# Origin of N-Glycan Site-Specific Heterogeneity

> **NIH NIH R01** · UNIVERSITY OF GEORGIA · 2020 · $845,521

## Abstract

PROJECT SUMMARY
Cell surface and secreted glycoproteins form a complex interface with the extracellular environment that
influences cellular differentiation, physiology, and pathology. Very little is known about how glycan diversity is
controlled to produce distinct sets of glycan structures in different cell types or on individual glycoproteins.
Glycan structures are synthesized by the action of glycosyltransferases (GTs) that yield heterogeneous
ensembles of glycan structures on each site of a given glycoprotein. Challenges remain in defining the
underlying `rules' that specify selective, site-specific modification of glycoproteins, including: 1) deciphering
how individual glycoenzyme active sites act as templates to specify regiospecific substrate recognition and 2)
determining how the context and steric constraints of individual glycosylation sites (glycosites) can limit or
restrict access to tune the diversity of glycan structures produced. We assembled an integrated research team
with expertise in glyco-enzymology, recombinant glycoprotein expression, glyco-analytical chemistry, protein
structural biology, bioinformatics, and chemo-enzymatic glycan synthesis to leverage our unique toolsets and
expertise to identify the essential features that govern site-specific glycan diversity. Our aims include (Aim 1)
determining how glycoenzyme active sites provide templates for glycan modification. We will pursue structural
studies on enzymes in complex with donor analogs and synthetic glycan acceptors and leverage bioinformatic
analyses to generate new hypotheses regarding the evolution of glycoenzyme substrate recognition and
specificity. These hypotheses will be tested by mutagenesis, protein redesign, and enzyme activity toward
acceptor substrates. In Aim 2 we will determine the structural basis for site-specific modification of glycoprotein
acceptors by examining the efficiency of enzymatic modification through the use of MS-based glycopeptide
mapping approaches. Structural data for the respective reporter glycoproteins will be used to compare
glycosite modification with steric constraints for individual glycosites. Hypotheses regarding glycosite
accessibility will be tested by mutagenesis of enzyme active sites and regions that flank the glycosites on the
glycoprotein reporters. In Aim 3 we will test our hypotheses for site-specific glycan modifications by reporter
expression in cultured cells. Site-specific glycoforms produced on the glycoprotein reporters in the mammalian
secretory pathway will be examined to determine if biosynthetic `rules' identified from in vitro studies will extend
to glycan modifications in the more complex environment of the cellular secretory pathway. The proposed
studies will provide fundamental knowledge on how glycoenzymes act as templates for the creation of diverse
glycan structures and how the steric constraints of their substrates tune those specificities to provide
predictable glycan diversity on individual gly...

## Key facts

- **NIH application ID:** 9830659
- **Project number:** 5R01GM130915-02
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** Natarajan Kannan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $845,521
- **Award type:** 5
- **Project period:** 2018-12-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9830659

## Citation

> US National Institutes of Health, RePORTER application 9830659, Origin of N-Glycan Site-Specific Heterogeneity (5R01GM130915-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9830659. Licensed CC0.

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