Leukocyte type core 2 {beta}1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Gal{beta}1,3[GlcNAc{beta}1,6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Gal{beta}1,3GalNAc-O-Ser/Thr), respectively.
The primary objective of the work described in this thesis is to shed light on the structure, catalytic mechanism and substrate specificity of C2GnT-L. Since glycosyltransferases are membrane bound glycoproteins that possess disulphide bonds, the first challenge was to produce sufficient quantities of C2GnT-L for biochemical and structural characterization. To this end, C2GnT-L and various active site mutants were expressed and purified from stably transformed mammalian cell lines. The x-ray crystal structure of wild-type C2GnT-L was solved, in both its apo and acceptor substrate complexed forms. The structures, along with revealing the structural basis for acceptor substrate specificity, showed that C2GnT-L belongs to the GT-A glycosyltransferase fold type. This was a surprising result given that, to this day, C2GnT-L is the only GT-A glycosyltransferase that does not possess a DXD motif and does not require a divalent metal ion for catalysis. The mechanism of the metal ion-independent C2GnT-L activity, within the context of the metal ion-dependent GT-A fold, was probed further using site directed mutagenesis in conjuncition with x-ray crystallography, enzyme assays, and frontal affinity chromatography. It was found that positively charged side chains in C2GnT-L functionally replace the divalent metal ion found in other GT-A glycosyltransferases, providing evidence for a convergence of metal ion-independent activity between GT-A and GT-B glycosyltransferase fold types.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32033 |
| Date | 18 January 2012 |
| Creators | Pak, John |
| Contributors | Rini, James |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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