A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.     

Crystal structure of beta1,4-galactosyltransferase complex with UDP-Gal reveals an oligosaccharide acceptor binding site

The crystal structure of the catalytic domain of bovine beta1,4-galactosyltransferase (Gal-T1) co-crystallized with UDP-Gal and MnCl(2) has been solved at 2.8 A resolution. The structure not only identifies galactose, the donor sugar binding site in Gal-T1, but also reveals an oligosaccharide acceptor binding site. The galactose moiety of UDP-Gal is found deep inside the catalytic pocket, interacting with Asp252, Gly292, Gly315, Glu317 and Asp318 residues. Compared to the native crystal structure reported earlier, the present UDP-Gal bound structure exhibits a large conformational change in residues 345-365 and a change in the side-chain orientation of Trp314. Thus, the binding of UDP-Gal induces a conformational change in Gal-T1, which not only creates the acceptor binding pocket for N-acetylglucosamine (GlcNAc) but also establishes the binding site for an extended sugar acceptor. The presence of a binding site that accommodates an extended sugar offers an explanation for the observation that an oligosaccharide with GlcNAc at the non-reducing end serves as a better acceptor than the monosaccharide, GlcNAc. Modeling studies using oligosaccharide acceptors indicate that a pentasaccharide, such as N-glycans with GlcNAc at their non-reducing ends, fits the site best. A sequence comparison of the human Gal-T family members indicates that although the binding site for the GlcNAc residue is highly conserved, the site that binds the extended sugar exhibits large variations. This is an indication that different Gal-T family members prefer different types of glycan acceptors with GlcNAc at their non-reducing ends.     

Novel Ca2+‐independent carbohydrate recognition of the C‐type lectins,SPL‐1 and SPL‐2, from the bivalve Saxidomus purpuratus

Novel Ca²⁺‐independent C‐type lectins, SPL‐1 and SPL‐2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL‐2 composed of two B‐chains) or distinct (SPL‐1 composed of A‐ and B‐chains) polypeptide chains, and show affinity for N‐acetylglucosamine (GlcNAc)‐ and N‐acetylgalactosamine (GalNAc)‐containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C‐type lectin family. X‐ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate‐recognition domain (CRD) of the C‐type lectin family. Nevertheless, these lectins (especially SPL‐2) showed Ca²⁺‐independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL‐2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate‐recognition motifs among the C‐type CRD (the QPD [Gln‐Pro‐Asp] and EPN [Glu‐Pro‐Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate‐binding specificities of individual A‐ and B‐chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α‐glycosidic linkages with slightly different specificities.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers sulfate to the C-6 position of non-reducing N-acetylglucosamine (GlcNAc) residues. We cloned human and mouse cDNAs encoding a novel GlcNAc6ST, designated as GlcNAc6ST-4, which showed sequence identities of 26 to 41% to other GlcNAc6STs. Human organs with strong expression of the enzyme mRNA were the heart, spleen, and ovary, while in the mouse strong expression was detected in the kidney. The enzyme expressed in CHO cells preferentially acted on mannose-linked GlcNAc, while a core 2 mucin-type oligosaccharide and an N-acetyllactosamine oligomer also served as acceptors. The distribution and the specificity of GlcNAc6ST are different from those of GlcNAc6STs identified previously.     

Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin

Phaseolin, the major storage protein of the common bean (Phaseolus vulgaris), is a glycoprotein which is synthesized during seed development and accumulates in protein storage vacuoles or protein bodies. The protein has three different N-linked oligosaccharide side chains: Man9(GlcNAc)2, Man7(GlcNAc)2, and Xyl-Man3(GlcNAc)2 (where Xyl represents xylose). The structures of these glycans were determined by 1H NMR spectroscopy. The Man9(GlcNAc)2 glycan has the typical structure found in plant and animal glycoproteins. The structures of the two other glycans are shown below. (Formula; see text) Phaseolin was separated by electrophoresis on denaturing gels into four size classes of polypeptides. The two abundant ones have two oligosaccharides each, whereas the less abundant ones have only one oligosaccharide each. Polypeptides with two glycans have Man7(GlcNAc)2 attached to Asn252 and Man9(GlcNAc)2 attached to Asn341. Polypeptides with only one glycan have Xyl-Man3(GlcNAc)2 attached to Asn252. Both these asparagine residues are in canonical glycosylation sites; the numbering starts with the N-terminal methionine of the signal peptide of phaseolin. The presence of the Man7(GlcNAc)2 and of Xyl-Man3(GlcNAc)2 at the same asparagine residue (position 252) of different polypeptides seems to be controlled by the glycosylation status of Asn341. When Asp341 is unoccupied, the glycan at Asn252 is complex. When Asn341 is occupied, the glycan at Asn252 is only modified to the extent that 2 mannosyl residues are removed. The processing of the glycans, after the removal of the glucose residues, involves enzymes in the Golgi apparatus as well as in the protein bodies. Formation of the Xyl-Man3(GlcNAc)2 glycan is a multistep process that involves the Golgi apparatus-mediated removal of 6 mannose residues and the addition of 2 N-acetylglucosamine residues and 1 xylose. The terminal N-acetylglucosamine residues are later removed in the protein bodies. The conversion of Man9(GlcNAc)2 to Man7(GlcNAc)2 is a late processing event which occurs in the protein bodies. Experiments in which [3H]glucosamine-labeled phaseolin obtained from the endoplasmic reticulum (i.e. precursor phaseolin) is incubated with jack bean alpha-mannosidase show that the high mannose glycan on Asn252, but not the one on Asn341, is susceptible to enzyme degradation. Incubation of [3H] glucosamine-labeled phaseolin obtained from the Golgi apparatus with jack bean beta-N-acetylglucosaminidase results in the removal of the terminal N-acetylglucosamine residues from the complex chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

The identification and characterization of xenoantigenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney

The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the alpha-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.     

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galbeta1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with beta-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.     

Very-high-field n.m.r. studies of bovine lung heparan sulphate oligosaccharides produced by nitrous acid deaminative cleavage. 13C-n.m.r. study of methylene resonances: degree and positions of C-6 sulphation.

Oligosaccharides with the general structure UA-(GlcNAc-GlcUA-)m-aManOH (m = 1-5) (where UA represents uronic acid, GlcNAc N-acetylglucosamine, GlcUA glucuronic acid and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by nitrous acid deaminative cleavage followed by reduction. Analysis of the methylene signals in the 100 MHz 13C-n.m.r. spectrum of the tetrasaccharide (m = 1) shows that, whereas the extent of C-6 O-sulphation in the GlcNAc is approx. 65%, in the aManOH [formerly a GlcNSO3 (N-sulphoglucosamine) residue in the parent heparan sulphate] it is only approx. 10%. In the higher oligosaccharides (m = 2-5) the gross extent of C-6 O-sulphation of GlcNAc residues falls systematically with increasing oligosaccharide size, whereas that in the aManOH residues remains below 10%. There is also evidence that the C-6 O-sulphation of the GlcNAc residues is confined to the GlcNAc residue adjacent to the non-reducing terminal uronic acid residue. It is therefore tentatively proposed that the GlcNAc in the sequence -GlcNSO3-UA-GlcNAc- might be a favoured substrate for the 6-O-sulphotransferase. It is concluded that in the low-sulphated heparan sulphates GlcNSO3 residues that do not occur in (GlcNSO3-UA-)n blocks tend to have a significantly smaller extent of C-6 O-sulphation than do GlcNAc residues that occur in -GlcNSO3-UA-GlcNAc-GlcUA-GlcNSO3-sequences.     

Interactions of wheat-germ agglutinin with GlcNAc beta 1,6Gal sequence

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.     

Mannose-specific lectin from the mushroom Hygrophorus russula

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5?kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528?bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Protein matrix effects on glycan processing by mannosidase II and sialyl transferase from rat liver

The effect of the protein environment on the reaction sequence and the relative rates of two two-step reactions involved in the biosynthesis of complex glycans in glycoproteins has been explored by comparing the processing of biotinylated substrates either free or bound to avidin. By use of biotinyl and biotinamidohexanoyl derivatives, the display of the glycan in a proximal and distal association with the avidin surface could also be assessed. Mannosidase II removes two Man residues from the substrate GlcNAcMan5GlcNAc2-R to yield GlcNAcMAn3GlcNAc2-R. The NMR spectra of the substrate, intermediate, and product showed that the first Man is removed from the 6-arm of the substrate. The rate constants for the first and second step (estimated by direct analysis of the reactants by anion-exchange chromatography with a pulsed amperometric detector) were determined to be about 0.05 and 0.08 min-1, respectively, for the free substrates. In the proximal complex k1 was reduced 80-fold, and the k2 step could not be observed under the same conditions. In the distal complex both k1 and k2 were reduced about 8-fold. Sialyl transferases transfer Sia from CMP-Sia to the biantennary substrate Gal2GlcNAc2-Man3GlcNA2-R to yield the product Sia2Gal2-GlcNAc2Man3GlcNAc2-R with the Sia linked either 2-3 or 2-6 to the Gal residues. The NMR spectra showed that the first step involved the Gal on the 3-arm of the substrate and that both Sia residues were added 2-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycan Microarray Analysis of P-type Lectins Reveals Distinct Phosphomannose Glycan Recognition

The specificity of the cation-independent and -dependent mannose 6-phosphate receptors (CI-MPR and CD-MPR) for high mannose-type N-glycans of defined structure containing zero, one, or two Man-P-GlcNAc phosphodiester or Man-6-P phosphomonoester residues was determined by analysis on a phosphorylated glycan microarray. Amine-activated glycans were covalently printed on N-hydroxysuccinimide-activated glass slides and interrogated with different concentrations of recombinant CD-MPR or soluble CI-MPR. Neither receptor bound to non-phosphorylated glycans. The CD-MPR bound weakly or undetectably to the phosphodiester derivatives, but strongly to the phosphomonoester-containing glycans with the exception of a single Man7GlcNAc2-R isomer that contained a single Man-6-P residue. By contrast, the CI-MPR bound with high affinity to glycans containing either phospho-mono- or -diesters although, like the CD-MPR, it differentially recognized isomers of phosphorylated Man7GlcNAc2-R. This differential recognition of phosphorylated glycans by the CI- and CD-MPRs has implications for understanding the biosynthesis and targeting of lysosomal hydrolases.     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.     

The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation.

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.     

Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.     

Characterization of a 24 kD parasitism-specific hemolymph protein from pharate pupae of the caribbean fruit fly,Anastrepha suspensa

A parasitism-specific protein was originally identified in the hemolymph of the Caribbean fruit fly Anastrepha suspensa parasitized by the braconid wasp Diachasmimorpha (Biosteres) longicaudata using single-dimensional (1-D) sodium dodecyl sulfate (SDS) PAGE. We now show that the protein is comprised of two closely migrating species both of which are glycoproteins of ≈? 24,000 Daltons (24 kD). The proteins were poorly resolved from whole hemolymph by 1-D SDS PAGE, but were well resolved by two-dimensional (2-D) PAGE and isoelectric focusing. They have pl's of ≈? 6.3 and 6.7 and contain Man residues, based on their affinity for concanavalin A (Con A). The presence of GlcNAc, NeuAc, and GalNAc residues in both proteins was implicated by their binding to wheat germ agglutinin (WGA). The proteins bound WGA more intensely following mannosidase treatment which eliminated their affinity to Con A and further implicated the presence of internal GlcNAc residues. However, binding of the proteins to WGA in the presence of competing GlcNAc (1 M) was reduced but not eliminated and suggested that in addition to GlcNAc, other WGA-binding sugar moieties, possibly NeuAc, a Sia, were present. To evaluate the presence of NeuAc, we treated the hemolymph with Vibrio cholerae neuraminidase which specifically cleaves terminal Sia. Samples of the neuraminidase-digested proteins were evaluated by WGA binding and Western blotting with the use of an anti-24 kD rabbit polyclonal serum to determine whether desialation eliminated the proteins' affinity to WGA or their immunoreactivity. Our results show that partial digestion of the 24 kD proteins with Vibrio cholerae neuraminidase resulted in two immunoreactive bands in Western blots of 1-D gels but only one of these, the upper undigested 24 kD band, bound WGA. This confirmed the presence of Sia residues in the proteins and demonstrated that desialation increased their relative electrophoretic mobilities. © 1994 Wiley-Liss, Inc.     

Molecular comparison of apocrine released and cytoplasmic resident carbonic anhydrase II

We have previously shown that carbonic anhydrase II usually described as a cytoplasmic resident isoform (cCAH II) is secreted by the rat coagulating gland (sCAH II) via the apocrine secretion mode. To get more detailed information why CAH II is cytoplasmic resident in some organs and secreted in others we cloned and sequenced the cDNA of rat coagulating gland sCAH II. The sequence of the secretory form was found to be completely identical with the cCAH II. Therefore, a signal peptide targeting sCAH II for apocrine secretion can be excluded. Considering the fact that other apocrine secreted proteins are glycosylated, cCAH II and sCAH II were analyzed for carbohydrate substitutions. As expected for a cytoplasmic protein, no glycan modification could be identified in cCAH II. In contrast, sCAH II carried exclusively Gal, GlcNAc and Fuc residues in a molar ratio of 1:0.8:0.5. Carbohydrate linkage analyses demonstrated the presence of terminal Fuc, terminal, 3-substituted and 3,6-disubstituted Gal as well as 4-substituted and 3,4-disubstituted GlcNAc. The composition of the glycan constituents as well as deglycosylation experiments clearly proved that sCAH II carries neither conventional mammalian-type N-glycans nor mucin-type O-linked sugar chains. Lacking a signal peptide for ER translocation, glycosylation of sCAH II must occur within the cytoplasmic compartment. Further studies have to elucidate whether or not glycosylation of sCAH II is essential for the apocrine release of the protein.