Human Natural Antibodies Recognizing Glycan Galß1-3GlcNAc (LeC).

The level of human natural antibodies of immunoglobulin M isotype against LeC in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors' blood using LeC-Sepharose (LeC is Galß1-3GlcNAcß). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with LeC, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10-9 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galß1-3GlcNAcß disaccharide, indicates that the target epitope of anti-LeC antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

Allergy-Protective Arabinogalactan Modulates Human Dendritic Cells via C-Type Lectins and Inhibition of NF-κB.

Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study was to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3 -: grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif -: containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs.

Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.

Klebsiella pneumoniae ST258 is a globally disseminated, extremely drug resistant, nosocomial clone with limited treatment options. We show that the vast majority of ST258 isolates express modified d-galactan-I lipopolysaccharide O-antigen, termed hereinafter as D-galactan-III. The genetic determinant required for galactan-III synthesis was identified as a distinct operon adjacent to the rfb (wb) locus encoding D-galactan-I synthesis. The three genes within the operon encode predicted glycosyltransferases. Testing an isogenic transformant pair revealed that expression of D-galactan-III, in comparison to D-galactan-I, conferred improved survival in the presence of human serum. Eighty-three percent of the more than 200 ST258 draft genome sequences currently available carries the corresponding operon and hence these isolates are predicted to express galactan-III antigens. A D-galactan-III specific monoclonal antibody (mAb) was shown to bind to extracted LPS from a panel of ST258 isolates. The same mAb confirmed accessibility of galactan-III in surface staining of ST258 irrespective of the distinct capsular antigens expressed by both clades described previously. Based on these data, the galactan-III antigen may represent an attractive target for active and passive immunization approaches against K. pneumoniae ST258.

Modulating Effects of Arabinogalactans from Plant Gum Exudates on Human Complement System.

Gum arabic and cashew nut tree gum exudate polysaccharide (CNTG) are plant polysaccharides composed of galactose and arabinose known as arabinogalactans (AGs). Although these fractions are used in food and pharmaceutical industry, cases of allergic reactions were described in clinical reports. As AGs were reported as modulators of the classical (CP) and alternative pathways (AP) of complement system (CS), in the present work, we investigate whether gum arabic and CNTG have an effect on both CS pathways. The complement fixation tests were performed with (CP-30 and AP-30) and without pre-incubation (CP-0 and AP-0). For CP-30, CNTG and gum arabic (833 µg/ml) showed a reduction of 28.0% (P = 0.000174) and 48.5% (P = 0.000143), respectively, on CP-induced haemolysis. However, no effect was observed for CP-0 in the CP-induced haemolysis. For AP-30, both CNTG and gum arabic (833 µg/ml) showed 87% reduction on the CP-induced haemolysis, with IC50 values of 100 and 7 µg/ml, respectively. For AP-0, a reduction of 11.3% for gum arabic and no effect for the CNTG on the CP-induced haemolysis were observed. These results suggested that gum arabic and CNTG could be acting as activators of the CS. Thus, this effect on the CS, especially on the AP, which accounts for up to 80-90% of total CS activation, indicates that both fractions may be harmful because of their potential pro-inflammatory action. Considering that CS activation induces inflammatory response, further studies confirming this immunomodulatory effect of these fractions are required to insure their safe use.

The Deconstruction of Pectic Rhamnogalacturonan I Unmasks the Occurrence of a Novel Arabinogalactan Oligosaccharide Epitope.

Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1→4)-linked galactose and (1→5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.

Anti-GalNAcß: a novel anti-glycan autoantibody associated with pregnancy loss in women with antiphospholipid syndrome and in a mouse experimental model.

OBJECTIVES: We evaluated the presence of anti-glycan antibodies (aGA) in patients with antiphospholipid syndrome (APS), and associations between aGA and clinical features of the disease. METHODS: Sera from APS patients and healthy controls were analyzed for aGA levels by ELISA. Analysis of the association of specific aGA with clinical manifestations of APS was performed. Selected aGA were affinity-purified and injected intravenously into naive mice which were tested for fetal loss. Matrigel invasion assay was performed for detection of choriocarcinoma cells (JAR) invasion and proliferation in the presence of selected aGA. Culture fluid of JAR invasion assays was analyzed for the presence of MMP2 and MMP9. RESULTS: High levels of several aGA were found in APS sera, of which anti-GalNAc-ß was significantly associated with recurrent pregnancy loss. Naive mice infused intravenously with anti-GalNAc-ß developed increased fetal loss. Anti-GalNAc-ß significantly inhibited the in-vitro percentage of JAR invasiveness and the secretion of MMP2 and MMP9 by human JAR cells. CONCLUSIONS: APS sera contain significant levels of aGA directed against several glycans. Anti-GalNAc-ß Ab is specifically associated with recurrent pregnancy loss both in human patients and experimental mouse model. The pathogenic effects of anti-GalNAc-ß include inhibition of JAR cells invasiveness accompanied by decreased MMP2 and MMP9 secretion.

A new allergen from ragweed (Ambrosia artemisiifolia) with homology to art v 1 from mugwort.

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.

Galactan-binding antibodies. Diversity and structure of idiotypes.

A group of eight IgM hybridoma proteins induced with beta(1,6)-D-galactan-containing antigens has been characterized in terms of primary amino acid sequence and idiotype expression. The H chain amino acid sequences reveal very strong homology in the VH segment although several substitutions are seen that suggest the occurrence of somatic mutation in these IgM molecules. Significant sequence variation was observed in CDR-3, the region generated by the D segment, and the two recombination events, VH-D and D-JH. The number of amino acids in this region contributed by the D segment was found to vary from two to six, yet the overall length of CDR-3 was precisely maintained by the addition of amino acids on either side of D during the recombination processes. These additional amino acids are suggested to result from nucleotide addition by repair enzymes. Idiotypic analysis of these proteins, in conjunction with an assessment of the H chain sequences, has permitted an identification of the molecular basis of both cross-reacting and unique idiotypic determinants expressed by these molecules.

Amino acids at the site of V kappa-J kappa recombination not encoded by germline sequences.

Murine V kappa-J kappa recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain VH-D-JH assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both kappa and heavy chain genes. We have examined the origin of an unusual amino acid, Ile, found at the site of V kappa-J kappa recombination in antigalactan antibodies, by sequence analysis of the corresponding rearranged and germline genes. Results indicate that the Ile codon can be generated by use of a single nucleotide 3' of the V kappa segment in combination with the second and third nucleotides of the first codon of J kappa 5 or J kappa 4. However, several antigalactan antibodies express Ile in combination with J kappa 2. An Ile codon cannot be generated by recombination in any reading frame between germline V kappa and J kappa 2 segments. These results suggest that the origin of the Ile codon in lines using J kappa 2 may represent a novel even in murine light chain assembly, possibly similar to the de novo addition of nucleotides observed in heavy chain gene recombination.

Immunochemical and molecular characterization of regulatory idiotopes expressed by monoclonal antibodies exhibiting or lacking beta 2-6 fructosan binding activity.

Hybridomas secreting antibodies bearing the ABPC48 (A48) regulatory idiotype (Id) were generated from BALB/c mice treated at birth or as adults with minute amounts of anti-A48-Id antibodies. The majority of these antibodies were recognized by the syngeneic monoclonal anti-A48-Id and anti-UPC-10-Id antibodies, IDA10 and 10-1, respectively. In Northern blotting experiments, most of these hybridomas were shown to use VH (heavy chain variable region) genes related to the 441-4 germline VH gene that encodes the A48 VH region. Hybridization was detected between polyadenylated H chain mRNA, isolated from the majority of the hybridomas, and the VH probe. Southern blots confirmed these results by showing a rearrangement of VH-related sequences to the JH (H chain joining segment) clusters on these same hybridomas. The antibodies from all of the hybridomas that derived from neonatal mice and half of those derived from adult mice showed specificity for fructosan determinants that, in most cases, was different from the beta 2-6 fructosan linkage specificity of A48. Surprisingly, several of the non-fructosan-binding hybridomas generated from the adult mice and the MOPC-173 myeloma demonstrated a clear specificity for the beta 1-6-D-galactan determinant. Of four galactan-binding myeloma proteins studied. XRPC 44 alone shared idiotypy with the UPC-10 myeloma. These findings suggest a possible clonal crossreactive regulation mediated by regulatory idiotopes. The crossreactive regulation concept is discussed.

Inducing inflammatory response in RAW264.7 and NK-92 cells by an arabinogalactan isolated from Ferula gummosa via NF-κB and MAPK signaling pathways.

The polysaccharide isolated from F. gummosa (FGP) was found homogenous with a weight average molecular weight (Mw) of 50.0 × 103 g/mol and radius of gyration (Rg) of 105.3 nm. The FGP was an arabinogalactan with a backbone formed of →6)-ß-Galp-1→ residues having random branching points at C-3 extended with either ß-Galp-(1→3)-ß-Galp-(1→ or α-Araf-(1→ side chain residues. FGP exhibited proliferative effect on RAW264.7 cells and induced macrophages to exert proinflammatory response releasing NO and up-regulating the transcription of cytokines including TNF-α, IL-1ß, IL-6 and IL-12. The FGP induced NK-92 cells to up-regulate the expressions of TNF-α, IFN-γ, granzyme-B, perforin, NKG2D and FasL. The presence of p-NF- κB, p-ERK, p-JNK and p-p38 in RAW264.7 and NK-92 cells indicated their activation through NF-κB and MAPKs signaling pathways. These findings suggested that polysaccharides from F. gummosa are potent in boosting immune system and thus may be considered for further studies of biomedical applications.

A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants.

We have identified a family of abundant peripheral plasma membrane glycoproteins that is unique to flowering plants. They are identified by a monoclonal antibody, MAC 207, that recognizes an epitope containing L-arabinose and D-glucuronic acid. Immunofluorescence and immunogold labeling studies locate the MAC 207 epitope to the outer surface of the plasma membrane both in protoplasts and in intact tissues. In some cells MAC 207 also binds to the vacuolar membrane, probably reflecting the movement of the plasma membrane glycoproteins in the endocytic pathway. The epitope recognized by MAC 207 is also present on a distinct soluble proteoglycan secreted into the growth medium by carrot (Daucus carota) suspension culture cells. Biochemical evidence identifies this neutral proteoglycan as a member of the large class of arabinogalactan proteins (AGPs), and suggests a structural relationship between it and the plasma membrane glycoproteins. AGPs have the property of binding to beta-glycans, and we therefore propose that one function of the AGP-related, plasma membrane-associated glycoproteins may be to act as cell surface attachment sites for cell wall matrix polysaccharides.

Bifidobacterium bifidum presents on the cell surface a complex mixture of glucans and galactans with different immunological properties.

The chemical structure of cell surface polysaccharides isolated from Bifidobacterium bifidum strain PRI1, an important member of the gut microbiota of breast-fed infants, has been elucidated by chemical and NMR spectroscopy analysis. Results demonstrated that the bacterium produces a complex mixture of polysaccharides that could be classified in two main groups: a phospho-glycero-ß-galactofuranan, PGßG, and a mixture composed of four neutral polysaccharides named as (CSGG), composed of ß-(1 → 6)-glucan, ß-(1 → 4)-galactan, ß-(1 → 6)-galactan, ß-galactofuranan and starch. These two fractions exerted different immune responses when assayed on dendritic cells: PGßG enhanced pro-inflammatory immune responses by increasing interferon-γ levels while CSGG induced immunosuppressive regulatory T cells and interleukin-10. These findings demonstrate that bacterial polysaccharides have a distinct role depending on their chemical structure in regulation of the host/bacterium interaction. Our findings suggest that polysaccharides may differentially regulate the host immunity depending on the composition of this complex mixture, either enhancing immunity or inducing immune tolerance.

Chemical and immunological modifications of an arabinogalactan present in tea preparations of Phyllanthus niruri after treatment with gastric fluid.

An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated and presented immunological properties when tested with peritoneal mice macrophages. AG was now submitted to acidic and neutral gastric conditions using human gastric fluids and aq. HCl solution. Since the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These were analyzed using (13)C NMR, HPSEC, and GC-MS for monosaccharide composition and methylation analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S presented the monosaccharides released from the native polymer and some oligosaccharides as shown by methylation data. AG-P contained larger molecular fragments comprising the internal units from AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3-fold, at 250 microg/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response was observed, revealing a structure-activity relation. The significance of the results is that most plant extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of phytomedicine.

Spatial control of cell envelope biosynthesis in mycobacteria.

The mycobacterial cell envelope is a complex multilayered structure that provides the strength to the rod-shaped cell and creates the permeability barrier against antibiotics and host immune attack. In this review, we will discuss the spatial coordination of cell envelope biosynthesis and how plasma membrane compartmentalization plays a role in this process. The spatial organization of cell envelope biosynthetic enzymes as well as other membrane-associated proteins is crucial for cellular processes such as polar growth and midcell septum formation. We will highlight metabolic enzymes involved in the localized biosynthesis of envelope components such as peptidoglycan, arabinogalactan and outer/inner membrane lipids. The known and potential roles of cytoskeletal and coiled coil proteins in driving subcellular protein localization will also be summarized. Finally, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain, recently revealed by biochemical fractionation and fluorescence microscopy. We consider how this dynamic and multifunctional membrane microdomain contributes to the subcellular localization of membrane proteins and spatially restricted cell envelope biosynthesis in mycobacteria.

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.).

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.