A Comprehensive Guide to Crucial Attributes of Therapeutic Proteins in Biological Pharmaceuticals
With this book, Dr. Raju offers a valuable resource for professionals involved in research and development of biopharmaceutical and biosimilar drugs. This is a highly relevant work, as medical practitioners have increasingly turned to biopharmaceutical medicines in their search for safe and reliable treatments for complex diseases, while pharmaceutical researchers seek to expand the availability of biopharmaceuticals and create more affordable biosimilar alternatives.
Readers receive a thorough overview of the major co-translational modifications (CTMs) and post-translational modifications (PTMs) of therapeutic proteins relevant to the development of biotherapeutics. The majority of chapters detail individual CTMs and PTMs that may affect the physicochemical, biochemical, biological, pharmacokinetic, immunological, toxicological etc. properties of proteins. In addition, readers are guided on the methodology necessary to analyze and characterize these modifications. Thus, readers gain not only an understanding of CTMs/PTMs, but also the ability to design and assess their own structure-function studies for experimental molecules. Specific features and topics include:
- Discussion of the research behind and expansion of biopharmaceuticals
- Twenty chapters detailing relevant CTMs and PTMs of proteins, such as glycosylation, oxidation, phosphorylation, methylation, proteolysis, etc.
- Each chapter offers an introduction and guide to the mechanisms and biological significance of an individual CTM or PTM, including practical guidance for experiment design and analysis
- An appendix of biologic pharmaceuticals currently on the market, along with an assessment of their PTMs and overall safety and efficacy
This volume will prove a key reference on the shelves of industry and academic researchers involved in the study and development of biochemistry, molecular biology, biopharmaceuticals and proteins in medicine, particularly as biopharmaceuticals and biosimilars become ever more prominent tools in the field of healthcare.
Table of Contents
Preface xv
About the Author xix
Abbreviations xxi
1 Introduction to Co- and Post-translational Modifications of Proteins 1
Brief Introductions to Individual Chapters 8
Chapter 2: Acetylation of Proteins 8
Chapter 3: C-Terminal Lys or Arg Clipping of Proteins 8
Chapter 4: Cysteinylation of Proteins 8
Chapter 5: Deamidation of Proteins 8
Chapter 6: Glycation of Proteins 9
Chapter 7: Glycosylation of Proteins 9
Chapter 8: N-glycosylation of Proteins 9
Chapter 9: O-glycosylation of Proteins 10
Chapter 10: Hydroxylation of Proteins 10
Chapter 11: Methylation of Proteins 10
Chapter 12: Oxidation of Proteins 11
Chapter 13: Phosphorylation of Proteins 11
Chapter 14: Prenylation of Proteins 11
Chapter 15: Proteolysis of Proteins 11
Chapter 16: Selenylation of Proteins 12
Chapter 17: Signal Peptides of Proteins 12
Chapter 18: Sulfation of Proteins and Glycoproteins 12
Chapter 19: SUMOylation 12
Chapter 20: Ubiquitination 13
Chapter 21: Other PTMs 13
References 13
2 Acetylation of Proteins 17
Introduction 17
Mechanism of N-acetylation at the N-termini of Proteins 19
Mechanism of N-acetylation and N-deacetylation of Lysine Residues 21
Mechanism of O-acetylation of Sugar Residues 21
Biological Significance of Protein Acetylation 22
Acetylation in Recombinant Therapeutic Proteins 23
Methods to Analyze Acetylation in Proteins and Carbohydrates 23
References 24
3 C-terminal Lys or Arg Clipping in Proteins 31
Introduction 31
Biological Significance of C-terminal Lys or Arg Clipping in Proteins 31
Analysis of C-terminal Lys or Arg Clipping in Proteins 32
References 32
4 Cysteinylation of Proteins 35
Introduction 35
Biological Significance of Cysteinylation of Proteins 35
Cysteinylation and Trisulfide Bonds in Recombinant Therapeutic Proteins 36
Analysis of Cysteinylation of Proteins 36
References 37
5 Deamidation of Proteins 39
Introduction 39
Mechanism of Deamidation of Proteins 40
Physicochemical Characteristics of Deamidated Proteins 42
Biological Significance of Deamidation of Proteins 43
Deamidation and Immunogenicity 43
Deamidation and Pharmacokinetics Properties of Proteins 44
Deamidation in Recombinant Therapeutic Proteins 44
Methods for the Analysis of Deamidation in Proteins 44
References 45
6 Glycation of Proteins 51
Introduction 51
Mechanism of Protein Glycation 52
Glycation of Proteins in Human 54
Protein Glycation and Human Diseases 55
Glycation in Recombinant Therapeutic Proteins 56
Methods to Analyze Protein Glycation 57
References 58
7 Glycosylation of Proteins 63
Introduction 63
Glycans and Aglycans 64
Glycosidic Bonds 64
Aldoses and Ketoses 66
Anomeric Groups: α- and β-Configurations 69
Natural Diversity of Glycans 70
Glycans and Enzymes 71
N-glycosylation 71
O-glycosylation 72
Phospho-Serine Glycosylation 72
GPI-Anchors (Glypiation) 72
C-mannosylation 73
References 73
8 N-glycosylation of Proteins 77
Introduction 77
Mechanism of N-glycosylation of Proteins 81
Biosynthesis of N-Glycans 81
Biosynthesis of Lipid-linked Precursor Oligosaccharide 81
En Bloc Transfer of the Precursor Oligosaccharide to Nascent Polypeptide Chain 82
Processing of the Glycan 82
Additional Processing of Oligosaccharide Unit for Chain Elongation and/or Modifications 83
Microheterogeneity of N-Glycans 84
Species-Specific N-glycosylation 85
Functions of N-glycans 87
Physicochemical Functions of N-glycans 87
Biological Functions of N-glycans 88
Impact of N-Glycans on Pharmacokinetic Properties of Proteins 88
N-Glycans and Human Diseases 89
N-glycosylation of RTPs 89
Methods to Analyze N-Glycans 90
References 92
9 O-glycosylation of Proteins 101
Introduction 101
Biosynthesis of O-Glycans 103
Biosynthesis of Mucin Type O-Glycans 103
Biosynthesis of O-linked GlcNAc on Proteins 105
Biosynthesis of O-linked Fucose on Proteins 106
Biosynthesis of O-linked Glc Residues 107
Biosynthesis of O-linked Gal Residues 107
Biosynthesis of O-linked Man Residues 107
O-Glycans on Hydroxyproline Residues 108
Physicochemical Properties of O-Glycosylated Proteins 108
Biological Functions of O-Glycans 108
O-glycosylation in RTPs 110
Analysis of O-Glycans 111
References 113
10 Hydroxylation of Proteins 119
Introduction 119
Mechanism of Hydroxylation 120
Mechanism of Hydroxylation in Organic Molecules 120
Mechanism of Hydroxylation in Biomolecules 121
Prolyl 4-Hydroxylase 123
Prolyl 3-Hydroxylase 123
Lysyl 5-Hydroxylase 123
Phenylalanine Hydroxylase 123
Tyrosine Hydroxylase 124
Biological Significance of Hydroxylation 124
Hydroxylation in RTPs 126
Analysis of Hydroxylation 126
References 127
11 Methylation of Proteins 133
Introduction 133
Mechanism of Protein Methylation 135
Chemical Methylation Reactions 135
Biological Methylation Reactions 135
Methylation of Arg Residues 135
Methylation of Lys Residues 137
Methylation of Prenylcysteine Residues 137
Methylation of Protein Phosphatase 2A 137
Methylation of Isoaspartyl Residues 138
O-Methylation of Sugar Residues 138
Physicochemical and Biological Significance of Methylation of Proteins 139
Methylation in RTPs 140
Methods to Analyze Methylation in Proteins and Glycoproteins 140
References 141
12 Oxidation of Proteins 147
Introduction 147
Mechanism of Protein Oxidation 149
Methionine (Met) Oxidation 149
Cysteine (Cys) Oxidation 150
Tryptophan (Trp) Oxidation 151
Tyrosine (Tyr) Oxidation 153
Oxidation of Other Amino Acid Residues and Protein Backbone 155
Oxidation in RTPs 155
Met Oxidation in mAbs 157
Analytical Methods to Measure Protein Oxidation 157
References 158
13 Phosphorylation of Proteins 163
Introduction 163
Mechanism of Protein Phosphorylation in Living Cells 164
Mg2+ Mediated Mechanism of Protein Phosphorylation 165
Protein Kinase-Based Mechanism of Protein Phosphorylation 166
Mechanism of Dephosphorylation of Proteins by Phosphatases 166
Protein Kinases 167
Serine Kinases 167
Tyrosine Kinases 168
Physicochemical and Biological Functions of Protein Phosphorylation 169
Phosphorylation in RTPs 169
Methods to Analyze Protein Phosphorylation 170
Gel Electrophoresis 170
Mass Spectrometry 171
Enrichment of Phosphoproteins 171
Enrichment of Phosphorylated Peptides 171
References 171
14 Prenylation of Proteins 177
Introduction 177
Mechanism of Protein Prenylation 177
Biological Significance of Prenylation of Proteins 179
Analysis of Prenylation of Proteins 179
References 179
15 Proteolysis of Proteins 183
Introduction 183
Chemical Proteolysis 184
Enzymatic Proteolysis 185
Biological Significance of Proteolysis 187
Post-translational Processing of Proteins by Proteolysis 187
Intracellular and Extracelluar Degradation of Proteins by Proteases 189
Proteolysis and Food Digestion 190
Role of Proteases in Apoptosis 190
Role of Proteolysis in Human Diseases 191
Use of Proteases in Laboratories 191
Sources of Proteases 193
Proteolysis in RTPs 193
Chemical Proteolysis in RTPs 193
Enzymatic Proteolysis in RTPs 194
Analytical Methods for the Detection of Proteolysis of Proteins 195
References 196
16 Selenylation 203
Introduction 203
Biological Significance of Selenylation of Proteins 203
References 205
17 Signal Peptides 207
Introduction 207
Biological Significance of Signal Peptides 207
Signal Peptides in RTPs 208
References 208
18 Sulfation of Proteins and Glycoproteins 211
Introduction 211
Biosynthesis of PAPS 212
Sulfation Reactions in the Cytosol 213
Sulfation in the Golgi Compartments 215
Sulfation of Tyrosine Residues 215
Mechanism of Tyrosine Sulfation 216
Biological Functions of Tyr Sulfation 216
Sulfation of Glycosaminoglycans (GAGs) and Proteoglycans 217
Biological Functions of GAGs 219
Sulfation in RTPs 220
Analysis of Sulfation in Biomolecules 221
Analysis of Tyr Sulfation 222
Analysis of Sulfated Glycoconjugates 222
Colorimetric Methods to Analyze Sulfated Glycoconjugates 222
Electrophoretic Methods to Analyze Sulfated Glycoconjugates 223
Chromatographic Methods to Analyze Sulfated Glycoconjugates 223
Analysis of Sulfated Glycoconjugates by Mass Spectrometry 224
References 224
19 SUMOylation 231
Introduction 231
Mechanism of SUMOylation 232
Biological Significance of SUMOylation 232
References 232
20 Ubiquitination 235
Introduction 235
Mechanism of Ubiquitination 235
Biological Significance of Ubiquitination 236
References 236
21 Other CTMs and PTMs of Proteins 239
Adenylylation or AMPylation 239
ADP-Ribosylation 239
Amidation 239
Arginylation 240
Butyrylation 240
Carbamylation 240
Carbonylation 240
γ-Carboxylation 241
Citrullination 241
Diphthamide 241
Formylation 241
Glypiation 242
Hypusine Formation 242
Iodination 242
Lipoylation 243
Malonylation 243
Myristoylation 243
Neddylation 243
Palmitoylation 244
Polyglutamylation 244
Polyglycylation 244
Propionylation 245
Pupylation 245
Pyroglutamate Formation 245
S-Glutathionylation 245
S-Nitrosylation 247
References 247
Appendix A 253
Index 271
