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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM086039-03
Application #
7893613
Study Section
Special Emphasis Panel (ZRG1-F04A-T (20))
Program Officer
Marino, Pamela
Project Start
2008-08-18
Project End
2011-06-01
Budget Start
2010-08-18
Budget End
2011-06-01
Support Year
3
Fiscal Year
2010
Total Cost
$41,594
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Chen, Wentao; Enck, Sebastian; Price, Joshua L et al. (2013) Structural and energetic basis of carbohydrate-aromatic packing interactions in proteins. J Am Chem Soc 135:9877-84
Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim et al. (2012) Correction to Context-Dependent Effects of Asparagine Glycosylation on Pin WW Folding Kinetics and Thermodynamics. J Am Chem Soc 134:4450-4451
Price, Joshua L; Culyba, Elizabeth K; Chen, Wentao et al. (2012) N-glycosylation of enhanced aromatic sequons to increase glycoprotein stability. Biopolymers 98:195-211
Price, Joshua L; Powers, David L; Powers, Evan T et al. (2011) Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts. Proc Natl Acad Sci U S A 108:14127-32
Price, Joshua L; Powers, Evan T; Kelly, Jeffery W (2011) N-PEGylation of a reverse turn is stabilizing in multiple sequence contexts, unlike N-GlcNAcylation. ACS Chem Biol 6:1188-92
Culyba, Elizabeth K; Price, Joshua L; Hanson, Sarah R et al. (2011) Protein native-state stabilization by placing aromatic side chains in N-glycosylated reverse turns. Science 331:571-5
Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim et al. (2010) Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics. J Am Chem Soc 132:15359-67