| The family of UDP-N-acetyl-D-galactosamine: Polypeptide N-acetylgalactosaminyltransferases (ppGaNTase) initiates the synthesis of ‘O-linked’ glycan chains on serine or threonine residues. While many of the biological interactions of glycan chains involve sugar residues at or near the terminal ends of the chain, initiation of these chains is an obligate pre-requisite to their formation. The biological functions of O-glycans are both important and diverse. Examples of their function include recognition, structure, signal transduction and cell trafficking.; The work described in this thesis relates to the cloning and characterization of ppGaNTase isoform-2 (ppGaNTase-T2) from mouse. Using standard phage cDNA library screening techniques, ppGaNTase-T2 was successfully cloned and shown to be highly conserved with the human homologue. Northern blot analysis indicated that this isoform is widely expressed in adult murine tissues, which is also true for ppGaNTase-T1. In situ hybridization data indicated that ppGaNTase-T2 transcripts are more widely expressed than the ppGaNTase-T1 message in the 16.5 day mouse embryo, with a predominantly complimentary expression pattern to ppGaNTase-T1. Enzyme activity assays were conducted to compare the rate of sugar transfer by the recombinantly expressed ppGaNTase isoforms on several different peptide substrates. Activity measurements indicated that the substrate specificity of ppGaNTase-T2 is unique, as is true for all of the isoforms identified to date. The Michaelis constants of ppGaNTase-T1, -T2 and -T3 for the sugar donor substrate, UDP-GalNAc, are similar within a factor of five of one another in the micromolar range. The Km values of these same isoforms for each of the acceptor peptide substrates: MUC2 (PTTTPISTTTMVTPTPTPTC), MUC1b (PDTRPAPGSTAPPAC) and EPO-T (PPDAATAAPLR) are within a factor of 2.5 in the millimolar range. In addition, evidence was generated that murine ppGaNTase-T1, -T2 and -T3 utilize both UDP-GalNAc and UDP-Gal as sugar donors. It was also demonstrated that the UDP-Galactose: polypeptide galactosyltransferase activity of ppGaNTase, has different acceptor substrate specificity than the N-acetylgalactosaminyltransferase activity of each isoform. Future investigations will focus on the complex interplay of multiple ppGaNTase family members in the glycosylation of multiply glycosylated substrates. |
