NMR analysis of human salivary mucin (MUC7) derived O‐linked model glycopeptides: comparison of structural features and carbohydrate–peptide interactions

Abstract

Two series of glycopeptides with mono- and disaccharides, [GalNAc and Galbeta (1-3)GalNAc] O-linked to serine and threonine at one, two or three contiguous sites were synthesized and characterized by 1H NMR. The conformational effects governed by O-glycosylation were studied and compared with the corresponding non-glycosylated counterparts using NMR, CD and molecular modelling. These model peptides encompassing the aa sequence, PAPPSSSAPPE (series I) and APPETTAAPPT (series II) were essentially derived from a 23-aa tandem repeat sequence of low molecular weight human salivary mucin (MUC7). NOEs, chemical shift perturbations and temperature coefficients of amide protons in aqueous and nonaqueous media suggest that carbohydrate moiety in threonine glycosylated peptides (series II) is in close proximity to the peptide backbone. An intramolecular hydrogen bonding between the amide proton of GalNAc or Galbeta (1-3)GalNAc and the carbonyl oxygen of the O-linked threonine residue is found to be the key structure stabilizing element. The carbohydrates in serine glycosylated peptides (series I), on the other hand, lack such intramolecular hydrogen bonding and assume a more apical position, thus allowing more rotational freedom around the O-glycosidic bond. The effect of O-glycosylation on peptide backbone is clearly reflected from the observed overall differences in sequential NOEs and CD band intensities among the various glycosylated and non-glycosylated analogues. Delineation of solution structure of these (glyco)peptides by NMR and CD revealed largely a poly L-proline type II and/or random coil conformation for the peptide core. Typical peptide fragments of tandem repeat sequence of mucin (MUC7) showing profound glycosylation effects and distinct differences between serine and threonine glycosylation as observed in the present investigation could serve as template for further studies to understand the multifunctional role played by mucin glycoproteins.

Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods

“…Sophisticated, multivariate algorithms based upon AA sequences rather than com-position have confirmed experimental findings that all SCPP are unfolded to a large degree (Naganagowda et al, 1999;Fisher et al, 2001;Syme et al, 2002;Buchko et al, 2008;Schedlbauer et al, 2008;Aichmayer et al, 2005;Delak et al, 2009;Holt et al, 2009) with a predominance of the poly-l-proline type II (PP-II) secondary structure (Supplementary File, section S4.1; http://dx.doi.org/10.3168/jds.2013-6831).…”

MUC7 Haplotype Analysis: Results from a Longitudinal Birth Cohort Support Protective Effect of the MUC7*5 Allele on Respiratory Function

“…Although the mechanisms of interaction between MUC7 and bacteria have mostly been shown to be mediated by the N-terminal domain of MUC7, carbohydrate structures have been implicated (Groenink et al 1996;Prakobphol et al 1999), and the shorter glycoprotein encoded by the MUC7 * 5 allele will contain fewer carbohydrate side chains. The loss of the 23 amino acids from the fourth repeat unit (Kirkbride et al 2001), which has a unique combination of serines, threonines and di-proline domains (amino acid sequence APP), could have an effect on the conformation of the MUC7 molecules, which is likely to influence their function (Gururaja et al 1998;Naganagowda et al 1999).…”

Conformational consequences of protein glycosylation: Preparation of O‐mannosyl serine and threonine building blocks, and their incorporation into glycopeptide sequences derived from α‐dystroglycan

“…Here, a strong negative band at 202 nm, with a weak positive band at 224 nm and another weak positive band at 188 nm, suggests an ordered structure, and is reminiscent of what is observed for an ordered PPII (polyproline II) helical structure. 26 There are obvious differences in the primary structures between what is being studied here and a canonical polyproline construct, [27][28][29] with other interactions at play here, particularly interactions between the GalNAc amide and the peptide backbone. The CD does point out the conformational differences resulting from different sugar modifications to the same peptide sequence.…”