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

The conformational features of a chemically synthesized 23-residue glycopep tide construct (II) carrying Gal-beta-(1,3)-alpha -GalNAc and its deglycosy lated counterpart (I; Gal: galactose; GalNAc: N-acetyl galactosamine) deriv ed from the C-terminal domain of human salivary mucin (MUC7). were investig ated using CD spectroscopy as well as molecular dynamic simulation studies. The corresponding deglycosylated peptide (I) was essentially used to compa re and study the influence of the sugar moiety on peptide backbone conforma tion. CD measurements in aqueous medium revealed that the apopeptide (I) co ntains significant populations of beta -strand conformation while the glyco peptide (II) possess, partly, helical structure. This transition in the sec ondary structure upon glycosylation from beta -strand to helical conformati on clearly demonstrates that the carbohydrate moiety exerts significant inf luence on the peptide backbone. On the other hand, upon titrating structure stabilizing organic cosolvent, triflouroethanol (TFE), both the peptides s howed pronounced helical structure. However the propensity fbr helical stru cture formation is less pronounced in glycopeptide compared to apopeptide s uggesting that the bulky carbohydrate moiety possibly posing steric hindran ce to the formation of TFE-induced secondary structure in II. Energy-minimi zed molecular model for the glycopeptide revealed that the preferred helix conformation in aqueous medium appears to be stabilized by the hydrogen-bon ded salt bridge like interaction between carbohydrate -OH and Lys-10 side c hain -N+H3 group. Size exclusion chromatographic analysis of both (glyco)pe ptides I and If showed an apparent Kd of 2.3 and 0.52 muM, respectively, in dicating that glycopeptide (II) has greater tendency for self-association. Due to high amphipathic character as well as due to the presence of a leuci ne Zipper motif (similar to LLYMKNLL similar to), which is known to increas e the stability at the coiled-coil interface via hydrophobic interactions, we propose therefore that, this domain could be one of the key elements inv olved in the self-association of intact MUC7 in vivo. Profound conformation al effects governed by glycosylation exemplified herein could have implicat ions in determining structure-function relationships of mucin glycoproteins . (C) 2001 John Wiley & Sons, Inc.