# Deciphering Membrane-Associated Glycan Assembly and Transfer

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $349,740

## Abstract

Membrane proteins make up over 30% of the human proteome and are critical in biological functions,
including transport and the transduction of cellular information. Membrane proteins also feature in the
multistep pathways that lead to glycoproteins. For example, the human pathway for protein N-glycosylation,
occurs exclusively at cellular membranes and is part of the essential process that ultimately affords all cell-
surface and secreted N-linked glycoproteins. Likewise, bacterial, glycoproteins are generated through
stepwise, membrane-associated glycan assembly pathways, which culminate in the biosynthesis of important
virulence-associated glycoproteins. Despite the widespread importance of multistep membrane pathways in
human health and disease and the extensive knowledge on the enzymes that make up these pathways, our
understanding of how the enzymes function together and in an ordered sequence are greatly challenged by
technical issues associated with the amphiphilic nature of membranes and the properties of associated
membrane-bound proteins. Functional studies on membrane proteins are often simplified by extraction into
detergent micelles. However, this treatment is highly perturbing and under these conditions, all but the most
stable multiprotein complexes will dissociate and the cryptic information that is programmed in the native
membrane will be lost. Therefore, a major current challenge is to develop strategies for understanding how
proteins are recruited into functional complexes at cellular membranes. This challenge demands the application
of synergistic in vivo and in vitro experimental approaches.
 The proposed research will investigate the membrane-associated protein N-glycosylation pathway of the
Gram-negative enteropathogen Campylobacter jejuni, which shares the logic of the more complex mammalian
pathway. The research has three aims. In Aim 1 we will define the membrane protein interactome for bacterial
N-glycosylation using styrene maleic acid lipoparticles (SMALP). SMALP will enable definition of a complete
molecular description of the local membrane environment around target membrane proteins in vivo. In Aim 2
we will leverage the interactome information for in vitro studies in lipid bilayer Nanodiscs (NDs), which provide
a native-like model membrane of defined composition, and enable in vitro experimental approaches to
understand the rules defining the membrane protein interactions and coordinated function. In Aim 3 we will
implement crosslinking studies to address a key question concerning the timing of glycan transfer to protein.
Ultimately, the studies will deliver detailed information regarding the membrane environment and protein
interaction network that supports efficient N-glycosylation in a representative bacterial pathway.
If successful, the research will provide insight into the molecular logic underpinning the processes that lead to
glycoprotein biosynthesis in all living systems and will inform on multidiscipli...

## Key facts

- **NIH application ID:** 9853792
- **Project number:** 5R01GM039334-32
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Barbara Imperiali
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $349,740
- **Award type:** 5
- **Project period:** 1988-02-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9853792

## Citation

> US National Institutes of Health, RePORTER application 9853792, Deciphering Membrane-Associated Glycan Assembly and Transfer (5R01GM039334-32). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9853792. Licensed CC0.

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