Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.     

Carbohydrate epitope structural elucidation by 1H-NMR spectroscopy of a new Mycobacterium kansasii phenolic glycolipid antigen.

The complete primary structure of the carbohydrate moiety of a new phenolic glycolipid antigen namely PheGl K-IV from Mycobacterium kansasii was successfully established from only one- and two-dimensional 1H-NMR data. Among the scalar two-dimensional techniques, correlated spectroscopy with a 45 degree mixing pulse and phase-sensitive double-quantum-filtered correlated spectroscopy were selected, combined with two-dimensional dipolar techniques (nuclear Overhauser effect). These techniques using milligram of quantities native PheGl K-IV allowed the following monoacetylated tetrasaccharide to be proposed for its carbohydrate part: 4-O-Me-alpha-Manp-(1----3)-4-O-Ac-2-O-Me-alpha-Fucp-(1----3) -2-O-Me-alpha-Rhap- (1----3)-2,4-di-O-Me-alpha-Rhap. The PheGl K-IV shares, with the other phenolic glycolipids isolated from M. kansasii (K-I, K-II), a common core assigned to the lipid aglycone glycosylated by the monoacetylated trisaccharide part. It differs in the structure of the distal monosaccharide residue.     

Evidence for cross-linking in tomato cutin using HR-MAS NMR spectroscopy

Cutin is a polyester biopolymer component of plant leaf and fruit cuticles, most often associated with waxes and cuticular polysaccharides, and sometimes with another aliphatic biopolymer called cutan. Insolubility of these cuticular biopolymers has made it difficult to apply traditional analytical techniques for structure determination, because most techniques providing molecular level details require solubility. By using the relatively new technique of one and two-dimensional high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, with added information from solid-state 13C NMR spectroscopy, detailed through-bond connectivities and assignments are made for cutin from Lycopersicon esculentum (tomato) fruit. Based on the data obtained, tomato cutin is found to be predominantly an aliphatic polyester with some olefinic and aromatic moieties, consistent with previous studies that employed various degradative approaches. Aside from esters, there are free primary and secondary alcohol groups, as well as free fatty acids. A significant finding is the presence of alpha-branched fatty acids/esters. Mid-chain hydroxyls appear to be generally unesterified, but esters of mid-chain hydroxyls have been identified. The alpha-branched fatty acids/esters and esters of mid-chain hydroxyls could point towards cross-linking.     

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.     

Spectroscopic methods for analysis of protein secondary structure

Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.     

GM1 oligosaccharide efficacy against α-synuclein aggregation and toxicity in vitro

Fibrillary aggregated α-synuclein represents the neurologic hallmark of Parkinson's disease and is considered to play a causative role in the disease. Although the causes leading to α-synuclein aggregation are not clear, the GM1 ganglioside interaction is recognized to prevent this process. How GM1 exerts these functions is not completely clear, although a primary role of its soluble oligosaccharide (GM1-OS) is emerging. Indeed, we recently identified GM1-OS as the bioactive moiety responsible for GM1 neurotrophic and neuroprotective properties, specifically reverting the parkinsonian phenotype both in in vitro and in vivo models.Here, we report on GM1-OS efficacy against the α-synuclein aggregation and toxicity in vitro. By amyloid seeding aggregation assay and NMR spectroscopy, we demonstrated that GM1-OS was able to prevent both the spontaneous and the prion-like α-synuclein aggregation. Additionally, circular dichroism spectroscopy of recombinant monomeric α-synuclein showed that GM1-OS did not induce any change in α-synuclein secondary structure. Importantly, GM1-OS significantly increased neuronal survival and preserved neurite networks of dopaminergic neurons affected by α-synuclein oligomers, together with a reduction of microglia activation.These data further demonstrate that the ganglioside GM1 acts through its oligosaccharide also in preventing the α-synuclein pathogenic aggregation in Parkinson's disease, opening a perspective window for GM1-OS as drug candidate.     

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.     

Sequence-specific 1H NMR assignments and secondary structure of porcine motilin

The solution structure of the 22-residue peptide hormone motilin has been studied by circular dichroism and two-dimensional 1H nuclear magnetic resonance spectroscopy. Circular dichroism spectra indicate the presence of alpha-helical secondary structure in aqueous solution, and the secondary structure can be stabilized with hexafluoro-2-propanol. Sequence-specific assignments of the proton NMR spectrum of porcine motilin in 30% hexafluoro-2-propanol have been made by using two-dimensional NMR techniques. All backbone proton resonances (NH and alpha CH) and most of the side-chain resonances have been assigned by using double-quantum-filtered COSY, RELAYED-COSY, and NOESY experiments. Simulations of NOESY cross-peak intensities as a function of mixing time indicate that spin diffusion has a relatively small effect in peptides the size of motilin, thereby allowing the use of long mixing times to confidently make assignments and delineate secondary structure. Sequential alpha CH-NH and NH-NH NOESY connectivities were observed over a significant portion of the length of the peptide. A number of medium-range NOESY cross-peaks indicate that the peptide is folded into alpha-helix from Glu9 to Lys20, which agrees favorably with the 50% helical content determined from CD measurements. The intensities of selected NOESY cross-peaks relative to corresponding diagonal peaks were used to estimate a rotational correlation time of approximately 2.5 ns for the peptide, indicating that the peptide exists as a monomer in solution under the conditions used here.     

Structural studies on the oligosaccharide moiety of the lipopeptidophosphoglycan from Trypanosoma cruzi

The structure of the carbohydrate moiety of the lipopeptidophosphoglycan from Trypanosoma cruzi was studied by 13C NMR spectroscopy and by methylation analysis of the original and of an acid-degraded sample. An oligosaccharide, consisting of 2-O-substituted and 6-O-substituted mannoses, which is linked to the ceramide, was separated by partial acid hydrolysis from an external chain that contained 3-O-substituted mannopyranosyl residues. beta-D-Galactofuranosyl terminal units are attached to position 3 of (1----2)-linked mannopyranose. Besides the previously reported monosaccharide components (mannose, galactose, glucose and glucosamine), ribose was identified in a partial acid hydrolysate of the lipopeptidophosphoglycan. The last three sugars are minor components and their organization into the overall structure of the lipopeptidophosphoglycan has not been determined.     

Novel carbohydrate-induced modification of peptides: crystal structure and NMR analysis of ester-bridged bicyclic galactose-enkephalin adduct containing imidazolidinone moiety.

Comparative studies based on x-ray crystallography and NMR spectroscopy were used for structural characterization of the novel minor, imidazolidinone moiety containing, product 2b of the Maillard reaction obtained in vitro by using the galactose-modified endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) 1. The x-ray analysis uniquely defined the molecular structure as cyclo-(N-(12-[-4)-D-galacto-pentitol-1-yl]-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1 --> O]acetyl]glycyl-L-phenylalanyl-L-leucyl-] (3), having an 18-membered ring with an ester bond between the secondary (C4') hydroxyl group of a D-galacto-pentitolyl residue and the C-terminal carboxy group of leucine-enkephalin. The absolute configuration of the new chiral centre at the imidazolidinone moiety was established as C2(S), indicating a cis arrangement of C2 and C4 substituents at the 5-membered heterocyclic ring. The NMR analysis of compound 2b carried out in CH3CN-d3 and DMSO-d6, indicated the existence of two isomers in solution, differing only in the position of the ester group in the molecule. NMR data for the minor isomer (13%-16%) are in agreement with structure 3. The migratory tendency of the peptidyl group from the primary (2b) to the secondary hydroxyl group (3) of a D-galacto-pentitolyl residue in methanol/water solution was confirmed by RP HPLC analysis.     

A 1H-NMR study of the solution conformation of secretin. Resonance assignment and secondary structure

The solution conformation of the 27 residue polypeptide hormone secretin has been investigated by 1H-NMR spectroscopy under conditions where it adopts a fully ordered structure as judged by circular dichroism spectroscopy, namely in an aqueous solution of 40% (v/v) trifluoroethanol. Using a combination of two-dimensional NMR techniques the 1H-NMR spectrum of secretin is completely assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. It is shown that under these conditions secretin adopts a conformation consisting of an N-terminal irregular strand (residues 1-6) followed by two helices (residues 7-13 and 17-25) connected by a 'half-turn' (residues 14-16); the last two residues (26 and 27) are again irregular. This conformation is shown to be very similar to that of glucagon in perdeuterated dodecylphosphocholine micelles and to that of the active 1-29 fragment of growth hormone releasing factor in 30% (v/v) trifluoroethanol:     

Structural analysis of the lipooligosaccharide from the commensal Haemophilus somnus genome strain 129Pt

The structure for the carbohydrate moiety of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 129Pt was elucidated. The structure of the core oligosaccharide and O-deacylated LOS was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the major fully extended carbohydrate glycoform of the LOS was determined on the basis of the combined data from these experiments. [Carbohydrate structure: see text]. In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and PEtn is phosphoethanolamine. Minor amounts of glycoforms containing nonstoichiometric substituents glycine and phosphate at the distal heptose residue were also identified.     

CSSI-PRO: a method for secondary structure type editing,assignment and estimation in proteins using linear combination of backbone chemical shifts

Estimation of secondary structure in polypeptides is important for studying their structure, folding and dynamics. In NMR spectroscopy, such information is generally obtained after sequence specific resonance assignments are completed. We present here a new methodology for assignment of secondary structure type to spin systems in proteins directly from NMR spectra, without prior knowledge of resonance assignments. The methodology, named Combination of Shifts for Secondary Structure Identification in Proteins (CSSI-PRO), involves detection of specific linear combination of backbone ¹H^α and ¹³C′ chemical shifts in a two-dimensional (2D) NMR experiment based on G-matrix Fourier transform (GFT) NMR spectroscopy. Such linear combinations of shifts facilitate editing of residues belonging to α-helical/β-strand regions into distinct spectral regions nearly independent of the amino acid type, thereby allowing the estimation of overall secondary structure content of the protein. Comparison of the predicted secondary structure content with those estimated based on their respective 3D structures and/or the method of Chemical Shift Index for 237 proteins gives a correlation of more than 90% and an overall rmsd of 7.0%, which is comparable to other biophysical techniques used for structural characterization of proteins. Taken together, this methodology has a wide range of applications in NMR spectroscopy such as rapid protein structure determination, monitoring conformational changes in protein-folding/ligand-binding studies and automated resonance assignment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

One- and two-dimensional 90.5-MHz 13C-NMR spectroscopy of the N-linked triantennary oligosaccharide units of calf fetuin

Complete assignments of all anomeric resonances in the proton and carbon spectra of the N-linked oligosaccharide units of fetuin were made using one- and two-dimensional NMR spectroscopy. We are able to confirm the presence of microheterogeneity in the N-acetylneuraminic acid linkages to the galactose residues and the presence of a unique triantennary structure which carries a side chain: NeuAc alpha(1-3)Gal beta(1-3)GlcNac beta(1-4)-. Anomeric carbon chemical shifts changes resulting from long-range conformational effects were observed.     

Lipopolysaccharides possessing two L-glycero-D-manno-heptopyranosyl-alpha -(1-->5)-3-deoxy-D-manno-oct-2-ulopyranosonic acid moieties in the core region. The structure of the core region of the lipopolysaccharides from Burkholderia caryophylli.

The carbohydrate backbone of the core-lipid A region was characterized from the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli. For the first time, the presence of two moieties of l-glycero-d-manno-heptopyranosyl-alpha-(1-->5)-3-deoxy-d-manno-oct-2-ulopyranosonic acid was identified in a core region, which is of particular interest with regard to the biosynthesis of this and of LPSs in general. The LPSs of B. caryophylli were degraded by mild hydrazinolysis (de-O-acylation), treatment with 48% aqueous HF at 4 degrees C (cleavage of phosphate groups and destruction of the O-specific polysaccharides), reduction with NaBH4, and de-N-acylation utilizing hot KOH. The major oligosaccharide representing the carbohydrate backbone of the core region and lipid A was isolated by high-performance anion-exchange chromatography. Its analysis employing compositional and methylation analyses, matrix-assisted laser desorption/ionization mass spectrometry, and (1)H and (13)C NMR spectroscopy applying various one-dimensional and two-dimensional experiments identified the following structure. Structure I All sugars are pyranoses and alpha-linked, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transferrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The 1H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional 1H-1H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503-506).     

Structural elucidation of the Brucella melitensis M antigen by high-resolution NMR at 500 MHz

The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-alpha-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers of the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz 1H and 125-MHz 13C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent beta-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants. Structural and serological considerations in conjuction with the sodium dodecyl sulfate banding pattern of Brucella A LPS suggest that its biosynthesis differs appreciably from that of the M antigen, which appears to be synthesized by regulated assembly of preformed oligosaccharide repeating units. Temperature, lysogenic phage may be responsible for such biosynthetic and structural variations.     

Structure elucidation of glycoprotein glycans and of polysaccharides by NMR spectroscopy

The applicability of 1H-NMR spectroscopy for the determination of the primary and tertiary structure of carbohydrate-containing molecules is demonstrated. For classes of known compounds the characterization can be based on chemical shifts observed in 1D NMR spectra with or without the aid of a computer database. For more complex structure determinations 2D NMR techniques are required. Here the application of 2D NMR is demonstrated for the primary structure determination of two bacterial exopolysaccharides, for the spatial structure determination of a disaccharide and a glycoprotein hormone.     

Structural analysis of the lipopolysaccharide from the nontypable Haemophilus influenzae strain SB 33.

The structure of the core region of the lipopolysaccharide (LPS) from the nontypable Haemophilus influenzae strain SB 33 was elucidated. The LPS was subjected to a variety of degradative procedures. The structures of the derived oligosaccharide products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. These analyses revealed a series of related phosphocholine (PCho) containing structures differing in the number of hexose residues. The results pointed to each species containing a conserved phosphoethanolamine (PEtn) substituted heptose-containing trisaccharide inner-core moiety. The major LPS glycoforms were identified as 2-Hex, 3-Hex and 4-Hex species according to the number of hexose residues present.     

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.