About one-third of the proteome of eukaryotes traverses the cellular secretory pathway, and the majority of these proteins are N-glycosylated. Within the secretory pathway there is an elaborate network of chaperones, folding enzymes, and degradation machinery dedicated to maintaining glycoprotein homoeostasis, or glycoproteostasis. Failures of glycoproteostasis, either because of mutations in N-glycoproteins themselves or defects in the glycoproteostasis network, are responsible for many diseases, including cystic fibrosis. N-glycans affect glycoproteostasis through intrinsic mechanisms, by directly stabilizing glycoproteins and/or inhibiting their aggregation, and through extrinsic mechanisms, by mediating their interactions with the glycoproteostasis network. We have considerable experience studying the extrinsic role of N-glycans in glycoproteostasis maintenance through our studies of the folding and trafficking of glycoproteins associated with lysosomal storage diseases. We have also investigated in depth the intrinsic effects of N-glycans on protein folding and have carefully studied the effects of local sequence on the efficiency of protein N-glycosylation, and their influence on the N-glycan structures produced. In this proposal, we will fuse these areas of expertise to study how N- glycans intrinsically and extrinsically affect folding and trafficking vs. degradation decisions by the glycoproteostasis network.
In Specific Aim 1, we will examine how the initial N-glycosylation event by oligosaccharyl transferase (OST) influences downstream trafficking vs. degradation (i.e., quality control) decisions by the glycoproteostasis network. We will explore the effect of N- glycosylation by OSTSTT3A vs. OSTSTT3B (where STT3A and STT3B are the two paralogs of the catalytic subunit of OST) on folding and trafficking vs. degradation decisions, by determining the effect of co-translational folding on substrate selectivity by OSTSTT3A vs OSTSTT3B, and by characterizing the interactomes of nascent glycoproteins and the various isoforms of OST itself.
In Specific Aim 2, we will determine how the conformational properties of the N-glycoprotein determine the processing of N-glycans by glycoproteostasis network components. Many components of the glycoproteostasis network bind to N-glycoproteins in a bidentate fashion, interacting with both the N-glycan and the protein. This binding mode enables them to sense both the folding status of the protein and the mode and extent of N-glycan trimming, but it is unclear to what extent this sensing is a function of the immediate protein neighborhood of the N- glycan (neighborhood-local), the entire domain to which the N-glycan is attached (domain-local), or the domains that are remote from the N-glycosylation site (non-local).

Public Health Relevance

About one-third of the proteome of eukaryotes traverses the secretory pathway, and the majority of these proteins are N-glycosylated. Failures of glycoprotein homoeostasis, or 'glycoproteostasis', either because of mutations in N-glycoproteins themselves or defects in the network of cellular pathways tasked with maintaining glycoproteostasis, are responsible for many diseases. In this proposal, we will combine cell biology and biophysical approaches to study the factors that affect trafficking vs. degradation decisions by the glycoproteostasis network.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK106582-01
Application #
8946941
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Haft, Carol R
Project Start
2015-07-27
Project End
2019-06-30
Budget Start
2015-07-27
Budget End
2016-06-30
Support Year
1
Fiscal Year
2015
Total Cost
$426,375
Indirect Cost
$201,375
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037