The structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

Yeast cell-wall synthesis

1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.     

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.     

Synthesis and conformational features of human salivary mucin C-terminal derived peptide epitope carrying Thomsen-Friedenreich antigen: implications for its role in self-association

The conformational features of a chemically synthesized 23-residue glycopeptide construct (II) carrying Gal-beta-(1,3)-alpha-GalNAc and its deglycosylated counterpart (I; Gal: galactose; GalNAc: N-acetyl galactosamine) derived from the C-terminal domain of human salivary mucin (MUC7) were investigated using CD spectroscopy as well as molecular dynamic simulation studies. The corresponding deglycosylated peptide (I) was essentially used to compare and study the influence of the sugar moiety on peptide backbone conformation. CD measurements in aqueous medium revealed that the apopeptide (I) contains significant populations of beta-strand conformation while the glycopeptide (II) possess, partly, helical structure. This transition in the secondary structure upon glycosylation from beta-strand to helical conformation clearly demonstrates that the carbohydrate moiety exerts significant influence on the peptide backbone. On the other hand, upon titrating structure stabilizing organic cosolvent, trifluoroethanol (TFE), both the peptides showed pronounced helical structure. However, the propensity for helical structure formation is less pronounced in glycopeptide compared to apopeptide suggesting that the bulky carbohydrate moiety possibly posing steric hindrance to the formation of TFE-induced secondary structure in II. Energy-minimized molecular model for the glycopeptide revealed that the preferred helix conformation in aqueous medium appears to be stabilized by the hydrogen-bonded salt bridge like interaction between carbohydrate --OH and Lys-10 side--N(+)H(3) group. Size exclusion chromatographic analysis of both (glyco)peptides I and II showed an apparent Kd of 2.3 and 0.52 microM, respectively, indicating that glycopeptide (II) has greater tendency for self-association. Due to high amphipathic character as well as due to the presence of a leucine zipper motif ( approximately LLYMKNLL approximately ), which is known to increase the stability at the coiled-coil interface via hydrophobic interactions, we propose therefore that, this domain could be one of the key elements involved in the self-association of intact MUC7 in vivo. Profound conformational effects governed by glycosylation exemplified herein could have implications in determining structure-function relationships of mucin glycoproteins.     

Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation

Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.     

Structure of the tryptic glycopeptide isolated from rabbit transferrin.

The structure of the tryptic glycopeptide isolated from rabbit transferrin was elucidated by use of sequential Edman degradations, specific exoglycosidases, endo-beta-N-acetylglucosaminidases, methylation analyses, and periodate oxidation studies. The glycopeptide consists of a heteropolysaccharide, AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 3[AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 6]-Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc, attached to a peptide, Asn-Ser-Ser-Leu-Cys, via a linkage involving N-acetyl-glucosamine and asparagine. The stoichiometry of this glycopeptide is 2 mol/mol of protein, indicating that rabbit transferrin contains two structurally identical glycopeptide segments.     

Localization and overall structure of a mannose-rich glycopeptide from a pathologic immunoglobulin

The structure of a mannose-rich glycopeptide from a human pathological IgM has been investigated. It belongs to the group I (simple) glycopeptides and contains only mannose and N-acetylglucosamine residues in a molar ratio of 10:2. The structures of its oligosaccharide moiety and peptide chain have been determined: its molecular localization is specified and the relation between its biosynthesis and the oligosaccharide structure determine is discussed. Based on the alpha- and beta-mannosidase digestions and permethylation studies for the oligosaccharide moiety, and on the results obtained after sequential analysis of the peptide chain, the following structure is proposed for the mannose-rich IgM Du glycopeptide: (Formula: see text). The recovery of one molecule of this glycopeptide per molecule of heavy chain and the determination of the amino acid sequence have led us to locate this glycopeptide on asparagine 402 of the Fc portion of the heavy chain mu of IgM Du.     

The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6. Unique structural requirement for the antigenic specificity provided by a glycosylhexapeptide

Previously, monoclonal antibody FDC-6 was established, which defines a structure specific for fibronectins isolated from fetal and malignant cells and tissues. The presence of the FDC-6-defined structure at type III connecting segment (III CS) is characteristic of oncofetal fibronectin (onf-FN), and its absence is characteristic of normal fibronectin (nor-FN) (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). Hepatoma fibronectin was sequentially digested by various proteases, followed by subsequent chromatography on an FDC-6 affinity column and reverse-phase columns at each step of digestion. A single strongly active glycosylhexapeptide (glycopeptide 1) and an inactive glycosylpentapeptide (glycopeptide 3) were isolated from glycopeptide A containing 35 amino acid residues. The minimum essential structure required for the FDC-6 activity was found to be a hexapeptide sequence Val-Thr-His-Pro-Gly-Tyr having NeuAc alpha 2----3Gal beta 1----3GalNAc or its core (Gal beta 1----3GalNAc or GalNAc) linked at threonine. Various synthetic peptides including the Val-Thr-His-Pro-Gly-Tyr sequence and a glycopeptide having the Val-Thr-His-Pro-Gly pentapeptide with the same glycosylation at threonine were all inactive. Elimination of sialic acid slightly increased the activity, and subsequent elimination of galactose did not alter the activity; however, removal of the Gal beta 1----3GalNAc residue by endo-alpha-N-acetylgalactosaminidase from desialylated glycopeptide A resulted in total inactivation of the reactivity with FDC-6 antibody. Thus, a single glycosylation at a defined threonine residue of the III CS region may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. This finding opens the possibility that a number of other oncofetal epitopes consist of a peptide and a common O-linked carbohydrate and that the combination produces a conformation specific to cancer or to a stage of development.     

Glycoprotein of the wall of sycamore tissue-culture cells

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.     

Characterization of Solubilized Products from the Cell Wall of Streptococcus pneumoniae during Autoplast Formation

The structure of the cell wall of Streptococcus pneumoniae R36a was investigated by means of chemical analysis of the solubilized products formed during autoplast formation. By autoplast formation, almost all of the cell wall components were solubilized as the end products within several hours. Analysis of the solubilized products revealed that the pneumococcal cell wall consists of three macromolecular components, teichoic acid-glycopeptide I (TA-GP I), teichoic acid-glycopeptide II (TA-GP II), and glycopeptide (GP III). The molecular size of TA-GP I was larger than that of TA-GP II. TA-GP I and TA-GP II were constituted of similar components, galactosamine, 2-acetamido-4-amino-2, 4, 6-trideoxyhexose, glucose, ribitol, choline, phosphate, and peptidoglycan components, but the ratio of teichoic acid to glycopeptide in TA-GP II was higher than that in TA-GP I. TA-GP II was solubilized more slowly than TA-GP I and GP III during autoplast formation. The assembly of the cell wall by TA-GP I and II, and GP III is discussed in connection with the action of autolysin.     

O-Linked glycopeptides retain helicity in water.

A 17-residue O-linked glycopeptide model incorporating a central alpha-mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.     

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.     

Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane.

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.     

Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galβ1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and β-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis – HGP33-5GN-α and -β. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the α and β forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-α and -β. Therefore, the obtained result might indicate that HGP25-5GN-α was an isomer of HGP25-5GN-β differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.     

'Wave-type' structure of a synthetic hexaglycosylated decapeptide: A part of the extracellular domain of human glycophorin A

The three-dimensional structure of a glycopeptide, His-Thr*-Ser*-Thr*-Ser*-Ser*-Ser*-Val-Thr-Lys, with 2-acetamido-2-deoxy--D-galactose (GalNAc) residues linked to six adjacent amino acids from Thr-10 to Ser-15, was studied by NMR spectroscopy and molecular dynamics (MD) simulations. The hexaglycosylated decapeptide is part of the extracellular domain of human glycophorin A and shows an extended structure of the peptide backbone due to O-glycosylation. Furthermore, each GalNAc residue exhibits one and only one NOE contact from the NHAc proton to the backbone amide proton of the amino acid that the sugar is directly bound to. This indicates a strong preference for the orientation of all GalNAc residues towards the N-terminus. NOE build-up curves were used to determine 42 inter-proton distances that, in connection with angles of the peptide backbone obtained from 3J-coupling constants, resulted in constraints for a MD simulation in water. The NMR data and the MD simulations show a preference for an extended backbone structure. The GalNAc residues are located alternatingly on opposite sides of the backbone and reduce the flexibility of the peptide backbone. The conformation of the molecule is relatively rigid and shows a 'wave-type' 3D structure of the peptide backbone within the glycosylation cluster. This new structural element is also supported by the unusual CD spectrum of the glycopeptide.     

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O‐glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O‐glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn‐antigen (GalNAc‐O‐Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding‐competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution‐state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre‐selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.     

A peptide binding chromogenic assay for detecting glycopeptide antibiotics

Summary A solid-phase peptide binding assay, based on the mechanism of action of glycopeptide antibiotics, was developed for detecting this chemical class of metabolites. Utilizing a pentapeptide (l-alanyl-d-isoglutaminyl-l-lysyl-d-alanyl-d-alanine)-bovine serum albumin conjugate immobilized on the wall of microtiter wells, the binding of the vancomycin-alkaline phosphatase to the peptide could be demonstrated by subsequently monitoring the enzyme activity. The presence of glycopeptides in fermentation broths could be detected and quantified with a competitive binding assay. Peptides with ad-alanyl-d-alanine carboxyl terminus were necessary for the binding of these glycopeptides, thus confirming the mode of action of this class of antibiotics.     

In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains

A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.     

D-galactofuranose in the N-linked sugar chain of a glycopeptide from Ascobolus furfuraceus

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced beta-elimination of a small oligosaccharide. Evidence for the O-glycosidic linkage was provided by increase in absorbance at 240 nm, decrease in threonine and serine content after the alkaline treatment and detection of tritiated oligosaccharide following alkaline NaB3H4 reduction. Mannose is the sugar involved in the O-glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N-glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo-beta-N-acetyl-glucosaminidase. Sequential NaB3H4 reduction and acid hydrolysis gave [3H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis.     

NMR-based determination of the binding epitope and conformational analysis of MUC-1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3.

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 (MUC-1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O-alpha-D-GalNAc)RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build-up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference (STD) NMR spectroscopy. The peptide's epitope is predominantly located in the N-terminal PDT segment whereas the C-terminal RP segment has fewer interactions with the protein. In contrast, the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N-acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N-terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well.