Asparagine (N)-linked glycosylation is a biologically significant protein modification system found in eukaryotes and prokaryotes. The modification of proteins through this process can induce a broad range of effects on protein function, and ultimately alter cellular processes such as cell signaling, protein complex assembly and adherence. Given that this pathway targets a diverse, but specific, group of proteins, it has become evident that cellular mechanisms are in place to direct this specificity. A major determinant of acceptor protein selectivity in eukaryotic N-linked glycosylation is the tight coupling of this process with protein translocation into the endoplasmic reticulum lumen. In this process, proteins are glycosylated in an unfolded state, making the glycosylation consensus sequences available as they emerge from the Sec translocon complex. In contrast, many fundamental questions remain for the N-linked glycosylation pathway in bacteria, specifically pertaining to the structural state - folded or unfolded - of acceptor proteins and whether glycosylation and protein translocation to the periplasm are coupled. Therefore, characterization of this glycosylation process at a molecular level is required. For this analysis, we will utilize previously identified N-linked glycosylation system, pgl, and a protein targeted by this system, PEB3, from Campylobacter jejuni. This study aims to identify the structural conformation of acceptor proteins, the relative position of the glycosylation consensus sequence and the protein-protein interactions that contribute to glycosylation efficiency. In the first part of this study, we plan to focus on the conformational state that PEB3 adopts during the glycosylation process using in vitro and in vivo glycosylation assays. Next, we propose to investigate the structural context of the glycosylation consensus sequence using a site-directed mutagenesis approach to generate PEB3 mutants containing the glycosylation consensus sequence in selected structured and unstructured regions of the protein. Glycosylation efficiencies of the PEB3 mutants will be measured quantitatively in vitro and in vivo to assess the structural requirements of acceptor proteins. We will also address the spatial localization of components of the N-linked glycosylation pathway with the Sec translocon in an effort to identify the mechanisms that permit efficient glycosylation of acceptor proteins in vivo. This will be achieved through both protein-protein interactions studies and an analysis of the subcellular localization of the oligosaccharyl transferase and Sec translocon by fluorescence microscopy. In addition to elucidating the basic molecular mechanisms for N-linked glycosylation efficiency in eukaryotes and prokaryotes, these studies will be useful for understanding how glycan modifications alter protein stability and function, and for the development of recombinant glycoproteins for therapeutics such as hormones, antibodies and cytokines.

Public Health Relevance

The asparagine (N)-linked glycosylation is important for both eukaryotic and prokaryotic organisms and can induce a wide array of effects on protein function. While this protein modification is known to be essential for eukaryotic cellular processes and contributes to bacterial pathogenicity, many fundamental aspects of the N- linked glycosylation pathway remain poorly understood. To further our understanding of the N-linked glycosylation pathway, this study aims to identify molecular factors that influence efficient glycosylation of acceptor proteins using the model N-linked glycosylation system of C. jejuni.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Ranallo, Ryan
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Silverman, Julie Michelle; Imperiali, Barbara (2016) Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons. J Biol Chem 291:22001-22010