Biochemical and biophysical investigations of N-linked glycosylation pathways in archaea

• Main Author: Chang, Michelle M. (Michelle Miran)
• Other Authors: Barbara Imperiali.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2015
• Subjects: Chemistry.
• Online Access: http://hdl.handle.net/1721.1/97981

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February 2015. === Cataloged from PDF version of thesis. "December 2014." === Includes bibliographical references. === Asparagine-linked glycosylation is an abundant and complex protein modification conserved among all three domains of life. Much is known about N-glycan assembly in eukaryotes and selected bacteria, in which the oligosaccharyltransferase (OTase) carries out the en bloc transfer of glycans from polyprenyl-PP-linked donors onto asparagine side chains of acceptor proteins. The first aim of this thesis is to elucidate the biochemical details of archaeal N-linked glycosylation, specifically through in vitro analysis of the polyprenyl-P-dependent pathway of the methanogenic archaeon Methanococcus voltae. The archaeal OTase, known as AglB, utilizes a-linked dolichyl-P-trisaccharide substrate as the glycosyl donor for transfer to the acceptor protein. This dolichyl-P-glycan is generated by an initial retaining glycosyltransferase (AglK) and elaborated by additional glycosyltransferases (AglC and AgIA) to afford Dol-P-GlcNAc- Glc-2,3-diNAcA-ManNAc(6Thr)A. Despite the homology to other bacterial or eukaryotic OTases that exploit polyprenyl-PP-linked substrates, the M. voltae AglB efficiently transfers disaccharide to model peptides from the Dol-P-GlcNAc-Glc-2,3-diNAcA monophosphate. While this archaeal pathway affords the same asparagine-linked P-glycosyl amide products generated in bacteria and eukaryotes, these studies provide the first biochemical evidence revealing that despite the apparent similarities of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life. A second focus of this thesis involves biophysical studies to probe structural features and conformational dynamics of AglB. An intramolecular LRET experimental system was developed to report on substrate binding and the resulting structural transformations in AgIB. There is a strong need for detailed studies on the mechanistic and functional significance of archaeal adaptations of N-linked glycosylation, especially exploring differences between AglB and other OTases that allow AglB to utilize these unique polyprenyl-P-linked substrates. Lastly, a cell-free expression system was established for the efficient synthesis of Alg5, a yeast dolichyl-phosphate glucosyltransferase that shares high sequence similarity to AglK, the first glycosyltransferase in the M. voltae pathway. Dol-P-Glc was generated and examined to unambiguously characterize the stereochemistry of the product of Alg5. === by Michelle M. Chang. === Ph. D.