robust, fully automatable technology platform that includes computer software for the detailed analysis of low femtomoles of N-linked sugars released from glycoproteins. Features include (i) sample immobilization in 96-well plates, glycan release, and fluorescent labeling; (ii) quantitative HPLC analysis, including monosaccharide sequence, linkage, and arm-specific information for charged and neutral glycans; (iii) automatic structural assignment of peaks from HPLC profiles via web-based software that accesses our database (GlycoBase) of more than 350 N-glycan structures, including 117 present in the human serum glycome; and (iv) software (autoGU) that progressively analyzes data from exoglycosidase digestions to produce a refined list of final structures. The N-glycans from a plate of 96 samples can be released and purified in 2 or 3 days and profiled in 2 days. This strategy can be used for (i) identification and screening of disease biomarkers and (ii) monitoring the production of therapeutic glycoproteins, allowing optimization of production conditions. This technology is also suitable for preparing released glycans for other analytical techniques. Here we demonstrate its application to rheumatoid arthritis using 5 microl of patient serum.

Separation-based glycoprofiling approaches using fluorescent labels.

Glycoprotein analysis is essential within the biopharmaceutical industry, as the structure of the different glycans present can affect the safety and efficacy of products. However analysis of cleaved glycans presents a major analytical challenge, due to their inherent complexity, lack of chromophore and the existence of various isoforms (both position and linkage). In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants, so the released glycan pool contains a range of structures. Both normal phase chromatography and capillary gel electrophoresis offer excellent selectivity for the analysis of fluorescently labelled glycans. The normal phase (NP) chromatographic approach is sensitive, reliable and well established, with databases available for searching structures assigned relative to retention times. Capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) offers faster analysis times, though currently no databases are available to search mobilities against structures, therefore data has to be cross-correlated with either normal phase chromatography or mass spectrometry approaches when developing and validating methods. The principles of both methods are described and a review is presented that includes evaluation against a set of criteria established through consultation with the biopharmaceutical industry.

Human complement factor I glycosylation: structural and functional characterisation of the N-linked oligosaccharides.

Factor I (fI) is a key serine protease that modulates the complement cascade by regulating the levels of C3 convertases. Human fI circulates in plasma as a heavily N-glycosylated (25-27% w/w) heterodimer composed of two disulphide linked chains, each carrying three N-linked oligosaccharide chains. It had been suggested that the oligosaccharides may have both structural and functional roles in the interactions with the natural substrate and the cofactor during a catalysis. The N-linked glycans of each fI chain were characterised in detail and the analysis revealed a similar composition of the glycan pools with both chains heavily sialylated. Disialylated structures were in excess over monosialylated ones: 55% over 40% for the heavy chain and 62% over 35% for the light chain. The dominant type of glycan identified on both chains was A(2)G(2)S(2), a biantennary structure with chains terminating in sialic acid linked to galactose. The glycan characterisation facilitated a strategy for the partial deglycosylation of the enzyme. Assessment of the proteolytic activities of the native and partially deglycosylated forms of fI showed that both forms of the enzyme have very similar proteolytic activities against C3(NH(3)) indicating that the charged glycans of fI do not influence the fI-cofactor-substrate interactions.

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation.

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.

Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions.

In contrast to the linear sequences of protein and DNA, oligosaccharides are branched structures. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. Glycan analysis therefore requires high-resolution separation techniques that can provide detailed structural analysis, including both monosaccharide sequence and linkage information. This chapter describes how a combination of high-performance liquid chromatography (HPLC) and exoglycosidase enzyme array digestions can deliver quantitative glycan analysis of sugars released from glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel bands by matching HPLC elution positions with a database of standard glycans.

Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids.

TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED.

Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations.

Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB cell surface molecules. After 2 h of culture, bone marrow mononuclear cells had a phenotype of CD44(hi), CD29(hi), CD90(lo), CD11a/CD18(hi), and CD45RB(lo). Isolated mononuclear cells were analyzed by flow cytometry and RT-qPCR at 2, 7, 14, 21, and 30 days of culture. At all culture time points, gene expression was in agreement with cell surface protein expression. In established cultures of MPCs, cells remained robustly positive for CD44 and CD29. The proportion of positive cells and the mean fluorescence intensity of positive cells increased in CD90 expression as MPC cultures became more homogeneous. Inversely, the population of cells in culture decreased expression of CD11a/CD18 and CD45RB molecules over time. The decreased expression of the latter molecules makes these useful negative markers of established MPC cultures under normal expansion conditions. The results of this study demonstrate numerous dynamic changes in cell surface molecule expression during early establishment of MPC populations, which may aid to improve MPC isolation methods for research or therapeutic applications.

Differentiation between isomeric triantennary N-linked glycans by negative ion tandem mass spectrometry and confirmation of glycans containing galactose attached to the bisecting (beta1-4-GlcNAc) residue in N-glycans from IgG.

Negative ion tandem mass spectrometry (MS/MS) spectra of three isomeric triantennary N-linked glycans provided clear differentiation between the isomers and confirmed the occurrence of an isomer that was substituted with galactose on a bisecting GlcNAc (1 --> 4-substituted on the core mannose) residue recently reported by Takegawa et al. from N-glycans released from human immunoglobulin G (IgG). We extend this analysis of human serum IgG to reveal an analogue of the fucosylated triantennary glycan reported by Takegawa et al. together with a third compound that lacked both the sialic acid and the fucose residues. In addition, we demonstrate the biosynthesis of bisected hybrid-type glycans with the galactose modification, with and without core fucose, on the stem cell marker glycoprotein, 19A, expressed in a partially ricin-resistant human embryonic kidney cell line. It would appear, therefore, that this modification of N-linked glycans containing a galactosylated bisecting GlcNAc residue may be more common than originally thought. Negative ion MS/MS analysis of glycans is likely to prove an invaluable tool in the analysis and monitoring of therapeutic glycoproteins.

Changes of serum glycans during sepsis and acute pancreatitis.

Acute inflammatory response is a complex process associated with the production of both pro- and anti-inflammatory mediators. Although it is generally considered to be a single homeostatic mechanism, there are differences associated with the nature and the site of inflammation. We examined the changes of N-linked glycans released from the serum of a patient with sepsis and a patient with acute pancreatitis during the first eight days of the disease. Sera were taken from patients at the time of reporting to hospital and then three more times. The blood from a healthy individual was drawn on one occasion only. Glycans were released using N-glycosidase F and were subjected to normal phase and weak anion exchange high-performance liquid chromatography, exoglycosidase digestions, and mass spectrometry. The levels of identified structures have been followed through the course of disease and compared to the control levels. Changes in serum glycans were found to occur very early in acute inflammation. The most prominent differences include the increase in ratio of outer arm to core fucose, increase in the amount of tetrasialylated structures, changes in the levels of mannose structures, and in the degree of branching. The relative proportions of different glycans changed daily and some differences were also observed between sepsis and pancreatitis, probably reflecting that in these two conditions, the acute phase response is triggered by a different stimulus that is associated with different patterns of production of cytokines.

Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer.

Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.

Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.

Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions.

Human follicular lymphoma cells contain oligomannose glycans in the antigen-binding site of the B-cell receptor.

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Oligosaccharides modulate the apoptotic activity of glycodelin.

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.

A comparison of laryngoplasty and modified partial arytenoidectomy as treatments for laryngeal hemiplegia in exercising horses.

OBJECTIVE: To compare upper airway mechanics, arterial blood gases, and tracheal contamination in horses with induced left laryngeal hemiplegia (recurrent laryngeal neuropathy [RLN]) treated by laryngoplasty/vocal cordectomy (LPVC) or modified partial arytenoidectomy (MPA). STUDY DESIGN: Repeated measures under the following conditions: Control, RLN, LPVC, and MPA. ANIMALS: Six horses. METHODS: Two trials were conducted under all conditions at 80% and 100% of maximal heart rate (HR(max)). In Trial 1, arterial blood gases, tracheal and pharyngeal pressures, and laryngeal videoendoscopy were recorded. In Trial 2, upper airway pressure and airflow were determined. Tracheobronchial aspirates were performed after exercise to quantify airway contamination. RESULTS: Compared with control, RLN significantly increased inspiratory impedance and worsened exercise-induced hypoxemia. At 80% HR(max), LPVC restored most variables to control values. At 100% HR(max), LPVC improved all variables, but did not restore minute volume, arterial pH, and PaCO(2). At 80% HR(max), MPA restored all variables except bicarbonate to control values. At 100% HR(max), MPA improved all variables, but did not statistically restore minute ventilation or bicarbonate level. Only minor differences were noted between LPVC and MPA. Both resulted in equivalent tracheal contamination. CONCLUSIONS: Airway mechanics and arterial blood gas values were not restored to normal after either LPVC or MPA in horses exercising at HR(max). This does not affect ventilation at sub-maximal exercise, but has clinical implications at HR(max). Both procedures diminish normal laryngeal protective mechanisms. CLINICAL RELEVANCE: At sub-maximal exercise intensities both LPVC and MPA restore airway ventilation to normal. At maximal exercise the superiority of LPVC over MPA is slight.