Synthesis and conformational features of human salivary mucin C-terminal derived peptide epitope carrying Thomsen-Friedenreich antigen: implications for its role in self-association.

Abstract

The conformational features of a chemically synthesized 23-residue glycopeptide construct (II) carrying Gal-beta-(1,3)-alpha-GalNAc and its deglycosylated counterpart (I; Gal: galactose; GalNAc: N-acetyl galactosamine) derived from the C-terminal domain of human salivary mucin (MUC7) were investigated using CD spectroscopy as well as molecular dynamic simulation studies. The corresponding deglycosylated peptide (I) was essentially used to compare and study the influence of the sugar moiety on peptide backbone conformation. CD measurements in aqueous medium revealed that the apopeptide (I) contains significant populations of beta-strand conformation while the glycopeptide (II) possess, partly, helical structure. This transition in the secondary structure upon glycosylation from beta-strand to helical conformation clearly demonstrates that the carbohydrate moiety exerts significant influence on the peptide backbone. On the other hand, upon titrating structure stabilizing organic cosolvent, trifluoroethanol (TFE), both the peptides showed pronounced helical structure. However, the propensity for helical structure formation is less pronounced in glycopeptide compared to apopeptide suggesting that the bulky carbohydrate moiety possibly posing steric hindrance to the formation of TFE-induced secondary structure in II. Energy-minimized molecular model for the glycopeptide revealed that the preferred helix conformation in aqueous medium appears to be stabilized by the hydrogen-bonded salt bridge like interaction between carbohydrate --OH and Lys-10 side--N(+)H(3) group. Size exclusion chromatographic analysis of both (glyco)peptides I and II showed an apparent Kd of 2.3 and 0.52 microM, respectively, indicating that glycopeptide (II) has greater tendency for self-association. Due to high amphipathic character as well as due to the presence of a leucine zipper motif ( approximately LLYMKNLL approximately ), which is known to increase the stability at the coiled-coil interface via hydrophobic interactions, we propose therefore that, this domain could be one of the key elements involved in the self-association of intact MUC7 in vivo. Profound conformational effects governed by glycosylation exemplified herein could have implications in determining structure-function relationships of mucin glycoproteins.