Glycosylation is one of the most common post-translational modifications of eukaryotic proteins. Glycans play diverse roles in molecular recognition events inside the cell and at the cell surface, and aberrant protein glycosylation is associated with a number of diseases, including cancer. In addition, much evidence suggests that N-glycans (attached to the amide nitrogen of an Asn side-chain) play crucial roles in mediating protein folding and secretion by directly increasing protein folding rates and stability. These roles are important because premature degradation of slowly folding or improperly folded proteins is related to the human lysosomal storage diseases. The long-term goal of the proposed research is to understand, in atomic detail, how N-linked glycosylation affects protein-folding energetics, and ultimately to shed light on the relationship between N-glycosylation and secretion efficiency. Specifically, the proposed work seeks to provide insight into how a single N-linked N-acetyl-D-glucosamine (GlcNAc) affects the folding energy landscape of the (3-sheet glycoprotein CD2ad in vitro. Pursuit of this goal will involve varying the identity and stereochemical configuration of the GlcNAc functional groups as well as the amino acid sequence of CD2ad and observing the resulting effect on the folding energy landscape of CD2ad. Such experiments will require the chemical synthesis of completely homogeneous CD2ad glycoforms which contain any desired sugar, not just GlcNAc at the glycosylation site. To that end, the proposed work involves the following specific aims: (1) synthesis of a series of thiolfunctionalized Asn-linked sugars which differ from GlcNAc in the identity and/or stereochemical configuration of their functional groups;(2) analysis of the ability of these thiol-functionalized Asn-linked sugars to facilitate the chemical ligation of short glycopeptides to peptide thioesters at ligation junctions that would be useful for full-length CD2ad synthesis;(3) preparation of monoglycosylated wild type and mutant CD2ad glycoforms by a combination of sugar-assisted and expressed protein ligation using the Asn-linked sugars and ligation junctions identified in (1 )-(2);and characterization and comparison of the folding energy landscape of CD2ad analogs, attributing changes in energetic parameters to specific protein/sugar contacts.
The proposed research aims to provide insight into how protein glycosylation affects protein folding and stability. This work could ultimately shed light on the relationship between protein glycosylation and protein secretion efficiency, which is related to human lysosomal storage diseases.
