Content. Course (LV 21246): Fr. 12:00-14:00 16 Lectures

Transcription

1 Content Course (LV 21246): Fr. 12:00-14:00 16 Lectures Content 1: Overview Occurrence of carbohydrates in nature Glycopolymers and glycoconjugates Carbohydrates in animals Carbohydrates in bacteria and fungi Carbohydrates in plants 2: Carbohydrate synthesis and analysis Synthesis of complex oligosaccharides Synthesis of rare carbohydrates Carbohydrates as key building blocks in the total synthesis of natural products Analysis and characterization of carbohydrates by NMR, MS and other analytical methods

2 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

3 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

4 Enzymes

5 Proteases - Proteases catalyze the hydrolysis of amide bonds in peptides and proteins. - Proteases are used in industry (e.g. laundry detergents, bread improvers), medicine and as a basic biological research tool. - Serine, threonine and cysteine proteases use a nucleophilic residue in a (usually in a catalytic triad).

6 JM Berg, JL Tymockzo, L Stryer, Biochemistry 6th Ed. Catalytic Triade in Proteases

7 Glycocalix - The glycocalix consists of the carbohydrate moieties of glycoproteins and glycolipids and covers the surface of cells Rutledge et al., Nature Rev. Neph. 2010, 361.

8 Common Classes of Animal Glycans Essentials of Glycobiology Second Edition Chapter 1, Figure 6

9 Carbohydrate-Modifying Enzymes Glycosyltransferases Enzyme + Glycosidases Enzyme + H 2 O + Other carbohydrate modifying enzymes (e.g. Epimerases) Enzyme

10 Synthesis of Sugar Nucleotides Enzymatic synthesis of sugar nucleotides Enzyme Glycosyltransferase Examples of activated sugar nucleotides UDP-Glucose GDP-Fucose CMP-Neuraminic acid

11 Chemical Synthesis of Sugar Nucleotides Chemical synthesis of sugar monophosphates Chemical synthesis of sugar nucleotides Field et al., Nat. Prod. Res. 2009, 1172.

12 Biosynthesis and Interconversion of Monosaccharides Essentials of Glycobiology Second Edition Chapter 4, Figure 1

13 Carbohydrate-Modifying Enzymes Glycosyltransferases Enzyme + Glycosidases Enzyme + H 2 O +

14 Enzymatic Mechanism in Glycosyl Transfer and Hydrolysis Glycosyltransferases + Enzyme Glycosidases Enzyme + H 2 O +

15 Mechanism of Retaining Glycosyltransferases

16 Catalytic site of Bovine β1-4 Galactosyltransferase Inverting glycosyltransferase Essentials of Glycobiology Second Edition Chapter 5, Figure 8

17 Mechanism of Inverting and Retaining Glycosidases Retaining Transition state Inverting Kren et al., Trends in Biotechnology 2009, 199.

18 Glycosidase Inhibitors

19 Tom Wennekes, UBC Neuraminidase

20 Carbohydrate-Modifying Enzymes Glycosyltransferases Enzyme + Glycosidases Enzyme + H 2 O + Other carbohydrate modifying enzymes (e.g. Epimerases) Enzyme

21 Biosynthesis of UDP-Apiose and dtdp-dihydrostreptose Essentials of Glycobiology Second Edition Chapter 4 Figure 3

22 Nomenclature of the Consortium of Functional Glycomics

23 Common Classes of Animal Glycans (Glycoproteins) Essentials of Glycobiology Second Edition Chapter 1, Figure 6

24 Protein Glycosylation - There are two types of protein glycosylation: N-glycosylation and O-glycosylation

25 - The three main types of N-glycans N-Glycans

26 N-Glycans - Different species have different types of N-glycans

27 Animal Cell

28 Synthesis of Dolichol-P-P-GlcNAc 2 Man 9 Glc 3 Essentials of Glycobiology Second Edition Chapter 8, Figure 3

29 Processing and Maturation of an N-Glycan Essentials of Glycobiology Second Edition Chapter 8, Figure 4

30 Consensus Sequence for N-Glycosylation

31 Protein Glycosylation - There are two main types of protein glycosylation: N-glycosylation and O-glycosylation

32 Trends in Microbiology, Volume 20, Issue 1, 2012, 30. O-Glycans

33 Blood Group Antigens

34 Animal Cell

35 Biosynthesis of Core 1 and 2 O-GalNAc Glycans Essentials of Glycobiology Second Edition Chapter 9, Figure 3

36 Mucin 1 Glycosylation

37 Common Classes of Animal Glycans (GAGs) Essentials of Glycobiology Second Edition Chapter 1, Figure 6

38 Occurrence of Proteoglycans

39 Occurrence of Proteoglycans (Extracellular Matrix) Proteoglycan Collagen Hyaluronic acid

40 Biosynthesis of Heparan Sulfate Essentials of Glycobiology Second Edition Chapter 16, Figure 7

41 Common Classes of Animal Glycans (Glycolipids) Essentials of Glycobiology Second Edition Chapter 1, Figure 6

42 Glycolipids Essentials of Glycobiology Second Edition Chapter 10, Figure 1

43 Biosynthesis of Glycosphingolipids Essentials of Glycobiology Second Edition Chapter 10, Figure 2

44 Biosynthesis of Glycosphingolipids

45 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

46 Last Week - The glycocalix is a layer composed of oligo- and polysaccharides that surrounds all cells. - The oligo- and polysaccharides are synthesized by the action of carbohydrate-modifying enzymes (glycosyltransferases, glycosidases, epimerases, etc.). - Glycosyltransferases catalyze the reaction of sugar nucleotide donors (eg UDP-glucose) with glycosyl acceptors. - Glycosidases catalyze the hydrolysis of glycosidic bonds. - There are different types of oligo- and polysaccharides attached to proteins: N-glycans, O-glycans and glycosaminoglycans (proteoglycans). - N-Glycans are always attached to an asparagine of the protein backbone via a GlcNAc monosaccharide and O-Glycans to serine or threonine via a GalNAc. - N-Glycans are attached to proteins in the endoplasmatic reticulum as a conserved precursor (from lipid-conjugate) and further elaborated in the Golgi apparatus. O- Glycans are attached and elaborated in the Golgi (also glycans of glycolipids)

47 Last Week Essentials of Glycobiology Second Edition Chapter 13, Figure 1

48 Common Classes of Animal Glycans (GPI-Anchors) Essentials of Glycobiology Second Edition Chapter 1, Figure 6

49 GPI-Anchored Proteins

50 GPI-Anchored Proteins

51 Biosynthesis of GPI-Anchored Proteins

52 Bacteria

53 Bacteria

54 Bacteria - A pilus is a hairlike appendage found on the surface of many bacteria. Pili are primarily composed of oligomeric pilin proteins and important for the attachment of bacteria to host cells. - The bacterial flagellum is made up of the protein flagellin. They are helical filaments, each with a rotary motor at its base which can turn clockwise or counterclockwise. The rotary motor provides bacterial mobility.

55 Bacterial Adhesion Essentials of Glycobiology Second Edition Chapter 39, Figure 3

56 Bacteria Gram-negative Gram-positive

57 Gram-negative Bacteria

58 Cell wall of Gram-Negative Bacteria Essentials of Glycobiology Second Edition Chapter 20, Figure 1

59 Exemplary LPS-Structure

60 Biosynthesis of LPS

61 Capsular Polysaccharides

62 Biosynthesis of Capsular Polysaccharides Bentley SD, Aanensen DM, Mavroidi A, Saunders D, et al. (2006) Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes. PLoS Genet 2(3): e31. doi: /journal.pgen

63 Gram-positive Bacteria

64 Cell Wall of Gram-positive Bacteria Essentials of Glycobiology Second Edition Chapter 20, Figure 2

65 van Heijenoort J Microbiol. Mol. Biol. Rev. 2011;75: Structure of Peptidoglycan

66 Biosynthesis of Peptidoglycan Essentials of Glycobiology Second Edition Chapter 20, Figure 4

67 Antibiotic Vancomycin

68 Inhibition of Peptidoglycan Biosynthesis by Antibiotics

69 Griffey R H et al. PNAS 1999;96: Aminoglycosides

70 Aminoglycoside Mode of Action - Aminoglycosides are active against gram-negative bacteria - Aminoglycosides bind to RNA in the Ribosome and inhibit protein synthesis

71 Fungal Cell Wall Essentials of Glycobiology Second Edition Chapter 21, Figure 1

72 Biosynthesis of Fungal Cell Walls

73 O-Glycans in Fungi Essentials of Glycobiology Second Edition Chapter 21, Figure 3

74 Plant Cell Wall

75 Cellulose Microfibrils

76 Biosynthesis of Cellulose Bashline L et al. Ann Bot 2014;aob.mcu040 The Author Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please journals.permissions@oup.com

77 Plant Cell Wall Biosynthesis

78 Xylan Biosynthesis Glucuronoarabinoxylan UDP-GlcA UDP-Xyl UDP-Ara

79 Harholt et al., Plant Physiol Pectin

80 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

81 Glycolysis

82 Glycolysis

83 Glycolysis Overall reaction Glucose + 2 ADP + 2 P i + NAD + 2 Pyruvate + 2 ATP + NADH Reaction sequence

84 Glycolysis

85 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

86 Enzymatic Synthesis of Oligosaccharides

87 Expression of Proteins in Bacteria Affinity purification: His-Tag:

88 Enzymatic Synthesis of Glycopeptides

89 Enzymatic Synthesis of Glycopeptides

90 Enzymatic Synthesis of Glycopeptides

91 Enzymatic Synthesis of N-Glycans Isolierung aus Eigelb Asn Sialidase Asn

92 Enzymatic Synthesis of N-Glycans

93 Purification by Size-Exclusion Chromatography

94 Enzymatic Synthesis of O-Glycans

95 Enzymatic Synthesis of O-Glycans

96 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) Metabolic oligosaccharide engineering 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

97 Last Week - While in vertebrates only 9 different monosaccharides are present, bacterial glycans contain many different and also rare monosaccharides. - Bacteria can be devided into two classes: gram-positive and gram-negative bacteria. - Characteristic for gram-positive bacteria is a thick layer of peptidoglycan in the cell wall. Characteristic for gram-negative bacteria is an outer coat composed of lipopolysaccharide - The enzymatic machinery for glycan synthesis can be used for preparative oligosaccharide synthesis. Usually glcosyltransferases and glycosidases are employed to produce the oligosaccharide of choice. - The enzymatic reactions for elongation of glycans are performed by mixing the oligosaccharide acceptor with the respective sugar nucleotide donor and the glycosyltransferase in buffer. - The products of the enzymatic reactions can be purified either by reversed-phase HPLC or by size-exclusion chromatography.

98 Enzymatic Synthesis of Carbohydrates Expression of recombinant enzyme in bacteria, yeast, or mammalian cell lines Purification by affinity chromatography Enzymatic synthesis (sugar 1-phosphate + UTP) Chemical synthesis (sugar 1-phosphate + activated UMP) Enzymatic synthesis Chemical synthesis Isolation and purification

99 Enzymatic Synthesis of Carbohydrates

100 Chemo-Enzymatic Synthesis of Asymmetrical N-Glycans Wang et al., Science 2013, 341,

101 Chemo-Enzymatic Synthesis of Asymmetric N-Glycans Wang et al., Science 2013, 341,

102 Biosynthesis of Capsular Polysaccharides Bentley SD, Aanensen DM, Mavroidi A, Saunders D, et al. (2006) Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes. PLoS Genet 2(3): e31. doi: /journal.pgen

103 Enzymatic Synthesis of a Bacterial Antigen nature chemical biology vol 6 june

104 Occurrence of Proteoglycans

105 Biosynthesis of Heparan Sulfate Essentials of Glycobiology Second Edition Chapter 16, Figure 7

106 Enzymatic Sulfation of Heparan Sulfate Oligosaccharides

107 Glycosynthases

108 Glycosynthases Enzymatic synthesis of Xylo-oligosaccharides Enzymatic synthesis of Xyloglucan-oligosaccharides

109 Enzymatic Synthesis of Polysaccharides

110 Sialic acid

111 Chem. Sci., 2014, 5, Sialic acid

112 Overview: Enzymatic Oligosaccharide Synthesis

113 Content 3: Enzymatic reactions with carbohydrates Biosynthesis of carbohydrates Glycolysis Chemo-enzymatic synthesis of oligosaccharides (application of glycosidases and glycosyltransferases) Metabolic oligosaccharide engineering 4: General methods in Glycobiology Evaluation of carbohydrate-protein interactions using carbohydrate microarrays, ELISA, SPR, and STD-NMR 5: Carbohydrates in Immunology Lectins and anti-carbohydrate antibodies Carbohydrate-based vaccines

114 Molecular Imaging

115 Metabolic Glycan Engineering

116 Design and synthesis of Cu-free click chemistry reagents Baskin J M et al. PNAS 2007;104:

117 Metabolic labeling with Azide-Modified Monosaccharides

118 Metabolic Labeling of Sialic Acid

119 Metabolic Labeling of Cells

120 Metabolic Labeling in Zebrafish Laughlin S T, and Bertozzi C R PNAS 2009;106:12-17

121 Metabolic Labeling of Bacterial Polysaccharides Yi W et al. PNAS 2009;106:

122 Angew. Chem. Int. Ed. 2012, 51, 1 5 Metabolic Labeling of Bacterial Polysaccharides

123 Metabolic Labeling of Plant Polysaccharides