Lambertus Dorland

High-resolution, 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins

Publisher Summary This chapter discusses the application of high-resolution, 1H-nuclear magnetic resonance (NMR) spectroscopy to the structural analysis of carbohydrates related to glycoproteins. Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the O-glycosylic chains are linked to the hydroxyl group of amino acid residues such as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). The high-resolution, 1H-NMR spectroscopy technique, in conjunction with methylation analysis, is extremely suitable for the structural studies of N-, as well as on O-, glycosylic glycans. For the interpretation of the 1H-NMR spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structural reporter groups” was developed. This means that the chemical shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit the assigning of the primary structure. This chapter presents literature data on the high-resolution, 1H-NMR spectroscopy of carbohydrates derived from glycoconjugates. It also discusses the results for carbohydrates related to the glycoproteins of N-glycosylic type.

The structure of the asialo‐carbohydrate units of human serotransferrin as proven by 360 MHz proton magnetic resonance spectroscopy

Human serotransferrin contains two identical carbohydrate chains [l-3] about the primary Structure of which controversy exsists. Jamieson et al. [3] suppose a branched structure, wherein the branching mannose, glycosidically linked to N-acetylglucosamine /3l+Asn, bears two chains: one consisting of a mannotriose, the other of an N-acetylglucosamine residue and both terminated by an N-acetylneuraminyliV-acetyllactosamine unit. Spik et al. [ 1,2] propose a more symmetrical structure, built up from a mannotriosido-di-N-acetylchitobiose core substituted by two N-acetylneuraminyl-N-acetyllactosamine units. This structure, presented in fig. 1 differs essentially from the foregoing structure since it has only 3 mannose residues instead of 4. In-this paper the investigation by high-resolution ‘H nuclear magnetic resonance spectroscopy of the structure of the asialoglycopeptide (asialo-glycanAsn) isolated from serotransferrin is described. In particular the signals of the anomeric protons, the mannose-H-2 protons and the N-acetyl methyl groups were analysed. For the assignment of these signals, spectra of the corresponding asialo-agalacto-glycanAsn-Lys, the glyco-amino acids GlcNAcpl+Asn [4] and Mancz(1+6)Man/3(1+4)G1cNAc/3(1+4) [Fu&l+6)] GlcNA@l+Asn [5] and the trisaccharide Mancu(l+3) Mar@(l+4) GlcNAc [6] , representing partial structures of the asialo-glycan-Asn have been used as reference compounds. The monosaccharide units in these substances are numbered in correspondence to the numbering in the serotransferrin glycopeptides (see fig.1 and table 1).

A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow κ-casein

Abstract 360-MHz 1 H-NMR spectra were recorded of NeuAcα(2 → 3)Galβ (1 → 3)GalNAc-ol (I), Galβ(1 → 3)[NeuAcα(2 → 6)] GalNAc-ol (II) and NeuAcα(2 → 3)-Galβ(1 → 3) [NeuAcα(2 → 6)]GalNAc-ol (III). The chemical shifts and coupling constants of the anomeric protons, the H-3ax and H-3eq of NeuAc, the GalNAc-ol skeleton protons, the H-3 of Gal and the N -acetyl protons of GalNAc-ol and NeuAc provide conclusive evidence for the identification of the primary structures. Compound II represents a novel carbohydrate chain of κ-casein.

Congenital microcephaly and seizures due to 3-phosphoglycerate dehydrogenase deficiency : outcome of treatment with amino acids

Congenital microcephaly, intractable seizures and severe psycho-motor retardation characterize 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, a disorder of L-serine biosynthesis. The enzyme defect results in low concentrations of serine and to a variable degree of glycine in plasma and cerebrospinal fluid. Short-term beneficial effects have been reported of oral treatment with the deficient amino acids. In this paper, we report the first follow-up data of amino acid therapy in five patients treated for 3–7.5 years. Different treatment regimes were used, but a favourable response to amino acids was observed in all patients. A major reduction in seizure frequency occurred in all patients; two patients became free of seizures. Amino acids were well tolerated and no adverse effects were documented. A progress of psychomotor development was only observed in one patient, diagnosed early and treated with a high dosage of L-serine. A favourable outcome of 3-PGDH deficiency depends on early diagnosis and treatment.

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

The primary structure of the asialo-carbohydrate units of the first glycosylation site of human plasma α1-acid glycoprotein

The elucidation of the structures of the carbohydrate units linked to glycosylation site I of human plasma alpha 1-acid glycoprotein is described. These carbohydrate units can be grouped into compounds with bi- (class A) and triantennary (class B) structures and the triantennary structure with a fucose residue (class BF) (Fig. 1). The structural variability of the carbohydrate units of glycosylation site I and also of glycosylation sites II to V (Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206--5214) accounts largely for the microheterogeneity of alpha 1-acid glycoprotein.

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Abstract Partial in vitro sialylation of biantennary and triantennary glycopeptides of α1-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution 1H-NMR spectroscopic analysis of the isolated products enabled the assignment of the Galβ(1→4)GlcNAcβ(1→2)Man α(1→3) Man branch as the most preferred substrate site for sialic acid attachment. The Galβ(1→4)GlcNAcβ(1→2)Man α(1→6) Man branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal N -acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Dutch neonates

Four neonates with a positive phenylalanine screening test (Phe concentrations between 258 and 1250 μmol/L) were investigated further to differentiate between phenylalanine hydroxylase (PAH) deficiency and variant hyperphenylalaninaemia (HPA) forms. In patients 1 and 2 a tetrahydrobiopterin (BH4) load caused a significant decrease of the plasma Phe levels. A combined phenylalanine/BH4 loading test was performed in patients 2, 3 and 4. In the latter two patients, plasma Phe concentrations completely normalized within 8 h after the BH4 load (20 mg/kg). Basal urinary pterins were normal in all four patients. The activity of dihydropteridine reductase (DHPR) was normal in patients 1, 2 and 3 and 50% of control values in patient 4 (not in the range of DHPR-deficient patients). In patient 3 a subsequent phenylalanine loading test with concomitant analysis of plasma biopterins revealed a normal increase of biopterin, excluding a BH4 biosynthesis defect. Pterins and neurotransmitter metabolites in CSF of patients 1, 3 and 4 were normal. DNA mutations detected in the PAH gene of patients 1–4 were A313T, and L367fsinsC; V190A and R243X; A300S and A403V; R241C and A403V. The results are suggestive for mutant PAH enzymes with decreased affinity for the cofactor BH4.

G. NMR spectroscopy of sialic acids

High-resolution NMR spectroscopy has become an invaluable technique in the study of biopolymers and of their constituents. Using 1H or 13C nuclei as probes information can be obtained about primary structures, conformations and intermolecular interactions of biomolecules in solution. In particular, the possibility to record spectra of underivatized compounds in aqueous solutions allows to afford further insight into the way of action of biomolecules under physiological conditions. For general reviews of high-resolution NMR spectroscopy in the study of biological systems the reader is referred to the recent books of Berliner and Reuben (1978, 1980), Jardetzky and Roberts (1981), and Shulman (1979).

Structural studies of 4-O-acetyl-α-N-acetylneuraminyl-(2→3)-lactose, the main oligosaccharide in echidna milk

The main oligosaccharide (50%) in the milk of the Australian echidna (Tachyglossus aculeatus) has been identified unequivocally as 4-O-acetyl-α-N-acetylneur-aminyl-(2→3)-lactose. The 4-O-acetyl substituent of the sialic acid residue was characterised by g.l.c.-m.s. of the isolated (after mild, acid hydrolysis) and trimethyl-silylated/esterified sialic acid, and by m.s. (after derivatisation) and 500-MHz, 1H-n.m.r. spectroscopy of the intact oligosaccharide. Information about the glycosidic bonds was obtained by methylation analysis and 500-MHz, 1H-n.m.r. spectroscopy. This animal species is the third one known to produce 4-O-acetylated sialic acid.