Purification and characterization of alpha-L-fucosidase from Streptomyces species.

Streptomyces sp. 142, isolated from a soil sample, produced alpha-fucosidase when cultured in the presence of L-fucose. The enzyme was purified 700-fold with an overall recovery of 17% from a cell-free extract by cation exchange chromatography and gel filtration chromatography. The apparent molecular weight of the purified enzyme was 40,000 by gel filtration chromatography. The enzyme had a pH optimum of 6.0 and was stable at pH 4.5-7.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine as the substrate showed that the enzyme specifically hydrolyzed terminal alpha 1-3 and alpha 1-4 fucosidic linkages in the oligosaccharides but did not hydrolyze alpha 1-2 or alpha 1-6 fucosidic linkages or a synthetic substrate, p-nitro-phenyl alpha-L-fucoside. The purified enzyme released L-fucose from a fucosylated glycoprotein, alpha 1-acid glycoprotein. Thus, the substrate specificities of the Streptomyces alpha-fucosidase resembled those of alpha-fucosidases I and III isolated from almond emulsin rather than those of other microbial alpha-fucosidases.     

Chemical structure of the peptidoglycan of Vibrio parahaemolyticus A55 with special reference to the extent of interpeptide cross-linking.

The chemical structure of the cell wall peptidoglycan of Vibrio parahaemolyticus A55 was studied. Estimation of cross linkages between peptide subunits in the peptidoglycan by dinitrophenylation showed that about 30% of the total 2,6-diaminopimelic acid (A2pm) residues were involved in cross linkages. The presence of interpeptide bridges was also demonstrated by isolating bisdisaccharide peptide subunit dimers from Chalaropsis muramidase digests of the cell wall peptidoglycan by gel filtration followed by ion-exchange column chromatography, although most of the building blocks obtained were uncross-linked disaccharide peptide monomers. The chain length of a glycan moiety of the peptidoglycan obtained by treatment with the L-11 enzyme and gel filtration of the digest was also studied. The chain length varied from 7 to 44, but 30% of the glycan fragments had muramic acid at the reducing end and a chain length of 28 to 44. In conformity with the above structural study it was demonstrated that a particulate enzyme fraction obtained by differential centrifugation of a sonicated preparation of V. parahaemolyticus catalyzed a penicillin-sensitive transpeptidation reaction, using UDP-MurNAc-14C-pentapeptide and UDP-GlcNAc as substrates.     

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.     

The specific substance from Pneumococcus type 34. The configuration of the glycosidic linkages

1. The specific compound from Pneumococcus type 34 was isolated from capsular material by ion-exchange chromatography. This separated it from a substance with chemical and serological properties corresponding to those reported for C-substance. 2. The configuration of the two galactofuranosyl linkages in the repeating unit of S.34 was determined and the configurations previously assigned to the other glycosidic linkages were confirmed. 3. The dephosphorylated deacetylated repeating unit is thus O-beta-d-galactofuranosyl-(1-->3)-O-alpha-d-glucopyranosyl-(1-->2)-O-beta-d-galactofuranosyl-(1-->3)-O-alpha-d-galactopyranosyl- (1-->2)-ribitol.     

Fractionation of the beta-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were beta-linked as shown by H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Enzymatic polymerization of coniferyl alcohol in the presence of cyclodextrins

The dehydrogenative polymerization of coniferyl alcohol by horseradish peroxidase was performed in 0.10 M phosphate buffer at 27 degrees C. Dehydrogenative polymer (DHP) from coniferyl alcohol was characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. The ratio of 8-O-4':8-5':8-8' linkages was determined by the 1H NMR spectrum of DHP acetate which had good solubility. In "end-wise like" polymerization (the slow addition of hydrogen peroxide), addition of alpha-cyclodextrin to the medium led to DHP with increased 8-O-4' content and a decrease in 8-5' linkages. Under higher pH conditions, DHP with higher 8-O-4' and 8-5' content was obtained in the presence of alpha-cyclodextrin. In the end-wise polymerization (the slow additions of coniferyl alcohol and hydrogen peroxide), using alpha-cyclodextrin also gave DHP with a 8-O-4' richer structure than that prepared in no additive system. The analysis of thioacidolysis products from DHP supported the results of the alpha-cyclodextrin effects on the 8-O-4'-rich structure of DHP. The 8-O-4' structure in DHP prepared in the presence of alpha-cyclodextrin had racemic form as shown by ozonation.     

Effect of molecule branching and glycosidic linkage on the degradation of polydextrose by gut microbiota

Polydextrose is a randomly linked complex glucose oligomer that is widely used as a sugar replacer, bulking agent, dietary fiber and prebiotic. Polydextrose is poorly utilized by the host and, during gastrointestinal transit, it is slowly degraded by intestinal microbes, although it is not known which parts of the complex molecule are preferred by the microbes. The microbial degradation of polydextrose was assessed by using a simulated model of colonic fermentation. The degradation products and their glycosidic linkages were measured by combined gas chromatography and mass spectrometry, and compared to those of intact polydextrose. Fermentation resulted in an increase in the relative abundance of non-branched molecules with a concomitant decrease in single-branched glucose molecules and a reduced total number of branching points. A detailed analysis showed a preponderance of 1,6 pyranose linkages. The results of this study demonstrate how intestinal microbes selectively degrade polydextrose, and provide an insight into the preferences of gut microbiota in the presence of different glycosidic linkages.     

Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.     

Effect of Molecule Branching and Glycosidic Linkage on the Degradation of Polydextrose by Gut Microbiota

Polydextrose is a randomly linked complex glucose oligomer that is widely used as a sugar replacer, bulking agent, dietary fiber and prebiotic. Polydextrose is poorly utilized by the host and, during gastrointestinal transit, it is slowly degraded by intestinal microbes, although it is not known which parts of the complex molecule are preferred by the microbes. The microbial degradation of polydextrose was assessed by using a simulated model of colonic fermentation. The degradation products and their glycosidic linkages were measured by combined gas chromatography and mass spectrometry, and compared to those of intact polydextrose. Fermentation resulted in an increase in the relative abundance of non-branched molecules with a concomitant decrease in single-branched glucose molecules and a reduced total number of branching points. A detailed analysis showed a preponderance of 1,6 pyranose linkages. The results of this study demonstrate how intestinal microbes selectively degrade polydextrose, and provide an insight into the preferences of gut microbiota in the presence of different glycosidic linkages.     

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).     

Studies on the Antigenic Activities of Yeasts

In order to determine the antigenic determinant groups of the mannan of Candida albicans by the precipitation-inhibition test, several oligosaccharides were prepared by acetolysis of the polysaccharide. The manno-oligosaccharides, from biose to heptaose were separated by a charcoal-Celite chromatography and a subsequent cellulose column chromatography. The oligosaccharides thus separated were examined on the degrees of polymerization and the mode of the linkages, and evidence was obtained that the biose and triose were joined through α1→2 linkage only, while the tetraose, pentaose and hexaose contained α1→3 linkage in addition to α1→2 linkages. Heptaose was joined entirely through α1→2 linkage. In the precipitation-inhibition test, the inhibitory power of the oligosaccharides of acetolysis product was found to be the following order: hexaose>heptaose>pentaose>tetraose>triose>biose, and the amount for the 50% inhibitions were 0.025, 0.09, 0.12, 0.60, 3.96 and 5.84 μmoles respectively. On the other hand, the biose, triose and tetraose, which were isolated from the acid-hydrolysate of the mannan of Saccharomyces cerevisiae and joined through α1→6 linkage, showed poor or nearly no inhibitory power. The above facts provide an evidence that the consecutive α1→6 linkages were not located in a position that is responsible for antigenic specificity of the mannan of C. albicans.     

Characterization of the thermophilic isoamylase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Isoamylase catalyzes the hydrolysis of -1,6-glucosidic linkages of starch and related polysaccharides. In this study, the treX gene (GenBank accession no. AE006815 REGION: 9279 … 11435) encoding the thermophilic isoamylase was PCR-cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7lac promoter. Both wild-type and His-tagged isoamylases were expressed in Escherichia coli. The wild-type isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4 °C. They had an apparent optimal pH of 5 and an optimal temperature at 75 °C. The enzyme activities remained unchanged after a 2 h incubation at 80 and 75 °C for the wild-type and His-tagged enzymes, respectively. These thermophilic isoamylases showed a potential to be used in industry to degrade the branching points of starch at a high temperature.     

Purification and characterization of alpha-L-fucosidases from Streptomyces sp. OH11242

alpha-L-Fucosidases were found in the culture fluid of Streptomyces sp. OH11242 grown with porcine gastric mucin (PGM) as the sole carbon source. The alpha-L-fucosidases were purified by ammonium sulfate precipitation followed by chromatography on Sepharose CL-4B, hydroxyapatite, Resource Q and Mono Q. Two enzyme fractions, termed Fase-I and Fase-II, were obtained, each bearing different substrate specificity. Fase-I hydrolyzed fucose residues from fucose-containing oligosaccharide chains on PGM, but not p-nitrophenyl alpha-L-fucoside (Fucalpha-O-PNP). In contrast, Fase-II cleaved fucose from Fucalpha-O-PNP, but not fucose-containing oligosaccharides on PGM. Fase-I also hydrolyzed the alpha1-2 fucosidic linkage in various oligosaccharides, but not alpha1-3 and alpha1-4 fucosidic linkages. Fase-II was separated into two fractions, Fase-IIa and -IIb by Mono Q chromatography, Fase-IIb hydrolyzed alpha1-3 and alpha1-4 fucosidic linkages, but not alpha1-2 fucosidic linkages, while Fase-IIa hydrolyzed none of them. Fase-I was purified to homogeneity by SDS-polyacrylamide gel electrophoresis, the molecular mass was estimated to be approximately 59000 and 76000 Da by SDS-PAGE and gel-permeation chromatography, respectively. The optimum pH for Fase-I activity was 5.5-6.0. These fucosidases with different substrate specificities might be useful to reveal the physiological role of fucose-containing oligosaccharides in the gastric mucins.     

Characterization of cyanobacterial glycogen isolated from the wild type and from a mutant lacking of branching enzyme

Cyanobacteria produce glycogen as their primary form of carbohydrate storage. The genomic DNA sequence of Synechocystis sp. PCC6803 indicates that this strain encodes one glycogen-branching enzyme (GBE) and two isoforms of glycogen synthase (GS). To confirm the putative GBE and to demonstrate the presence of only one GBE gene, we generated a mutant lacking the putative GBE gene, sll0158, by replacing it with a kanamycin resistance gene through homologous recombination. GBE in sll0158(-) mutant was eliminated; the mutant strain produced less glucan, equivalent to 48% of that produced by the wild type. In contrast to the wild-type strain that had 74% of the glucan being water-soluble, the mutant had only 14% of the glucan water-soluble. Molecular structures of glucans produced by the mutant and the wild type were characterized by using high-performance size-exclusion and anion-exchange chromatography. The glycogen produced by the wild type displayed a molecular mass of 6.6 x 10(7) daltons (degree of polymerization (DP) 40700) and 10% branch linkages, and the alpha-D-glucan produced by the mutant displayed a molecular mass of 4.7-5.6 x 10(3) daltons (DP 29-35) with slight branch linkages. The results indicated that sll0158 was the major functional GBE gene in Synechocystis sp. PCC6803.     

Characterization of microsomal and cytosolic alpha-1,2-mannosidases from mung bean hypocotyls

Microsomal and cytosolic alpha-mannosidase activities, which hydrolyze alpha-1,2-mannosyl-mannose linkages in the Man5GlcNAc2 oligosaccharide, have been isolated from homogenates of mung bean hypocotyls. The alpha-1,2-mannosidase activities were readily distinguished from previously described aryl alpha-mannosidases by several criteria. They were optimally active in the presence of Ca2+ between pH 5.5 and 6, they were inhibited by Zn2+, and they had essentially no activity with p-nitrophenyl-alpha-mannoside. The microsomal and cytosolic alpha-1,2-mannosidases demonstrated specificity for oligosaccharides with terminal nonreducing alpha-1,2-mannosyl linkages, and they were inhibited by mannosyl-mannose disaccharides, with the inhibition decreasing in the order of alpha-1,2-greater than alpha-1,3-greater than alpha-1,6-mannosyl-mannose. The cytosolic alpha-1,2-mannosidase activity, which was present in the 100,000 g supernatant, was separated from the aryl alpha-mannosidase by ammonium sulfate precipitation. The microsomal alpha-1,2-mannosidase, which was tightly associated with the particulate fraction, was solubilized with Triton X-100 and 0.2 M KCl. The two alpha-1,2-mannosidase activities were readily differentiated by gel-filtration chromatography. The solubilized microsomal enzyme chromatographed in approximately the same position as a Mr 460,000 globular protein whereas the cytosolic enzyme was eluted in a retarded position, indicating a much smaller protein.     

Isolation and characterization of a galactose binding lectin with insulinomimetic activities. From the seeds of the bitter gourd Momordica charantia (Family Cucurbitaceae)

A galactose binding lectin was isolated from the seeds of the bitter gourd Momordica charantia by delipidation with petroleum ether, extraction with phosphate buffered saline, ammonium sulfate precipitation and affinity chromatography on lactogel. The lectin had a molecular weight of 124,000 and approximately 5% carbohydrate content. The molecular weights of the individual subunits were 37,000, 35,000 and 33,000. The lectin exhibited potent hemagglutinating activity. In addition, it demonstrated antilipolytic and lipogenic activities in isolated rat adipocytes although it did not possess intrinsic lipolytic activity. The antilipolytic activity was susceptible to destruction by heat, trypsin, chymotrypsin, glutathione and galactose, indicating that the integrity of the protein moiety, the disulfide linkages, and galactose, which is the sugar specifically bound by the lectin, all play an important role in interaction with the adipocyte leading to an expression of this insulin-like activity.     

Bonding of hydroxycinnamic acids to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl position of lignin, not the beta-position, in grass cell walls.

A suspension in dichloromethane-water (18:1, v/v) of various fractions containing hydroxycinnamic acid ester-ether bridges between lignin and polysaccharides prepared from cell walls of matured oat (Avena sativa L.) intemodes, and a solution of their acetates in the same solvent, were treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). This reagent selectively cleaves benzyl ether and ester linkages of negatively charged aromatic nuclei. The sample treated with DDQ was directly hydrolysed either under mild (1 M NaOH, overnight at 37 degrees C) or severe (4 M NaOH, for 2 h at 170 degrees C) conditions. The hydroxycinnamic acids released in the hydrolysate were methylated with diazomethane and analysed quantitatively using gas chromatography. Significant portions of ether linkages between hydroxycinnamic acids and lignin were cleaved with DDQ, which suggests that most of the hydroxycinnamic acids were ether-linked at the benzyl position, and not the beta-position, of the lignin side chain as previously claimed.     

Structure of an antifreeze polypeptide from the sea raven. Disulfide bonds and similarity to lectin-binding proteins.

The antifreeze polypeptide (AFP) from the sea raven, Hemitripterus americanus, is a member of the cystine-rich class of blood antifreeze proteins which enable survival of certain fishes at sub-zero temperatures. Sea raven AFP contains 129 residues with 10 half-cystine residues. We have analyzed these half-cystine residues and established that all 10 of the half-cystine residues appeared to be involved in disulfide bond formation and that disulfide bonds linked Cys7 to Cys18, Cys35 to Cys125, and Cys89 to Cys117. These assignments were established by extensive proteolytic digestions of native AFP using pepsin and thermolysin and purification of the peptides by Sephadex G-15 gel filtration chromatography, anion exchange chromatography, and C18 reverse-phase high performance liquid chromatography. Cystine-containing peptides were detected by a colorimetric assay using nitrothiosulfobenzoate. Disulfide-containing peptides were reduced and alkylated, purified, and analyzed by amino acid analysis. The unreduced disulfide-linked peptides were sequenced directly by automated Edman degradations to confirm the disulfide assignments. Possible arrangements of the two remaining disulfide bonds include linkages Cys69/111 to Cys100/101. The sea raven AFP shares structural similarity with pancreatic stone protein and several lectin-binding proteins, especially with respect to half-cystines, glycines, and bulky aromatic residues. Two of the disulfide linkages we determined for sea raven AFP: Cys7-Cys18 and Cys35-Cys125, are conserved in these proteins. These similarities in covalent structure suggest that the sea raven AFP, pancreatic stone protein, and several lectin-binding proteins comprise a family of proteins which may possess a common fold.     

Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI-mass spectrometry

Alginate with long strictly alternating sequences of mannuronic (M) and guluronic (G) acid residues, F(G) = 0.47 and F(GG) = 0.0, was prepared by incubating mannuronan with the recombinant C-5 epimerase AlgE4. By partial acid hydrolysis of this PolyMG alginate at pH values from 2.8 to 4.5 at 95 degrees C, alpha-L-GulpA-(1-->4)-beta-D-ManpA (G-M) linkages were hydrolyzed far faster than beta-D-ManpA-(1-->4)-alpha-L-GulpA (M-G) linkages in the polymer chain. The ratio of the rates (kG-M/kM-G) decreased with increasing pH. The dominant mechanism for hydrolysis of (1-->4)-linked PolyMG in weak acid was thus proved to be an intramolecular catalysis of glycosidic cleavage of the linkages at C-4 by the undissociated carboxyl groups at C-5 in the respective units. The higher degradation rate of G-M than M-G glycosidic linkages in the polymer chain of MG-alginate at pH 3.5 and 95 degrees C was exploited to make oligomers mainly consisting of M on the nonreducing and G on the reducing end and, thus, a majority of oligomers with an even number of residues. The ratio of the rate constants kG-M/kM-G at this pH was 10.7. The MG-hydrolysate was separated by size exclusion chromatography and the MG oligosaccharide fractions analyzed by electrospray ionization-mass spectrometry together with 1H and 13C NMR spectroscopy. Chemical shifts of MG-oligomers (DP2-DP5) were elucidated by 2D 1H and 13C NMR.     

Determination of the disulfide bond arrangement of Newcastle disease virus hemagglutinin neuraminidase. Correlation with a beta-sheet propeller structural fold predicted for paramyxoviridae attachment proteins

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(196) linkages and that HN ectodomains dimerize via Cys(123). Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cys(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subsequent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cys(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys(344)-Cys(461) and Cys(455)-Cys(465) linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.