The beta1 2-d-xylose and alpha1 3-l-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

Abstract

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 'H-NMR showed that the major component was the fucosecontaining heptasaccharide Mancr3(Mancr6)(Xylfl2)Manfl4GlcNAcfl4(Fucc3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components.The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Mana3(Mana6)Manfl4GlcNAcfl4GlcNAc] by (1) a D-xylose residue linked 61 -2 to the P-mannosyl residue and (2) an L-fucose residue linked a 1 4 3 to the reducing terminal N-acetylglucosamine residue.The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.All species of the genus Erythrina tested contain Nacetyllactosamine-specific lectins that are remarkably similar in their properties [l, 21. These molecules are all glycoproteins (3 -10% neutral carbohydrate by weight) with M , approximately 60000 and are composed of two identical or nearly identical subunits. Their amino acid composition is very similar and a high degree of homology exists in the N-terminal sequences. [20] and more recently for sycamore cell culture laccase [21] and Caesalpinia pulcherrimu protease inhibitor [22].The simultaneous presence of xylose and fucose therefore appears to be a common feature of many plant glycoproteins. This combination has only been described in glycoproteins of animal origin for Helix pomatiu a-haemocyanin [23].In this paper we describe the isolation of the asparaginelinked oligosaccharides of E. cristagalli lectin, their separation by high-resolution gel permeation chromatography and highvoltage electrophoresis and their characterisation by a combination of methylation analysis, nuclear magnetic resonance and sequential exoglycosidase digestion. We also compare the oligosaccharides of E. cristagalli lectin with those of the lectins from four other species of Erythrina (E. corallodendron, E. caffra, E. latissima and E. lysistemon) and the unrelated legumes Lonchocarpus capassa and Sophora japonica.

The utility of ETD mass spectrometry in proteomic analysis

“…(B) The tandem mass spectrum generated from a histone H3.1 peptide eluting 6 seconds later. Here the c-type ion series indicates the N terminus is modified identical to the peptide shown in (A); however, the C terminus is monomethylated at K 37 , as opposed to being dimethylated at K 36 . Note the spectrum shown in (B) contains fragment ions derived from both species (co-elution) [30] Copyright (2005) National Academy of Sciences, USA.…”

“…The average mass of the corresponding glycopeptide is 3002 Da with the following known glycan structure Manα3 (Manα6)(Xylβ2)Manβ4GlcNAcβ4(Fucα3)GlcNAc [36] (Figure 6). The CAD spectrum for this triply charged glycopeptide ion contains information about the glycan structure, however, there is no fragmentation of the peptide backbone ( Figure 6A).…”

Characterization of a Xylose-Specific Antiserum That Reacts with the Complex Asparagine-Linked Glycans of Extracellular and Vacuolar Glycoproteins

“…The structure of a typical complex glycan, found on many plant glycoproteins (see, for examples, 1, 9, 24, 25, 27), is shown in Figure 7. We suggest that the anti-fF, antibodies bind to the xylose3, 1 Figure 7 is an excellent competitor, while Man3GlcNAc2 does not compete. The incomplete inhibition of the antibody antigen reaction with 5 mm ofcomplex glycopeptide may indicate that this polyclonal antiserum recognizes more than one epitope on the complex glycan, and that there is a differential affinity for these epitopes.…”

1H‐NMR structural determination of the N‐linked carbohydrate chains on glycopeptides obtained from the bean lectin phytohemagglutinin

“…This glycan structure has also been found attached to the lectins from Clerodendron trichotomum (Kitagaki-Ogawa et al, 1986), Erythrina cristugalli (Ashford et al, 1987), S. japonicu (Fournet et al, 1987), Ricinus communis (Kimura et al, 1988), and Artocurpus intergrifolia (Capon et al, 1990), and to the protease inhibitor from Cuesuljkiupulcherrimu (Hase et al, 1986). Related xylose-containing glycan structures were found attached to the protease bromelain from Ananus sutivus (Van Kuik et al, 1986), to the storage protein phaseolin from P .…”