Protein glycosylation in procyclic Trypanosoma brucei brucei: A genetic approach

摘要

Protein glycosylation is a very complicated and dynamic process. This process is determined by several factors, such as the cellular sugar nucleotide levels, the relative abundance of glycosyltransferases and glycosidases, the biosynthesis of precursors (i.e. dolichol and lipid-linked oligosaccharide precursors), the structure of a protein and the secretion fate of a protein. Although the process is greatly influenced by the physiological state and the environments of a cell (or epigenetic factors), it is also known that this process is predetermined by the genetic composition of a cell. Therefore, I have initiated a genetic approach to study protein glycosylation in procyclic Trypanosoma brucei.;Concanavalin A, a lectin cytotoxic to procyclic trypanosomes, was used to isolate two glycosylation mutants, Con A 1-1 and Con A 4-1. Analyses of procyclic acidic repetitive proteins, a major surface glycoprotein of procyclic trypanosomes suggested that the two Con A resistant cells have defects in N-linked glycosylation. Further biochemical analyses of these two mutants indicated that Con A 1-1 has a primary defect in reduction of polyprenol and Con A 4-1 has a primary defect in biosynthesis of an N-linked oligosaccharide precursor. These two cell lines are the first well characterized glycosylation mutants in trypanosomes. As in many other glycosylation mutants, these mutants can be used to clone novel genes involved in protein glycosylation of eucaryotes. Therefore, a genetic functional complementation, based on an trypanosome artificial chromosome, was set up.;This thesis is divided into five chapters and two supplements. The first two chapters provide background information. In Chapter One, I have described several of the alg (asparagine-linked glycosylation) mutants in Saccharomyces cerevisiae and LEC/Lec mutants in CHO cells. This is to demonstrate how genetic approaches are used to clone genes involved in N-linked glycosylation and to study assembly and functions of protein-linked complex glycans. In Chapter Two, I have reviewed some important aspects of the biology of Trypanosoma brucei and its major surface glycoproteins. In Chapter Three, isolation of Con A 1-1 and Con A 4-1 and alteration of PARP, were described. In Chapter Four, I have presented data on biochemical analyses of these glycosylation mutants and suggested experiments to further confirm the primary biochemical defects in these mutants. In Chapter Five, I have described the construction of a trypanosome artificial chromosome and a screening protocol for a functional complementation in Trypanosoma brucei. Supplement One contains my work on overexpression of GPI-PLC in procyclic cells. Supplement Two contains my contribution to a work in purification of a mitochondrial DNase from Crithidia fasciculata and two publications on this work.