Polyprenyl-dependent glycan assembly pathways in microbial pathogens
Name
743773645-MIT.pdf
Description
Full printable version
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30.49 MB
Format
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Checksum (MD5)
95e9396651e8d5c4148b24c112b9e2fc
Author(s)
Hartley, Meredith Diane
Advisor(s)
Barbara Imperiali.
Alternative Title
Polyprenyl-dependent assembly of protein glycans in microbial pathogens
Date Issued
2011
Publisher
Massachusetts Institute of Technology
Abstract
Polyisoprenyl-dependent glycan assembly pathways form the basis for the biosynthesis of many complex glycoconjugates. This thesis addresses key aspects of undecaprenyl-phosphate related processes; undecaprenol is the linear polyisoprenol that is utilized by virtually all known bacterial species. In the first chapter, a previously identified undecaprenol kinase from Streptococcus mutans is adapted for enzymatic phosphorylation of polyprenols as an alternate method to chemical phosphorylation. This chemoenzymatic method is shown to be particularly efficient in the biosynthesis of undecaprenyl-diphosphate linked glycans, since the kinase can be coupled to various glycosyltransferases to generate the complex products starting from undecaprenol and ATP. A second focus of this thesis involves the biochemical characterization of the undecaprenyl-dependent O-linked protein glycosylation pathway in Neisseria gonorrhoeae. The N. gonorrhoeae pathway is shown to produce UDP-N,N'-diacetylbacillosamine, which is the UDP-sugar donor in the first membrane-associated step. Furthermore, it is demonstrated that glycosyltransferases in N. gonorrhoeae assemble glycans on undecaprenyl-diphosphate prior to transfer to hydroxyl side chains of serine and threonine residues. The final glycan transfer is performed by an oligosaccharyltransferase (OTase), and specificity studies of O-linked and Nlinked bacterial OTases suggest that both enzymes prefer their native glycans under in vitro assay conditions. This work represents the first biochemical characterization of a polyprenyldependent pathway that produces O-linked glycoprotein in prokaryotes. The final three chapters present the foundations for a new experimental approach to the study of polyprenyl-dependent pathways by employing a model membrane system, termed Nanodiscs, which comprises a discoidal phospholipid bilayer encircled by a scaffold protein. In this thesis, the glycosyltransferases responsible for the first two committed membrane steps in the Campylobacter jejuni N-linked protein glycosylation pathway are co-incorporated into Nanodiscs. Importantly, radioactivity-based assays using a dual isotope-labeling strategy demonstrate the functional reconstitution of both proteins. In addition, efforts are described toward the development of FRET- and LRET-based methodology for future characterization of the protein-protein interactions between PglC and PglA. This work establishes a basis for future studies of polyprenols in the C. jejuni glycan assembly process and may be applicable to other essential polyprenyl-dependent processes.
Description
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Subjects
Chemistry.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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