GlcNAc Disaccharide Research Articles

Mitogenic leukoagglutinin from Phaseolus vulgaris binds to a pentasaccharide unit in N-acetyllactosamine-type glycoprotein glycans.

The carbohydrate binding specificity of leukoagglutinin (La; Phaseolus vulgaris isolectin L4) was studied by using quantitative precipitation and precipitation-inhibition. A series of purified glycopeptides and synthetic oligosaccharides were used as inhibitors. The minimal structural unit required for La binding was the disaccharide GlcNac(1 leads to beta 2)Man. Additions for this basic unit of different sugar residues gave a positive or negative contribution to binding. The most complementary structure was the pentasaccharide (formula: see text). This pentasaccharide units occurs in tetraantennary N-acetyllactosamine-type glycoprotein glycans. Glycoproteins containing such structures were accordingly precipitated by La. Selected glycopeptides and oligosaccharides were also tested as inhibitors of La-induced DNA synthesis in human lymphocytes. The pattern of inhibition was essentially the same as that obtained by precipitation-inhibition, indicating that binding to lymphocytes via the carbohydrate binding site of the lectin is an essential step in the activation process.

Fractionation of oligosaccharides containing N-acetyl amino sugars by reverse-phase high-pressure liquid chromatography

High-pressure liquid chromatography (hplc) of N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc) containing carbohydrates was performed on several reverse-phase silica columns. Nanomolar level detection was accomplished using far uv-absorbance monitoring. Baseline separations of the α and β anomers of GlcNAc, chitobiose, chitotriose, and chitotetraose were observed with water elution of the reverse-phase column. With the addition of up to 3% acetonitrile to the eluting solvent, similar resolution of chitin oligomers up to a chain length of seven was observed. Anomerization of the residue could be followed by isolation of either anomeric peak with subsequent rechromatography. Reduction of chitotriose with borohydride yielded a single sharp peak with a retention volume similar to that of the reducing trisaccharide. Semipreparative reverse-phase hplc allowed for the separation and identification by 13C NMR of the GlcNAc-α-1→6 GlcNAc disaccharide from the β-1→6 isomer. Methyl glycosides of GalNAc and GlcNAc were shown to have retention times much longer than the free sugar.

Substrate-induced evolution of lysozymes

Digestion by five different lysozymes (mucopeptide N-acetylmuramoylhydrolases, EC 3.2.1.17) of a soluble uncross-linked peptidoglycan secreted by Micrococcus luteus (Mirelman, D., Bracha, R. and Sharon N. (1974) Biochemistry 13, 5045–5053) was investigated. Hen egg-white and human lysozymes converted the peptidoglycan into the disaccharide GlcNAc- β(1 → 4)- N-acetylmuramic acid (-MurNAc), the tetrasaccharide (GlNAc- β(1 → 4)-MurNAc) 2 and the disaccharide-hexapeptide: ▪ With goose egg-white lysozyme, mainly the disaccharide and the disaccharidehexapeptide were produced, in approximately equimolar amounts. Papaya lysozyme afforded mainly products without peptide substitution, such as the disaccharide and tetrasaccharide, while T4 phage lysozyme gave only the disaccharide-hexapeptide. Lysozymes from different sources thus differ in their specificity requirements with respect to peptide substitution on the lactyl moiety of N-acetylmuramic acid. This pattern of specificity is discussed in terms which correlate it with the differences in the structures of their presumed natural substrates. We postulate that evolutionary changes in the substrate structure may have influenced the development of the active site of lysozyme so that it could function most efficiently with the particular natural substrate encountered by each species.

The Structures of Oligosaccharides Accumulating in the Liver of Gm1-Gangliosidosis, Type I

Abstract Structurally related oligosaccharides have been isolated from freshly frozen liver from cases of Gm1-gangliosidosis, type I, an inherited glycolipid storage disease. In the three major fractions of approximate molecular weight, 1400, 1800, and 2200, respectively, galactose, N-acetylglucosamine, and mannose were the main components. From periodate oxidation, proton magnetic resonance, optical rotation, and methylation studies, it was concluded that the molecular weight 1800 oligosaccharide had the structure [see PDF for equation] with 1 additional Gal β1→4 GlcNAc disaccharide unit linked β1→4 to either of the monosubstituted mannose residues. The molecular weight 1400 fraction lacked this additional disaccharide. In the molecular weight 2200 fraction the terminal N-acetylglucosamine was glycosidically linked. The anomeric configurations were confirmed by digestions with appropriate enzymes. The oligosaccharides likely derive from erythrocyte stromal glycoproteins whose normal breakdown is impaired during development by the deficient β-galactosidase activity present in this disease.