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

Abstract

The conformational behavior of the N-glycan Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta 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 glycopeptide 1H 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 NOESY 1H 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 Fuc alpha 1-3GlcNAc linkage was altered. In addition, the omega = 60 degrees (gt) rotamer of the Man alpha 1-6Man linkage seems to be present for a significant period of time, whereas in the glycopeptide the omega = -60 degrees (gg) conformation exists exclusively. Except for the Xyl beta 1-2Man linkage, the mobilities around the glycosidic linkages in the glycoprotein were reduced compared with those in the glycopeptide, especially concerning the Fuc alpha 1-3GlcNAc and Man alpha 1-6Man linkages. These findings might be the result of an interaction of the polypeptide chain with the Fuc alpha/Man alpha 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.