500 MHz 1H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-beta-galactosidase.

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)     

A computerized approach to the analysis of oligosaccharide structure by high-resolution proton n.m.r.

The 500 MHz proton-n.m.r. spectra of 21 oligosaccharides, predominantly of the lacto-N and lacto-N-neo series and their derivatives containing non-reducing terminal fucose, sialic acid or N-acetylgalactosamine and reduced-end hexitol or hexosaminitol, were examined with 2H2O as solvent. The chemical-shift data obtained from analysis of the spectra were collated with data from other laboratories who have used 250-500 MHz n.m.r. in the analysis of secreted and chemically synthesized oligosaccharides and of the O- and N-linked chains of glycoproteins. A referenced computer library was constructed that includes the chemical shifts of monosaccharides within oligosaccharide sequences that make up the majority of the carbohydrate structures found in mammalian glycoproteins. Examples are given of the computerized interrogation of this library for the assignment of possible structures of unknown oligosaccharides.     

cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression.

Lactotransferrin was highly purified from lysates of human neutrophilic leucocytes by immuno-affinity chromatography. A comparative analysis of the molar carbohydrate compositions of human leucocyte lactotransferrin and human milk lactotransferrin reveals that the glycans of leucocyte lactotransferrin differ essentially by the absence of fucose residues. Structural analysis combining methylation-mass spectrometry and 400 MHz 1H-n.m.r. spectrometry of oligosaccharide alditols released from human leucocyte lactotransferrin shows the presence of two disialylated and non-fucosylated biantennary glycans of the N-acetyl-lactosaminic type. These results question a previously proposed mechanism for hyposideraemia in which the leucocyte lactotransferrin was involved and in which the fucose residues played a key role.     

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.     

Chondroitinase ABC digestion of dermatan sulphate. N.m.r. spectroscopic characterization of the oligo- and poly-saccharides.

Dermatan sulphates, in which iduronate was the predominant uronate constituent, were partially digested by chondroitinase ABC to produce oligosaccharides of the following structure: delta UA-[GalNAc(4SO3)-IdoA]mGalNAc(4SO3) [where m = 0-5, delta UA represents beta-D-gluco-4-enepyranosyluronate, IdoA represents alpha-L-iduronate and GalNAc(4SO3) represents 2-acetamido-2-deoxy-beta-D-galactose 4-O-sulphate], which were fractionated by gel-permeation chromatography and examined by 100 MHz 13C-n.m.r. and 400/500 MHz 1H-n.m.r. spectroscopy. Experimental conditions were established for the removal of non-reducing terminal unsaturated uronate residues by treatment with HgCL2, and reducing terminal N-acetylgalactosamine residues of the oligosaccharides were reduced with alkaline borohydride. These modifications were shown by 13C-n.m.r. spectroscopy to have proceeded to completion. Assignments of both 13C-n.m.r. and 1H-n.m.r. resonances are reported for the GalNAc(4SO3)-IdoA repeat sequence in the oligosaccharides as well as for the terminal residues resulting from enzyme digestion and subsequent modifications. A full analysis of a trisaccharide derived from dermatan sulphate led to the amendment of published 13C-n.m.r. chemical-shift assignments for the polymer.     

Structural analysis of the carbohydrate chains of beta-N-acetylhexosaminidases from bovine brain.

The oligosaccharide structures of bovine brain beta-N-acetylhexosaminidases A and B (EC 3.2.1.30) were studied at the glycopeptide level by employing 500 MHz 1H-n.m.r. spectroscopy and methylation analysis involving g.l.c.-m.s. More than 90% of the chains were found to be of the oligomannoside type, containing, on average, five to six mannose residues. Biantennary N-acetyl-lactosamine-type chains terminated in N-acetylneuraminic acid were found to comprise the remaining 5-10% of the total carbohydrate. The isoenzyme forms A and B do not differ from each other in the structure of their carbohydrate moiety, but do deviate in carbohydrate content and, in consequence, in the number of carbohydrate chains per molecule.     

The structure of carbohydrate chains of riboflavin-binding glycoprotein from chicken egg protein. III. 1H-NMR spectroscopy (500 MHz) of neutral fucosylated oligosaccharides

Using LiBH4/ButOH treatment, oligosaccharides were cleaved off the hen egg white riboflavin-binding glycoprotein. HPLC led to the isolation of four fucose-containing oligosaccharide alditols, whose structure was elucidated by means of 1H NMR 500 MHz spectroscopy. The main fucosylated oligosaccharide, also present in hen ovomucoid, was found to be a biantennary carbohydrate chain of N-acetyllactosamine type.     

Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study.

Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted.     

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.     

13C n.m.r. studies of the carbohydrate portion of glucoamylase from Aspergillus oryzae

Proton-decoupled, natural abundance ¹³C n.m.r. spectroscopy was used to investigate the carbohydrate structure and content of glucoamylase from Aspergillus oryzae. We found α-d-mannopyranose was the dominant sugar present (⋍91 residues). The Elson-Morgan assay showed that hexosamine was also present as a minor component (2.6% of the total carbohydrate). The intermannose linkages appear to be random. Integration data suggest that 41 α-d-mannopyranose residues are O-2 and O-3 glycosylated and 17 α-d-mannopyranose residues are involved in O-4 glycosylation. Treatment of glucoamylase with α-mannosidase appeared to remove all the carbohydrate residues present.     

400 MHz 1H n.m.r. study of the conformation and self-association of peptidolipin NA

Peptidolipin NA is a lipocyclopeptide extracted from Nocardia Asteroides having the formula:

Its conformation and self-association properties and those of its l-Val(6) analogue have been investigated in three solvents of different polarities: chloroform, pyridine and dimethylsulphoxide, using 400 MHz ¹H n.m.r. A model of the conformation of peptidolipin NA in pyridine has been proposed in which the peptide backbone is folded into a γ-turn around the l-Pro residue. Conformational changes are caused by l-Ala(6) → l-Val(6) replacement and by changing the solvent polarity. Nevertheless, the general shape of the peptide ring seems to be maintained. In chloroform, self-association occurs involving the (2) and, to a lesser extent, the (6) residues. Peptidolipin NA could be representative of natural amphiphiles in which a long hydrophobic tail is bound to a polar peptide moiety.     

The assignment of carbonyl resonances in 13C-n.m.r. spectra of peracetylated mono- and oligo-saccharides containing D-glucose and D-mannose: an alternative method for structural determination of complex carbohydrates

In the present study, proton homonuclear (COSY) and 13C-1H heteronuclear shift-correlation, n.m.r. spectroscopies have been used to assign the carbonyl carbon resonances of peracetylated D-gluco- and D-mannopyranose monosaccharides and oligosaccharides containing residues of parent D-glucopyranose monomers. Chemical shifts of these assigned resonances, particularly those arising from acetyl groups near to aglycon substitution sites, were found to be sensitive to the position and configuration of glycosidic linkages present. In addition, evidence is presented that indicates that the shifts of these carbonyl carbon resonances depend on long-range interactions with other peracetylated pyranose monomers resulting from folding of the oligosaccharide chain. These results suggest that carbonyl carbon resonances of peracetylated carbohydrates may be useful probes of oligosaccharide structure.     

Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy

A series of nine closely related somatostatin analogues, containing the hexapeptide H-Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7-NH2 sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201-995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D-n.m.r. methods, in particular NOESY experiments and detection of long-range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' beta turn involving amino acids Phe3 to Thr6. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe1 and Thr8 (ol) plays a major role in stabilization of the active conformation.     

N.m.r. study of conformational changes in lysozyme around the thermal transition point

Natural abundance carbon-13 n.m.r. at 50.3 MHz has been used to further document the thermal transition that hen egg-white lysozyme undergoes in solution between 20 degrees and 30 degrees. The study focuses on the temperature sensitivity of more than 50 carboxylic, aromatic and aliphatic single carbon resonances for which unambiguous assignments to specific residues are known. The analysis of selective perturbations in chemical shifts indicates that residues located on both edges of the active site cleft and in the hydrophobic box are primarily involved in the temperature-induced conformational transition. N.m.r. results are compared with crystallographic data on low temperature (form A) and high temperature (form B) interconverting lysozyme crystals, taking advantage of the recent availability of quality high resolution maps for B form orthorhombic crystals. In most cases, a good correlation is found at the atomic level between residues involved in the thermal transition in solution and in the crystalline state. Discrete discrepancies are noted for some residues such as Trp-62 and His-15.     

1H-n.m.r. studies of squash seed trypsin inhibitor

1H-n.m.r. studies at 500 MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30 degrees, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-alpha CH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded beta sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a beta turn.     

13C-n.m.r. spectroscopy of cellulose ethers

Characteristics of the ¹³C-n.m.r. spectra of cellulose ethers (methyl, carboxymethyl, and hydroxyethyl) have been examined at 22.6 MHz. Partial depolymerization with acid or cellulase proved to be a requisite preliminary step. Strong deshielding of ¹³C nuclei bearing alkoxyl groups was clearly evident in these spectra, which permitted an assessment of the degree of substitution at individual positions of the d-glucose residues. Better resolved spectra, and more-detailed structural analyses, were afforded by complete hydrolysates of the polymers. The findings are wholly consistent with data obtained for these derivatives by other methods, showing that the reactivities of the hydroxyl groups of cellulose are OH-2>OH-6 ? OH-3. It is also shown that reducing-end residues liberated during enzymic hydrolysis of the cellulose derivatives are not substituted at the 2-position.     

The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study.

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5'-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3'-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a 'melting' temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3'-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5 X 10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.     

N.m.r. analyses of the histidine microenvironments in a human salivary proline-rich glycoprotein

The pKa's of the three histidine residues in a proline-rich glycoprotein from human parotid saliva (PRG) were determined by 360 MHz proton n.m.r. spectroscopy. The addition of calcium (0.64 mM) caused drops in the pKa's of all three histidines by approximately 0.25 units. When imidazole and cyclo)L-histidine-L-proline) were used as model compounds, corresponding concentrations of calcium had no effect on their pKa's. Also, the model compounds gave absolute pKa values in good agreement with similar chemical species reported in the literature. Exchange lifetime data and previously reported hydrogen----deuterium exchange experiments suggest that the PRG histidine N tau H protons are not involved in hydrogen-bonds. Collectively, these data imply that changes in PRG conformation occur upon the addition of calcium.     

Assignments of the 1H- and 13C-n.m.r. spectra of tobramycin at low and high pH

The 1H-n.m.r. spectra (200 MHz) of protonated (pD 3.9) and non-protonated (pD 11.9) tobramycin have been analysed completely by 2D methods. The 3JH,H values are consistent with an essentially undistorted 4C1 conformation for each of the three moieties and are practically independent of the state of protonation. The resonances in the 13C-n.m.r. spectrum (50 MHz) have been reassigned at both pD values on the basis of 2D1H-13C chemical-shift correlation and 1D selective INEPT measurements.     

Analysis of some partly and fully esterified oligogalactopyranuronic acids by p.m.r. spectrometry at 220 MHz

The p.m.r. spectra of mono-, di-, tri-, tetra-, and penta-galactopyranuronic acids (1–5), the corresponding fully esterified methyl esters (6–10), the partly esterified di- (11) and tri-galactopyranuronic acids (12, 13), and the unsaturated di-, tri-, and tetra-galactopyranuronic acids (14–16) were measured on solutions in D₂O at 220 MHz at a pH of 1 and 6. Observation of doublets (J 4 Hz) in the range δ 4.90–5.05 p.p.m. indicates the site of esterification in the non-reducing or reducing sugar residue. Esterification of the sugar residue at the non-reducing end can be deduced from both the presence of a methyl resonance peak at δ 3.80 and the indifference of the signal at δ 4.35 (H-4) to the change in pH. The δ values and coupling constants confirm that all the d-galacturonic acid residues have the CI conformation and are α-(1→4)-linked. In the unsaturated oligogalactopyranuronic acids, the double bond is located between C-4 and C-5 of the sugar unit at the non-reducing end. The 4-deoxyhex-4-enopyranosyluronic acid residue occurs in the ²H₁(d) conformation. Compound 11 was identified as O-(α-d-galactopyranosyluronic acid)-(1→4)-(methyl α,β-d-galactopyranuronate). Compounds 12 and 13 each consisted of a mixture of the three possible isomers; preference for the site of esterification decreases in the order reducing sugar unit, non-reducing sugar unit, sugar unit at the non-reducing end.