臺灣博碩士論文加值系統

Helicobacter pylori is a major gastroduodenal pathogen to cause gastric and duodenal ulcers. The infection is associated with atrophic gastritis, a precondition to gastric cancer and lymphoma. This pathogenic bacterium expresses fucose-containing Lewis antigens as a molecular mimicry to the cell surface glycoconjugates of gastric epithelial cells in order to avoid the detection of host immune system. Such mimicry also plays an important role in mucosal adhesion, immune evasion and persistent colonization.
Fucosyltransferases (FTs) are the enzymes responsible for the last steps in the biosynthesis of Lewis carbonhydrate determinants, including Lea, Leb, Lex, Ley, sLex and sLey. Expression of these antigens in H. pylori is phase variable, controlled by an on-and-off switching of specific genes involved in LPS biosynthesis. Among the enzymes involved in the biosynthesis, a1,3/4-FT catalyzes the last step of glycosylation. We have cloned the fut gene from H. pylori NCTC11639 and expressed the protein in E. coli for further studies. The enzyme was expressed at a high level but obtained in an insoluble form. Several C-terminal truncations were thus constructed to enhance the protein solubility and activity.
This thesis aims to investigate the substrate specificity of H. pylori a1,3-FT, with a special emphasis on longer oligosaccharides. The results were verified by mass spectrometry. Our finding indicates that this enzyme has broad substrate specificity with sialylation and sulfation tolerance. The essential groups are consistent of those in human a1,3/4-FTs.
Although H. pylori and human counterparts share low sequence similarity, they display several common features including the essential hydroxyl groups and substrate specificity. Therefore, H. pylori FT could be a useful model for studying the entire FT family. This information can be employed for the use in enzymatic synthesis of fucosylated glycoconjugates.

Table of contents I
Abstract IX

Chapter 1. Introduction 1
1. Helicobacter pylori 1
1.1 Persistent infection 1
1.2 Virulence factors 2
1.3 Lewis antigens 4
1.3.1 Structures of Lewis antigens 4
1.3.2 Lewis antigens in H. pylori 5
1.4 Phase variation and immune modulation 6
2. Fucosyltransferase 8
2.1 Introduction of fucosyltransferase 8
2.1.1 Fucosyltransferase in human 9
2.1.2 Fucosyltransferase in H. pylori 11
2.2 Substrate specificity study of fucosyltransferase 14
2.2.1 Substrate specificity of human FTs 14
2.2.2 Substrate specificity of H. pylori FTs 15
3. Specific aims 17
Chapter 2. Methods and Materials 21
Materials and instruments 21
Methods 22
1. DNA manipulation 22
1.1 Primer design 22
1.2 Mutant strand synthesis reaction 22
1.3 Dpn I digestion of the amplified gene product 23
2. Protein expression and purification 23
3. Protein identification 24
3.1 SDS-PAGE analysis 24
3.2 Immunoblot analysis 24
3.2 Protein molecular weight determination 24
4. Fucosyltransferase assay 24
5. Preparation of polyLacNAc derivatives 25
5.1 Preparation of neutral polyLacNAc from keratan sulfate I 26
5.2 Preparation of sulfated LacNAc from keratan sulfate I 26
5.3 Synthesis of sialyl oligosaccharides by a2,3-SiaT 26
5.3.1 Synthesis of CMP-sialic acid by NeuA 27
5.3.2 Sialylation of the oligosaccharides by the a2,3-SiaT 27
5.4 Preparation of GlcNAcb1,3Galb1,4Glc 28
6. Sample preparation for mass spectrometry 28
6.1 Separation of glycans by PGC solid phase extraction column 28
6.2 Permethylation of oligosaccharides 28
7. Mass spectrometry 29
7.1 MALDI-TOF 29
7.2 MALDI-Q-TOF 29
7.3 MALDI-TOF-TOF 30
7.4 CID-MS/MS interpretation 30
Chapter 3. Results 33
1. Plasmid constructs 33
2. Sequence alignment of H. pylori FTs 33
3. Protein expression and purification 34
4. Synthesis of sialyl oligosaccharides 35
5. Substrate specificity of C-45 FT 35
5.1 Disaccharide acceptors 36
5.2 Trisaccharide acceptors 36
5.3 Oligosaccharide acceptors 37
5.4 Chitose acceptors 38
6. Kinetic studies 38
7. Substrate specificity of C-115 FT 39
8. Structure characterization 39
8.1 11639FT is an exclusively a1,3-fucosyltransferase 39
8.2 Addition of Gal at C-4 of Lactose abolish fucosylation activity 40
8.3 Addition of sialic acid at C-6 of LacNAc diminish fucosylation activity 40
8.4 Preparation and fucosylation of GlcNAcb1,3Galb1,4Glc 41
8.5 Fucosylation of chitose acceptors 41
9. Separation of polyLacNAc from Keratan Sulfate I 42
10. Fucosylation of polyLacNAc 43
11. Fucosylation of sulfated oligosaccharides 43
Chapter 4. Discussion 71
1. C-45 FT has a1,3-fucosyltransferase exclusively 71
2. Extra sugar in non-reducing end influence a1,3-fucosyltransferase activity 72
2.1 C-2 or C-3 replacement enhance a1,3-fucosyltransferase activity 72
C-4 replacement inhibits a1,3-fucosyltransferase activity 72
2.2 Sialylated and sulfated oligosaccharides are acceptable 73
3. Essential groups for substrate recognition 73
3.1 Essential gropus on GlcNAc 73
3.1 Essential gropus on Gal 74
4. Preference of the fucosylation site in polyLacNAc 75
5. Comparison of the substrate specificity of human FT and H. pylori FT 75
6. Structure based insight of LacNAc binding site 77
Conclusion 79
Reference 80

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