Protein N-glycosylation, which is ubiquitous in eukaryotes, is a complex pathway involving numerous gene families. Early stages of the glycosylation pathway show a high degree of conservation among eukaryotes, yet diversification of the number and size of gene families involved in the later stages of the pathway has led to the evolution of increasingly complex N-glycan structures and functions in various organisms. The overall purpose of this research project has been to characterize the diversity within the α-mannosidase gene family of filamentous fungi. The α-1,2-mannosidases are involved in mannose removal in the intermediate stages of the N-glycosylation pathways, and diversification of this gene family may have provided the first significant divergence in these pathways among major lineages.
Four novel α-mannosidases were identified and characterized from the filamentous fungus Aspergillus nidulans. These genes were designated Class II α-mannosidase, Class I α-1,2-mannosidase IA, Class I α-1,2-mannosidase IB and Class I α-1,2-mannosidase IC, based on their similarity to other Class I and Class II α-mannosidase sequences. The Class II α-mannosidase was highly similar to the rat ER/cytosolic and yeast vacuolar Class II α-mannosidases, and these three proteins formed a phylogenetically distinct subgroup, Class IIC. The Class I enzymes were highly related to each other, and to other fungal Class I α-1,2-mannosidases. Phylogenetic analysis indicates these genes duplicated and diverged subsequent to the divergence of fungi from insects and mammals. In addition to this research on A. nidulans, a single Class I α-1,2-mannosidase was identified and characterized from the Dutch Elm pathogen, Ophiostoma novo-ulmi, which was highly related to the A. nidulans Class I α-1,2-mannosidase IA and IC enzymes, and less so to the A. nidulans Class I α-1,2-mannosidase IB.
Analysis of the function and/or biochemical properties of these enzymes was examined using several methods. Disruption and overexpression of the A. nidulans Class IIC α-mannosidase did not have any noticeable effect on the growth or morphology of the organism, indicating that this gene was not essential for growth. Biochemical characterization of the A. nidulans Class I α-1,2-mannosidase IC was initiated by recombinant secretion of the enzyme into culture media. Successful expression of the enzyme showed that the α-1,2-mannosidase IC did not exert any cytotoxic effects when overexpressed, suggesting that high levels of expression and purification should be feasible. Finally, disruption of the Class I α-1,2-mannosidase from O. novo-ulmi slightly altered the morphology of the organism, but was not lethal. The possible presence of multiple Class I α-1,2-mannosidases in this organism could explain the non-lethality of this mutation.
Elucidation of the N-glycosylation pathways of A. nidulans may be useful in host strain improvement for heterologous protein expression systems. Modulation of the N-glycosylation pathways to produce specific N-glycan structures would increase the utility of the host for the production of human therapeutic glycoproteins which require these N-glycans for efficacy. Additionally, investigation of the genetic components of the N-glycosylation pathways of the Dutch Elm pathogen may provide global antifungal targets with broad applicability in other fungi. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9431 |
| Date | 08 June 2018 |
| Creators | Eades, Caleb Joshua |
| Contributors | Hintz, William E. A. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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