Structural Studies of Mucin Glycoprotein Motifs

摘要

Protein glycosylation is the most prevalent and complex form of protein post-translational processing. The variety and dynamic aspects of these modifications add an additional dimension to the diversity of protein properties and function. Mucin glycoproteins comprise a major subset of the universe of glycoproteins. This class is characterized by a high density of S and T residues, often in clusters, whose sidechains serve as loci of glycosylation, either throughout the length of the protein or in subdomains. A number of prominent examples are found as domains in cell surface proteins. The distribution of glycosylation is determined through the initial attachment of an α-O-GalNAc residue to S or T residues as mediated by the particular member(s) of the repertoire of polypeptide GalNAc transferase enzymes that the cell is expressing at the time. Thus, the patterns and glycan structures reflect the condition of the cell in a time dependent way and can serve as biomarkers. These changes play an important role in modulating normal cellular interactions. Aberrant glycosylation of cell surface molecules often is a manifestation of diseases including cancer, [1] raising the prospect of using these unusual structures in targeted therapies. An understanding of the molecular recognition events of the normal and unusual forms of these molecules requires knowledge of their conformational properties at atomic resolution. Unfortunately, both the natural microheterogeneity of glycosylation on native glycoproteins, as well as their high molecular weight, has seriously limited this effort. Chemical synthesis, however, can provide homogeneous and well defined material that circumvents these problems, and solid phase synthesis has proven invaluable in preparing mucin glycopeptide motifs for our efforts to address the conformation of mucins by NMR [2]. Since glycosylated mucins naturally show extended structures, interactions with sequentially remote portion of the molecule are unlikely, and the short synthetic segments are expected to be subject to the same local forces that dominate the structure of the native glycoprotein. In this work we have characterized the conformations of a mucin glycoprotein motif derived from MUC2, systematically varying the distribution and density of a-O-GalNAc glycosylation. Several members of this series have also previously been examined as substrates for polypeptide GalNAc transferase enzymes, allowing, as well, for potential insights into the structure/function relationships of the enzymatic reactions [3].