Carbohydrate-binding properties of human neo-CRP and its relationship to phosphorylcholine-binding site

Binding characteristics of two types of ligands for human neo-C-reactive protein (neo-CRP), which is a conformationally altered but physiologically relevant form of CRP, were studied fluorometrically by probing CRP immobilized on a polystyrene surface with europium-labeled ligands. Two Eu-ligands used were bovine serum albumin derivatives that contain on average 40 residues of ligand structures, one derivative containing phosphorylcholine (PC) and the other lactosyl residues. The PC-containing ligands required the presence of calcium for binding, whereas galactose-containing derivatives bound in the absence of calcium. The optimal pH for the PC-dependent binding was broad (pH 6-8), whereas the best binding pH for the galactose-dependent binding was around 6. The carbohydrate-mediated binding is rather nonspecific: the binding site prefers galactose configuration, but other hexoses can be accommodated. The two best monosaccharide inhibitors at this site were galactose-6-phosphate and galacturonic acid, suggesting the importance of having a negatively charged group at C-6 position of galactose. In fact, the phosphate-binding site is common to both PC and sugar phosphates, and the choline- and the sugar-binding sites are probably located on either side of the phosphate-binding site. Binding characteristics of Eu-labeled PC-BSA to neo-CRP are quite similar to that found for native CRP in solution phase [Lee et al. (2002) J. Biol. Chem., 277, 225-232], whereas binding of sugar phosphates by neo-CRP shows considerably less stringent requirements compared to native CRP. For instance, galactose-alpha1-phosphate was not inhibitory at all in the native CRP binding assay, whereas it was a good inhibitor in the neo-CRP assay.     

Differential affinities of <Emphasis Type="Italic">Erythrina cristagalli</Emphasis> lectin (ECL) toward monosaccharides and polyvalent mammalian structural units

Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galβ1→4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 × 10⁴, 3.9 × 10³ and 2.4 × 10³ more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galβ1-related structures play critically important roles in lectin binding. Mapping all information available, it can be concluded that: [a] Galβ1→4GlcNAc (II) and some Galβ1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with Galβ1→4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear trisaccharide, Galβ1→4GlcNAcβ1→6Gal. These analyses should facilitate the understanding of the binding function of ECL. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galα1-4GalNAcβ1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galα1-4GalNAcβ1-3Gal (NOR-tri), and weakly by Galα1-4Gal. However, the type 1 antibodies are strongly inhibited by Galα1-4Galβ1-3Gal-R and weakly by Galα1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galα1-4GalNAcβ1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several α-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-αGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.     

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-αGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-³H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1 → 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory for antibody recognition. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Native and modified C-reactive protein bind different receptors on human neutrophils

Native C-reactive protein (CRP) is a planar pentamer of identical subunits expressed at high serum levels during the acute phase of inflammation. At inflammatory sites, an isomeric form termed modified CRP (mCRP) is expressed and reveals neoantigenic epitopes associated with modified monomeric CRP subunits. mCRP cannot assume the native pentameric conformation but rather forms a nonpentameric aggregate of monomers. While native CRP inhibits neutrophil movement in vitro and in vivo, the effect of mCRP on neutrophil movement has not been reported. To model the biological function and biochemical interaction of mCRP on neutrophils, in vitro chemotaxis and binding experiments were performed using mCRP. Reported here, mCRP effectively inhibited fMLP-induced chemotaxis similar to native CRP. Additionally, mCRP increased binding of labeled native CRP to neutrophils. This increased binding occurred by direct protein-protein interaction of the C-terminus thereby implicating the CRP(199-206) sequence. Binding of mCRP to neutrophils was blocked by anti-CD16 monoclonal antibody whereas native CRP was not. These results suggest that modified forms of CRP inhibit chemotaxis, a function similar to native CRP, but that mCRP and native molecule bind different receptors on human neutrophils.     

Mapping the binding areas of human C-reactive protein for phosphorylcholine and polycationic compounds. Relationship between the two types of binding sites.

We developed a fluorescence-based assay method for determining ligand binding activities of C-reactive protein (CRP) in solution. Using this method, we compared the phosphorylcholine (PC)- and polycation-based binding activities of human CRP. The PC-based binding required calcium, whereas a polycation (e.g. poly-l-lysine) was bound in the presence of either calcium or EDTA, the binding being stronger in the presence of EDTA. The published crystallographic structures of CRP and the CRP.PC complex show it to be a ring-shaped pentamer with a single PC-binding site per subunit facing the same direction. As expected from such a structure, binding affinity of a ligand increased tremendously when multiple PC residues were present on a macromolecular structure. In addition to PC-related structures, certain sugar phosphates (e.g. galactose 6-phosphate) are bound near the PC-binding site, and one of the sugar hydroxyl groups appears to interact with CRP. The best small ligands for the polycationic binding site were Lys-Lys and Lys4. Because of the presence of multiple Lys-Lys sequences, polylysines have tremendously enhanced affinity. Although PC inhibits both PC- and polycation-based binding, none of the amines that inhibit polylysine binding inhibits PC binding, suggesting that the PC and polycationic binding sites do not overlap.     

The Binding of Monomeric C-Reactive Protein (mCRP) to Integrins αvβ3 and α4β1 Is Related to Its Pro-Inflammatory Action

The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.     

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(±6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.     

Lectin Binding Assays for In-Process Monitoring of Sialylation in Protein Production

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.     

Conjugation of oligosaccharides by reductive amination to amine modified chondroitin oligomer and γ-cyclodextrin

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell–cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading

Primary open angle glaucoma (POAG) is a major blindness-causingdisease, characterized by elevated intraocular pressure dueto an insufficient outflow of aqueous humor. The trabecularmeshwork (TM) lining the aqueous outflow pathway modulates theaqueous outflow facility. TM cell adhesion, cell–matrixinteractions, and factors that influence Rho signaling in TMcells are thought to play a pivotal role in the regulation ofaqueous outflow. In a recent study, we demonstrated that galectin-8(Gal8) modulates the adhesion and cytoskeletal arrangement ofTM cells and that it does so through binding to β₁ integrinsand inducing Rho signaling. The current study is aimed at thecharacterization of the mechanism by which Gal8 mediates TMcell adhesion and spreading. We demonstrate here that TM cellsadhere to and spread on Gal8-coated wells but not on galectin-1(Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TMcells to Gal8-coated wells was abolished by a competing sugar,β-lactose, but not by a noncompeting sugar, sucrose. Also,a trisaccharide, NeuAc2-3Galβ1-4GlcNAc, which binds specificallyto the N-CRD of Gal8, inhibited the spreading of TM cells toGal8-coated wells. In contrast, NeuAc2-6Galβ1-4GlcNAc whichlacks affinity for Gal8 had no effect. Affinity chromatographyof cell extracts on a Gal8-affinity column and binding experimentswith plant lectins, Maakia Amurensis and Sambucus Nigra, revealedthat ₃β₁, ₅β₁, and _vβ₁ integrins are major counterreceptorsof Gal8 in TM cells and that TM cell β₁ integrins carrypredominantly 2-3-sialylated glycans, which are high-affinityligands for Gal8 but not for Gal1 or Gal3. These data lead usto propose that Gal8 modulates TM cell adhesion and spreading,at least in part, by interacting with 2-3-sialylated glycanson β₁ integrins.     

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)—based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galβ1-3GalNAcα- and Galβ1-3GalNAcβ-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcα-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC₅₀ values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 × 10⁻⁸ M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcβ and GalNAcβ1-3GalNAcβ ligands was found in human serum. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural and immunochemical identification of Lea, Leb, H type 1, and related glycolipids in small intestinal mucosa of a group O Le(a-b-) nonsecretor

Total nonacid glycosphingolipids were isolated from small intestine mucosal scrapings of a red cell blood group O Le(a-b-) nonsecretor cadaver. Glycolipids were extracted and fractionated into five fractions based on chromatographic and immunostaining properties. These glycolipid fractions were then analysed by thin-layer chromatography for Lewis activity with antibodies reactive to the type 1 precursor (Lec), H type 1 (Led), Lea and Leb epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and EI-MS/MS-TOF) and proton NMR spectroscopy. EI-MS/MS-TOF allowed for the identification of trace substances in fractions containing several other glycolipid species. Consistent with the red cell phenotype, large amounts of lactotetraosylceramide (Lec-4) were detected. Inconsistent with the red cell phenotype, small quantities of Lea-5, H-5-1 and Leb-6 glycolipids were immunochemically and structurally identified in the small intestine of this individual. By EI-MS/MS-TOF several large glycolipids with 9 and 10 sugar residues were also identified. The extensive carbohydrate chain elongation seen in this individual with a Lewis negative nonsecretor phenotype supports the concept that Lewis and Secretor blood group fucosylation may be a mechanism to control type 1 glycoconjugate chain extension. Abbreviations: FUT1, H gene; FUT2, Secretor gene, (gene bank accession no. U17894); FUT3, Lewis gene or Fuc-TIII gene, (gene bank accession no. X53578); FUT5, Fuc-TV gene; [Imm]+, immonium ion; Lea-5, Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Leb-6, Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lec-4, Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Led or H-5-1, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lex-5, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; MAb, monoclonal antibody; MS, mass spectrometry; CID, collision-induced dissociation; EI, electron impact ionisation; MS/MS-TOF, tandem mass spectrometry using a time-of-flight mass spectrometer as the second mass spectrometer: m/Cz, mass-to-charge ratio; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; TLC, (high performance) thin layer chromatography. Saccharide types are abbreviated to Hex for hexose, HexNAc for N-acetylhexosamine and dHex for deoxyhexose (fucose). Ceramide is abbreviated to Cer, and ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids This revised version was published online in November 2006 with corrections to the Cover Date.     

Exquisite binding specificity of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> lectin toward TF-related O-linked mucin-type glycans

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galβ1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to “α-anomers” but not the “β-anomers” of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.     

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal ß-Gal residueswas investigated by screening sulfotransferase activity presentin 37 human tissue specimens toward the following synthesizedacceptor moieties: Galß1,3GalNAc-O-Al, Galß1,4GlcNAcß-O-Al,Galß1,3GlcNAcß-O-Al, and mucin-type Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnstructures containing a C-3 methyl substituent on either Gal.Two distinct types of Gal: 3-O-sulfotransferases were revealed.One (Group A) was specific for the Galß1, 3GalNAc-linkage and the other (Group B) was directed toward the Galß1,4GlcNAcbranch ß1,6 linked to the blood group T hapten. Enzymeactivities found in breast tissues were unique in showing astrict specificity for the T-hapten. Galß-O-allylor benzyl did not serve as acceptors for Group A but were veryactive with Group B. An exainination of activity present insix human sera revealed a specificity of the serum enzyme towardß1,3 linked Gal, particularly, the T-hapten withoutß1,6 branching. Group A was highly active toward T-haptenlacrylamidecopolymer, anti-freeze glycoprotein, and fetuin O-glycosidicasialo glycopeptide; less active toward fetuin triantennaryasialo glycopeptide; and least active toward bovine IgG diantennaryglycopeptide. Group B was moderately and highly active, respectively,with the latter two glycopeptides noted and least active withthe first two. Competition experiments performed with Galß1,3GaLNAc-O-Aland Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnhaving a C-3 substituent (methyl or sulfate) on either Gal reinforcedearlier findings on the specificity characteristics of GroupA and Group B. Group A displayed a wider range of optimal activity(pH 6.0–7.4), whereas Group B possessed a peak of activityat pH 7.2. Mg²⁺ stimulated Group A 55% and Group B 150%, whereasMn⁺² stimulated Group B 130% but inhibited Group A 75%. Ca²⁺stimulated Group B 100% but inhibited Group A 35%. Group A andGroup B enzymes appeared to be of the same molecular size (<100,000Da) as observed by Sephacryl S-100 HR column chromatography.The following effects upon Gal: 3-O- sulfotransferase activitiesby fucose, sulfate, and other substituents on the carbohydratechains were noted. (1) A methyl or GlcNAc substituent on C-6of GalNAc diminished the ability of Galß1,3GalNAc-O-Alto act as an acceptor for Group A. (2) An 1,3-fucosyl residueon the ß1,6 branch in the mucin core structure didnot affect the activity of Group A toward Gal linked ß1,3to GalNAc-. (3) Lewis x and Lewis a terminals did not serveas acceptors for either Group A or B enzymes. (4) Eliminationof Group B activity on Gal in the ß1,6 branch owingto the presence of a 3-fucosyl or 6-sulfo group on GlcNAc didnot hinder any action toward Gal linked ß1,3 to GalNAc.(5) Group A activity on Gal linked ß1,3 to GalNAcremained imaffected by 3'-sulfation of the ß1,6 branch.The reverse was true for Group B. (6) The acceptor activityof the T-hapten was increased somewhat upon C-6 sulfation ofGalNAc, whereas, C-6 slalylation resulted in an 85% loss ofactivity. (7) A novel finding was that Galß1,4GlcNAcß-O-Aland Galß1,3GlcNAcß-O-M, upon C-6 sulfationof the GlcNAc moiety, became 100% inactive and 5- to 7-foldactive, respectively, in their ability to serve as acceptorsfor Group B. human tissues glycoprotein galactose:sulfotransferase specificities kinetic properties     

Conversion of native oligomeric to a modified monomeric form of human C-reactive protein

C-reactive protein (CRP) is a pentameric oligoprotein composed of identical 23 kD subunits which can be modified by urea-chelation treatment to a form resembling the free subunit termed modified CRP (mCRP). mCRP has distinct physicochemical, antigenic, and biologic activities compared to CRP. The conditions under which CRP is converted to mCRP, and the molecular forms in the transition, are important to better understand the distinct properties of mCRP and to determine if the subunit form can convert back to the pentameric native CRP form. This study characterized the antigenic and conformational changes associated with the interconversion of CRP and mCRP. The rate of dissociation of CRP protomers into individual subunits by treatment in 8 M urea–10 mM EDTA solution was rapid and complete in 2 min as assayed by an enzyme-linked immunofiltration assay using monoclonal antibodies specific to the mCRP. Attempts to reconstitute pentameric CRP from mCRP under renaturation conditions were unsuccessful, resulting in a protein retaining exclusively mCRP characteristics. Using two-dimensional urea gradient gel electrophoresis, partial rapid unfolding of the pentamer occurred above 3 M urea, a subunit dissociation at 6 M urea, and further subunit unfolding at 6–8 M urea concentrations. The urea gradient electrophoresis results suggest that there are only two predominant conformational states occurring at each urea transition concentration. Using the same urea gradient electrophoresis conditions mCRP migrated as a single molecular form at all urea concentrations showing no evidence for reassociation to pentameric CRP or other aggregate form. The results of this study show a molecular conversion for an oligomeric protein (CRP) to monomeric subunits (mCRP) having rapid forward transition kinetics in 8 M urea plus chelator with negligible reversibility.     

The study of the substrate specificity of rat-brain fucosyltransferase using synthetic acceptors

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Galβ1-4GlcNAcβ1-R fragment were subjected to enzymic fucosylation. The isomer with a 1–3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated, whereas Fucα1-2Galβ1-4GlcNAc displayed the same substrate properties as Galβ1-4GlcNAc. FT from the cerebellum and forebrain was shown to have a specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from the forebrain, the specificity profiles being similar. This communication is dedicated to the 70th birthday of Prof. A.Ya. Khorlin.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> mucoid strain 8830 binds glycans containing the sialyl-Lewis x epitope

Pseudomonas aeruginosa infection of patients with cystic fibrosis (CF) is a leading cause of their morbidity and mortality. Pathogenesis is initiated in part by molecular interactions of P. aeruginosa with carbohydrate residues in airway mucins that accumulate in the lungs of patients with this disease. To explore the nature of the glycans recognized by a stable, mucoid, alginate-producing strain P. aeruginosa 8830 we generated a genetically modified Pa8830 expressing green fluorescent protein (Pa3380-GFP). We tested its binding to a panel of glycolipids and neoglycolipids in which selected glycans were covalently attached to dipalmitoyl phosphatidylethanolamine and analyzed on silica gel surfaces. Among all glycans tested, Pa8830-GFP bound best to sialyl-Le^x-containing glycan NeuAc(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R and bound weakly to H-type blood group Fucα1-2Galβ1-4GlcNAc-R, sialyl-lactose, and Le^x, and exhibited little binding toward non-fucosylated derivatives. Interestingly, while Pa8830-GFP bound to the glycosphingolipid asialoGM1, it did not appear to bind to a wide variety of other glycosphingolipids including GM1, GM2, asialoGM2, and sulfatide. These results indicate that P. aeruginosa 8830 has preferential binding to sialyl-Le^x-containing glycans and has weak recognition of related fucose- and sialic acid-containing glycans. The finding that Pa8830 binds sialyl-Le^x-containing glycans, which occur at increased levels in mucins from CF patients, is consistent with studies of other strains of P. aeruginosa and further suggests that such glycans on CF mucins contribute to disease pathogenesis. Invited Submission from Dr. Subhash Basu, from the 7th International Symposium on Biochemical Roles of Eukaryotic Cell Surface Macromolecules in Puri, India, January, 2005.     

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA₁₂₀-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA₁₂₀ was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.