Conformational analysis of the xylose-containing N-glycan of pineapple stem bromelain as part of the intact glycoprotein

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    Abstract

    The conformational behavior of the N-glycan Manα1-6(Xylβ1-2)Manβ1- 4GlcNAcβ1-4(Fucα1-3)GlcNAcβ of stem bromelain as part of the intact glycoprotein was investigated and compared with that of the same N-glycan as part of a bromelain-derived glycopeptide. Proton chemical shifts of the glycoprotein N-glycan were determined by 2D HOHAHA and 2D NOESY measurements, making use of the glycopeptide1H NMR data. During each 2D NMR experiment about 4% of the glycoprotein denatured. Experimental data concerning interproton distances of the intact glycoprotein N-glycan were obtained by NOESY1H NMR spectroscopy. Several theoretical models for the N-glycan, obtained by molecular dynamics simulations of the glycopeptide, were investigated. Comparison of experimental and theoretical NOESY cross peak intensities was performed with the program CROSREL. In comparison with the glycopeptide, the distribution of populations between two main conformations of the Fucα1-3GlcNAc linkage was altered. In addition, the ω = 60° (gt) rotamer of the Manα1-6Man linkage seems to be present for a significant period of time, whereas in the glycopeptide the ω = -60° (gg) conformation exists exclusively. Except for the Xylβ1-2Man linkage, the mobilities around the glycosidic linkages in the glycoprotein were reduced compared with those in the glycopeptide, especially concerning the Fucα1-3GlcNAc and Manα1- 6Man linkages. These findings might be the result of an interaction of the polypeptide chain with the Fucα/Manα side of the N-glycan. A qualitative analysis of the NMR spectra showed a larger degree of mobility in the denatured glycoprotein N-glycan than in the intact glycoprotein.
    Original languageEnglish
    Pages (from-to)8196-8206
    Number of pages11
    JournalBiochemistry
    Volume34
    Issue number25
    DOIs
    Publication statusPublished - 16 Dec 1995

    Keywords

    • bromelain
    • glycan
    • glycoprotein
    • xylose
    • amino acid sequence
    • amino acid substitution
    • article
    • conformational transition
    • fruit
    • genetic heterogeneity
    • genetic linkage
    • glycosylation
    • molecular dynamics
    • nonhuman
    • priority journal
    • protein analysis
    • protein denaturation

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