Glycosylation of proteins in cell culture is affected by many factors. The host cell line determines the complement of glycosylation enzymes and resulting glycoprotein structures. Culture conditions like batch versus continuous processing and extracellular environment can also impact glycosylation. Understanding glycosylation is important because protein properties like pharmacokinetics, bioactivity, and antigenicity are influenced by glycans.
Glycosylation, the attachment of sugar moieties to proteins, is a post-translational modification (PTM) that provides greater proteomic diversity than other PTMs.
Protein glycosylation and its associated disordersSaranya Sankar
Protein glycosylation and its associate disorders. Glycosylation is one of the post translational modifications important for the normal function of the protein such as cell adhesion, signalling etc.. defect in this process leads to fatal disorder such as cancer, PNH....
Catalytic antibodies (abzymes) are monoclonal antibodies that exhibit enzymatic activity. They are produced by immunizing animals with transition state analogs that mimic the intermediate of chemical reactions. Abzymes function like enzymes by binding and stabilizing the transition state, lowering the activation energy of reactions and catalyzing them. Potential applications of abzymes include treating cancer, HIV, drug detoxification, controlling obesity, and targeting unwanted protein-protein interactions. One example is an abzyme that catalytically destroys the CD4 binding site on HIV, rendering the virus inert.
In this presentation i have explained about all the super secondary structure their types and their functions . The ppt has been made in such a way that it will clear out our basic concepts first and then it will go higher. I hope you like it
Post-translational modifications are chemical changes made to proteins after translation. Some key post-translational modifications include phosphorylation, glycosylation, ubiquitination, and acetylation. Phosphorylation involves adding phosphate groups and is important for processes like cell signaling. Glycosylation attaches carbohydrate groups and affects protein structure and function. Ubiquitination labels proteins for destruction, regulating processes like the cell cycle. Acetylation adds acetyl groups and is involved in gene regulation. These post-translational modifications are important for regulating protein activity, localization, and interactions in the cell.
Gene regulation is how a cell controls which genes, out of the many genes in its genome, are "turned on" (expressed). Thanks to gene regulation, each cell type in your body has a different set of active genes – despite the fact that almost all the cells of your body contain the exact same DNA. These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job. [Source: https://www.khanacademy.org/science/biology/gene-regulation/gene-regulation-in-eukaryotes/a/overview-of-eukaryotic-gene-regulation]
Glycosylation, the attachment of sugar moieties to proteins, is a post-translational modification (PTM) that provides greater proteomic diversity than other PTMs.
Protein glycosylation and its associated disordersSaranya Sankar
Protein glycosylation and its associate disorders. Glycosylation is one of the post translational modifications important for the normal function of the protein such as cell adhesion, signalling etc.. defect in this process leads to fatal disorder such as cancer, PNH....
Catalytic antibodies (abzymes) are monoclonal antibodies that exhibit enzymatic activity. They are produced by immunizing animals with transition state analogs that mimic the intermediate of chemical reactions. Abzymes function like enzymes by binding and stabilizing the transition state, lowering the activation energy of reactions and catalyzing them. Potential applications of abzymes include treating cancer, HIV, drug detoxification, controlling obesity, and targeting unwanted protein-protein interactions. One example is an abzyme that catalytically destroys the CD4 binding site on HIV, rendering the virus inert.
In this presentation i have explained about all the super secondary structure their types and their functions . The ppt has been made in such a way that it will clear out our basic concepts first and then it will go higher. I hope you like it
Post-translational modifications are chemical changes made to proteins after translation. Some key post-translational modifications include phosphorylation, glycosylation, ubiquitination, and acetylation. Phosphorylation involves adding phosphate groups and is important for processes like cell signaling. Glycosylation attaches carbohydrate groups and affects protein structure and function. Ubiquitination labels proteins for destruction, regulating processes like the cell cycle. Acetylation adds acetyl groups and is involved in gene regulation. These post-translational modifications are important for regulating protein activity, localization, and interactions in the cell.
Gene regulation is how a cell controls which genes, out of the many genes in its genome, are "turned on" (expressed). Thanks to gene regulation, each cell type in your body has a different set of active genes – despite the fact that almost all the cells of your body contain the exact same DNA. These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job. [Source: https://www.khanacademy.org/science/biology/gene-regulation/gene-regulation-in-eukaryotes/a/overview-of-eukaryotic-gene-regulation]
The document discusses various supersecondary structures of proteins, which are intermediate structures between secondary and tertiary protein structures. It describes several common motifs composed of two or more secondary structures, such as helix-turn-helix, helix-loop-helix, beta-hairpins, and the Rossmann fold. These motifs are building blocks that occur frequently in protein structures and are associated with specific functions like DNA binding. The document provides detailed examples and diagrams of different supersecondary structure motifs involving helices, strands, and their combinations.
post translational modifications of proteinAnandhan Ctry
Post-translational modifications (PTMs) are chemical modifications of proteins that occur after translation. PTMs play a key role in regulating protein function by modifying activity, localization, and interactions. The main types of PTMs discussed are phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and proteolysis. These modifications are identified through techniques like mass spectrometry, HPLC, radioactive labeling, and gel electrophoresis. PTMs are important for processes like cell signaling, growth, and apoptosis.
The MAP kinase pathway is a three-kinase signalling module that transmits signals from growth factor receptors to the nucleus. When Ras is activated by growth factors, it recruits and activates Raf, the first kinase in the cascade. Raf then activates MEK, which activates ERK. Active ERK translocates to the nucleus and phosphorylates transcription factors, influencing processes like cell proliferation. The three-kinase design allows for signal amplification and transmission from the membrane to the nucleus, while regulatory mechanisms maintain specificity.
The document discusses DNA binding proteins. It describes how DNA is wrapped around histone proteins to form nucleosomes, which resemble "beads on a string". There are five main types of histone proteins - H1, H2A, H2B, H3, and H4. Histone proteins can be modified through processes like acetylation and methylation, which affect gene expression. Other non-histone proteins use motifs like zinc fingers and helix-turn-helix to bind DNA in a sequence-specific manner and regulate transcription.
The document discusses methods for directed mutagenesis in DNA. It describes how early methods involved random mutagenesis using physical and chemical mutagens. Modern techniques now allow directed mutagenesis through recombinant DNA methods, allowing specific mutations even at the single nucleotide level. Several directed mutagenesis methods are outlined, including oligonucleotide-mediated mutagenesis and PCR-based mutagenesis. Important discoveries in directed mutagenesis include methods developed for prokaryotes and eukaryotes in the 1980s, for which Mario Capecchi and Oliver Smithies received the Nobel Prize.
- Glycoproteins are proteins that contain oligosaccharide chains covalently attached to their polypeptide backbones. They serve important biological roles.
- There are two main classes of glycoproteins: O-linked, where sugars attach to serine or threonine residues, and N-linked, where sugars attach to asparagine residues.
- Diseases can result from defects in glycoprotein biosynthesis, such as I-cell disease caused by a defect in N-linked glycosylation and congenital disorders of glycosylation caused by various defects impacting O-linked or N-linked glycosylation.
Yeast vectors are useful for expressing eukaryotic proteins due to yeasts' ability to perform post-translational modifications. Common yeast species used include Saccharomyces cerevisiae, Pichia pastoris, and Schizosaccharomyces pombe. Vectors include integrating, episomal, replicating, centromere, and artificial chromosome plasmids. Vectors are introduced into yeast via transformation or electroporation. Expression is controlled by inducible promoters like GAL or CUP1 in S. cerevisiae and AOX1 in P. pastoris.
Mechanism of vd(j) recombination and generation of antibody diversityKayeen Vadakkan
The document summarizes the mechanism of V(D)J recombination and generation of antibody diversity. It discusses:
1) How V(D)J recombination involves rearrangement of one V, D (only in heavy chains), and J gene segment in B and T lymphocytes, bringing them under the control of regulatory elements.
2) The recognition signals and rearrangement process, which involves double stranded breaks and joining of coding ends.
3) The four main stages of V(D)J recombination - synapsis, cleavage, hairpin opening and end processing, and joining.
4) The seven means by which antibody diversity is generated - multiple gene segments, combinatorial joining, junctional flexibility
Chapter 15 - principle of metabolic regulation - BiochemistryAreej Abu Hanieh
The document discusses metabolic pathways and their regulation in living cells. It notes that biochemical reactions are organized into metabolic pathways that have dedicated purposes like extracting energy, storing fuels, and eliminating waste. Pathways work to maintain homeostasis by keeping metabolite concentrations at a steady state. Regulation of pathway flux occurs through changing the number or activity of regulatory proteins in response to various factors. This allows pathways to rapidly adapt flux as needed to environmental changes while maintaining homeostasis.
Glycoproteins and proteoglycans are important biomolecules that function in the extracellular matrix. Glycoproteins have short carbohydrate chains without repeats attached to proteins, serving roles like structure, lubrication, and cell signaling. Proteoglycans contain long repeating disaccharide units that bind large amounts of water, functioning mainly as structural components. Both are synthesized in the Golgi and degraded in lysosomes, with defects causing diseases like mucopolysaccharidoses due to glycosaminoglycan accumulation.
The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
Serine proteases are digestive enzymes like trypsin, chymotrypsin, and elastase that differ in substrate specificity. Their catalytic mechanism involves a catalytic triad of serine, histidine, and aspartate residues. During catalysis, the serine hydroxyl performs a nucleophilic attack on the substrate peptide bond, forming a transient acyl-enzyme intermediate before hydrolysis releases the cleaved peptides. Chymotrypsin prefers substrates with aromatic or large hydrophobic residues at the cleavage site.
1. Proteins in eukaryotic cells are synthesized in the cytosol but must be targeted to various intracellular destinations like organelles. They use signal sequences and membrane receptors to direct their transport.
2. In the ER, proteins are modified through glycosylation and folding before being sent to the Golgi apparatus for further processing and sorting to their final locations like the plasma membrane or lysosomes.
3. Mitochondria and chloroplasts import proteins using signal sequences after full synthesis, while nuclear transport relies on non-cleaved NLS sequences and importin proteins.
4. Bacteria also use cleaved signal sequences and chaperones to transport proteins through membrane complexes. Cells import proteins through receptor-mediated
1. DNA replication is the process by which daughter DNA molecules are synthesized from a parental DNA template. It ensures the genetic information is transferred to the next generation with high fidelity.
2. Replication occurs semi-conservatively such that each new double helix contains one strand from the original parent DNA and one newly synthesized strand. It also occurs bidirectionally from an origin of replication.
3. DNA polymerases are the key enzymes that catalyze DNA synthesis. Other important enzymes and proteins include primase, helicase, topoisomerase, ligase, and single-stranded DNA binding proteins. Together they facilitate the initiation, elongation and termination of DNA replication.
Glycoproteins are proteins that contain carbohydrate chains covalently attached. They can be O-linked, N-linked or GPI-anchored. Glycoproteins play important structural and functional roles like cell adhesion and acting as receptors. They are synthesized through a complex process in the endoplasmic reticulum and Golgi apparatus. Congenital disorders of glycosylation can occur from mutations affecting glycoprotein synthesis. Blood groups are also determined by glycoproteins on red blood cell surfaces.
Protein-protein interactions are important for many biological processes. There are various types of interactions depending on their composition and duration. Methods to study interactions include yeast two-hybrid, co-immunoprecipitation, affinity chromatography, and chromatin immunoprecipitation. Databases such as IntAct and MINT provide repositories for protein interaction data.
- Cell signaling pathways regulate nearly all cellular functions through cascades of signaling events involving receptors, signal transducers, and effector proteins. Receptors include G-protein coupled receptors, receptor tyrosine kinases, cytokine receptors, and intracellular receptors.
- Signaling proteins that act as transducers include kinases, GTPases, adaptor proteins, and second messengers like cyclic nucleotides, calcium, lipids, and nitric oxide. These relay, integrate, and distribute signals within cells.
- Feedback loops allow cells to adapt their sensitivity to signaling and respond appropriately to their environment. Understanding cell signaling pathways is challenging due to their complexity, branching, and convergence.
lac operon is a negatively controlled inducible operon.E.coli can use lactose as a source of carbon.
The enzymes required for the use of lactose as a source of carbon are synthesised only when the lactose is available as carbon source.
The lac operon is an example of nagatively controlled inducible operon.
Structure
The lac operon consists of 5 structural units.
Promoter
Operator
Structural genes
CAP binding sites
Regulatory gene
Posttranslational modifications occur in different locations in the cell and serve various purposes. The main locations for modification include the nucleus, lysosome, mitochondria, Golgi, ER, cytosol, ribosome, plasma membrane, extracellular fluid, and extracellular matrix. Common types of modifications include acetylation, phosphorylation, glycosylation, amidation, and ubiquitination. These modifications influence protein stability, activity, localization, and signaling interactions.
Therapeutic Glycoproteins are budding medicine for the treatment of various life threatening diseases like cardiometabolic disorders, autoimmune diseases and cancer. Most of the approved therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cell line.
Its increasing demand has challenged the current production capacity. So as to increase the quality and quantity of these glycoproteins various attributes are being exploited such as the glycosylation patterns which is affected by the cell culture media components, manufacturing mode, culture system and operation, pH, temperature, different expression systems like mammalian, insect, plant, and animal expression system. The choice of a significant host system plays a crucial role in the resulting glycan produced. Glycosylation is highly regulated mechanism, a crucial protein quality attribute that can modulate the efficacy of therapeutic glycoprotein
The document discusses various supersecondary structures of proteins, which are intermediate structures between secondary and tertiary protein structures. It describes several common motifs composed of two or more secondary structures, such as helix-turn-helix, helix-loop-helix, beta-hairpins, and the Rossmann fold. These motifs are building blocks that occur frequently in protein structures and are associated with specific functions like DNA binding. The document provides detailed examples and diagrams of different supersecondary structure motifs involving helices, strands, and their combinations.
post translational modifications of proteinAnandhan Ctry
Post-translational modifications (PTMs) are chemical modifications of proteins that occur after translation. PTMs play a key role in regulating protein function by modifying activity, localization, and interactions. The main types of PTMs discussed are phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and proteolysis. These modifications are identified through techniques like mass spectrometry, HPLC, radioactive labeling, and gel electrophoresis. PTMs are important for processes like cell signaling, growth, and apoptosis.
The MAP kinase pathway is a three-kinase signalling module that transmits signals from growth factor receptors to the nucleus. When Ras is activated by growth factors, it recruits and activates Raf, the first kinase in the cascade. Raf then activates MEK, which activates ERK. Active ERK translocates to the nucleus and phosphorylates transcription factors, influencing processes like cell proliferation. The three-kinase design allows for signal amplification and transmission from the membrane to the nucleus, while regulatory mechanisms maintain specificity.
The document discusses DNA binding proteins. It describes how DNA is wrapped around histone proteins to form nucleosomes, which resemble "beads on a string". There are five main types of histone proteins - H1, H2A, H2B, H3, and H4. Histone proteins can be modified through processes like acetylation and methylation, which affect gene expression. Other non-histone proteins use motifs like zinc fingers and helix-turn-helix to bind DNA in a sequence-specific manner and regulate transcription.
The document discusses methods for directed mutagenesis in DNA. It describes how early methods involved random mutagenesis using physical and chemical mutagens. Modern techniques now allow directed mutagenesis through recombinant DNA methods, allowing specific mutations even at the single nucleotide level. Several directed mutagenesis methods are outlined, including oligonucleotide-mediated mutagenesis and PCR-based mutagenesis. Important discoveries in directed mutagenesis include methods developed for prokaryotes and eukaryotes in the 1980s, for which Mario Capecchi and Oliver Smithies received the Nobel Prize.
- Glycoproteins are proteins that contain oligosaccharide chains covalently attached to their polypeptide backbones. They serve important biological roles.
- There are two main classes of glycoproteins: O-linked, where sugars attach to serine or threonine residues, and N-linked, where sugars attach to asparagine residues.
- Diseases can result from defects in glycoprotein biosynthesis, such as I-cell disease caused by a defect in N-linked glycosylation and congenital disorders of glycosylation caused by various defects impacting O-linked or N-linked glycosylation.
Yeast vectors are useful for expressing eukaryotic proteins due to yeasts' ability to perform post-translational modifications. Common yeast species used include Saccharomyces cerevisiae, Pichia pastoris, and Schizosaccharomyces pombe. Vectors include integrating, episomal, replicating, centromere, and artificial chromosome plasmids. Vectors are introduced into yeast via transformation or electroporation. Expression is controlled by inducible promoters like GAL or CUP1 in S. cerevisiae and AOX1 in P. pastoris.
Mechanism of vd(j) recombination and generation of antibody diversityKayeen Vadakkan
The document summarizes the mechanism of V(D)J recombination and generation of antibody diversity. It discusses:
1) How V(D)J recombination involves rearrangement of one V, D (only in heavy chains), and J gene segment in B and T lymphocytes, bringing them under the control of regulatory elements.
2) The recognition signals and rearrangement process, which involves double stranded breaks and joining of coding ends.
3) The four main stages of V(D)J recombination - synapsis, cleavage, hairpin opening and end processing, and joining.
4) The seven means by which antibody diversity is generated - multiple gene segments, combinatorial joining, junctional flexibility
Chapter 15 - principle of metabolic regulation - BiochemistryAreej Abu Hanieh
The document discusses metabolic pathways and their regulation in living cells. It notes that biochemical reactions are organized into metabolic pathways that have dedicated purposes like extracting energy, storing fuels, and eliminating waste. Pathways work to maintain homeostasis by keeping metabolite concentrations at a steady state. Regulation of pathway flux occurs through changing the number or activity of regulatory proteins in response to various factors. This allows pathways to rapidly adapt flux as needed to environmental changes while maintaining homeostasis.
Glycoproteins and proteoglycans are important biomolecules that function in the extracellular matrix. Glycoproteins have short carbohydrate chains without repeats attached to proteins, serving roles like structure, lubrication, and cell signaling. Proteoglycans contain long repeating disaccharide units that bind large amounts of water, functioning mainly as structural components. Both are synthesized in the Golgi and degraded in lysosomes, with defects causing diseases like mucopolysaccharidoses due to glycosaminoglycan accumulation.
The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
Serine proteases are digestive enzymes like trypsin, chymotrypsin, and elastase that differ in substrate specificity. Their catalytic mechanism involves a catalytic triad of serine, histidine, and aspartate residues. During catalysis, the serine hydroxyl performs a nucleophilic attack on the substrate peptide bond, forming a transient acyl-enzyme intermediate before hydrolysis releases the cleaved peptides. Chymotrypsin prefers substrates with aromatic or large hydrophobic residues at the cleavage site.
1. Proteins in eukaryotic cells are synthesized in the cytosol but must be targeted to various intracellular destinations like organelles. They use signal sequences and membrane receptors to direct their transport.
2. In the ER, proteins are modified through glycosylation and folding before being sent to the Golgi apparatus for further processing and sorting to their final locations like the plasma membrane or lysosomes.
3. Mitochondria and chloroplasts import proteins using signal sequences after full synthesis, while nuclear transport relies on non-cleaved NLS sequences and importin proteins.
4. Bacteria also use cleaved signal sequences and chaperones to transport proteins through membrane complexes. Cells import proteins through receptor-mediated
1. DNA replication is the process by which daughter DNA molecules are synthesized from a parental DNA template. It ensures the genetic information is transferred to the next generation with high fidelity.
2. Replication occurs semi-conservatively such that each new double helix contains one strand from the original parent DNA and one newly synthesized strand. It also occurs bidirectionally from an origin of replication.
3. DNA polymerases are the key enzymes that catalyze DNA synthesis. Other important enzymes and proteins include primase, helicase, topoisomerase, ligase, and single-stranded DNA binding proteins. Together they facilitate the initiation, elongation and termination of DNA replication.
Glycoproteins are proteins that contain carbohydrate chains covalently attached. They can be O-linked, N-linked or GPI-anchored. Glycoproteins play important structural and functional roles like cell adhesion and acting as receptors. They are synthesized through a complex process in the endoplasmic reticulum and Golgi apparatus. Congenital disorders of glycosylation can occur from mutations affecting glycoprotein synthesis. Blood groups are also determined by glycoproteins on red blood cell surfaces.
Protein-protein interactions are important for many biological processes. There are various types of interactions depending on their composition and duration. Methods to study interactions include yeast two-hybrid, co-immunoprecipitation, affinity chromatography, and chromatin immunoprecipitation. Databases such as IntAct and MINT provide repositories for protein interaction data.
- Cell signaling pathways regulate nearly all cellular functions through cascades of signaling events involving receptors, signal transducers, and effector proteins. Receptors include G-protein coupled receptors, receptor tyrosine kinases, cytokine receptors, and intracellular receptors.
- Signaling proteins that act as transducers include kinases, GTPases, adaptor proteins, and second messengers like cyclic nucleotides, calcium, lipids, and nitric oxide. These relay, integrate, and distribute signals within cells.
- Feedback loops allow cells to adapt their sensitivity to signaling and respond appropriately to their environment. Understanding cell signaling pathways is challenging due to their complexity, branching, and convergence.
lac operon is a negatively controlled inducible operon.E.coli can use lactose as a source of carbon.
The enzymes required for the use of lactose as a source of carbon are synthesised only when the lactose is available as carbon source.
The lac operon is an example of nagatively controlled inducible operon.
Structure
The lac operon consists of 5 structural units.
Promoter
Operator
Structural genes
CAP binding sites
Regulatory gene
Posttranslational modifications occur in different locations in the cell and serve various purposes. The main locations for modification include the nucleus, lysosome, mitochondria, Golgi, ER, cytosol, ribosome, plasma membrane, extracellular fluid, and extracellular matrix. Common types of modifications include acetylation, phosphorylation, glycosylation, amidation, and ubiquitination. These modifications influence protein stability, activity, localization, and signaling interactions.
Therapeutic Glycoproteins are budding medicine for the treatment of various life threatening diseases like cardiometabolic disorders, autoimmune diseases and cancer. Most of the approved therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cell line.
Its increasing demand has challenged the current production capacity. So as to increase the quality and quantity of these glycoproteins various attributes are being exploited such as the glycosylation patterns which is affected by the cell culture media components, manufacturing mode, culture system and operation, pH, temperature, different expression systems like mammalian, insect, plant, and animal expression system. The choice of a significant host system plays a crucial role in the resulting glycan produced. Glycosylation is highly regulated mechanism, a crucial protein quality attribute that can modulate the efficacy of therapeutic glycoprotein
Proteoglycans are complex macromolecules consisting of a core protein with one or more glycosaminoglycan chains attached. They are found mainly in connective tissues and help modulate cellular development processes. Glycoproteins contain oligosaccharide chains covalently bonded to amino acids on their polypeptide side chains. They are found in cellular membranes and function in cellular recognition. Some examples of glycoproteins discussed are mucins, transferrins, fibrinogen, follicle-stimulating hormone, and erythropoietin.
INTRODUCTION
STRUCTURE
TYPES OF BONDS
N-LINKED GLYCOSYLATION
O-LINKED GLYCOSYLATION
AMOUNT OF CARBOHYDRATES PRESENT IN GLYCOPROTEIN
BIOLOGICAL SIGNIFICANCE AND FUNCTION
I CELL DISEASE
BIOLOGICAL ADVANTAGE OF ADDING OLIGOSACCHARIDES TO PROTEIN
CONCLUSION
REFERENCES
Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation.
Glycoproteins are proteins that contain carbohydrate chains. There are three main classes of glycoproteins defined by how the carbohydrate is linked to the protein: O-linked, N-linked, and glycosylphosphatidylinositol linked. N-linked glycoproteins are the major class and are distinguished by an asparagine-N-acetylglucosamine linkage. Glycans on glycoproteins are involved in processes like viral infection and immune response. Mucins are glycoproteins that form protective barriers and lubricate epithelial surfaces. Glycosaminoglycans are complex polysaccharides that bind water and form the extracellular matrix.
What is Glycoprotein ?:
Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains.
This process is known as glycosylation.
The carbohydrate is attached to the protein during the following modifications: Co-translational modification & Post-translational modification.
In proteins that have segments extending extracellularly, the extracellular segments are often glycosylated.
This document discusses glycoproteins, which are proteins that contain oligosaccharide chains covalently attached to their polypeptide side chains. There are three major classes of glycoproteins based on the nature of the linkage between the polypeptide and oligosaccharide chains. Glycoproteins play important roles in various biological functions and processes like fertilization. Several methods are used to study the structure and functions of glycoproteins, including using specific glycosidases and lectins.
golgi apparatus structure and function.pptxInamUlHaqKhan6
The Golgi apparatus is a membrane-bound organelle found in eukaryotic cells that packages and modifies proteins and lipids. It consists of stacked, flattened sacs called cisternae. Molecules enter the Golgi through the cis face and undergo processing and modification as they move through the cisternae towards the trans face. At the trans face, molecules are packaged into vesicles and transported throughout the cell. The Golgi apparatus plays key roles in protein modification, secretion, and sorting of molecules to their final destinations in the cell.
The document discusses the Golgi apparatus, which packages and modifies proteins and lipids within eukaryotic cells. It describes the Golgi apparatus as consisting of stacked sacs called cisternae with a cis-facing side that receives vesicles from the ER and a trans-facing side that exports vesicles. The Golgi apparatus modifies proteins through phosphorylation, sulfation, and glycosylation as they move between cisternae, and then sorts proteins into vesicles for transport to other parts of the cell through either the constitutive or regulated secretory pathways.
Glycoproteins are proteins that contain carbohydrate chains covalently attached to their polypeptide side chains. They can be N-linked, O-linked, or GPI-linked. Glycoproteins serve many important functions including structure, protection, reproduction, hormones, and enzymes. Alterations in cell surface glycoproteins can impact physiology, for example certain viruses use cell surface glycoproteins to infect cells. I-cell disease results from a deficiency in an enzyme involved in glycoprotein targeting, leading to severe developmental and neurological issues.
This document summarizes carbohydrates and glycobiology. It discusses the roles of polysaccharides, proteoglycans, glycoproteins, and glycolipids. Proteoglycans are composed of core proteins with attached glycosaminoglycan chains and function in cell adhesion, growth factor binding, and tissue structure. Glycoproteins contain oligosaccharide chains attached to proteins via N- or O- linkages and are important for protein targeting, solubility, and cell recognition. Glycolipids contain oligosaccharides attached to membrane lipids and are involved in cell signaling and immune response. Carbohydrates provide an informational code read by lectins and are critical for many cellular processes.
Golgi apparatus ppt (introduction structure and Function)Dryogeshcsv
The Golgi apparatus is a membrane-bound organelle found in eukaryotic cells that packages and modifies proteins and lipids. It consists of stacked, flattened sacs called cisternae. Proteins enter the Golgi at the cis face and undergo processing and modification as they move through the cisternae towards the trans face. At the trans face, proteins are selectively packaged into vesicles and transported to their final destinations within or outside the cell. The Golgi apparatus plays important roles in protein modification, secretion, and sorting of macromolecules.
Glycobiology is the study of carbohydrates and their attachment to other molecules. Carbohydrates are attached to proteins, lipids, nucleic acids, and other carbohydrates through glycosylation, which is catalyzed by glycosyltransferases. Major classes of glycoconjugates include glycoproteins, glycolipids, glycosaminoglycans, and proteoglycans. Glycoproteins are divided into O-linked and N-linked types based on whether the carbohydrate is attached to serine/threonine or asparagine. Glycolipids are lipids with attached carbohydrates and are found embedded in cell membranes. Together, glycoconjugates serve important structural, adhesive
Biological role of peptidoglycans, glycasoaminoglycans, lectins.pptKantiKondala
This document discusses carbohydrates and glycoconjugates. It covers the classes of glycoconjugates - proteoglycans, glycoproteins, and glycolipids. It describes the structure and functions of these molecules, including their roles in cell signaling, adhesion, and recognition. Lectins are proteins that bind carbohydrates and are involved in various cellular processes. The document also discusses how carbohydrates act as informational molecules on the cell surface and how their complex structures allow them to encode information.
This document discusses glycoproteins. It defines glycoproteins as proteins that contain oligosaccharide chains attached to their polypeptide side chains. There are three major classes of glycoproteins: N-linkage, O-linkage, and GPI linkage. Glycoproteins serve many functions including as structural molecules, enzymes, and in the immune system. Examples of glycoproteins mentioned include mucins, antibodies, and those found in platelets and hormones. The document also lists eight common sugars found in glycoproteins.
G-proteins are proteins that act as molecular switches inside cells and are involved in signal transduction. They have two classes: monomeric small GTPases and heterotrimeric G-protein complexes composed of alpha, beta, and gamma subunits. G-protein coupled receptors (GPCRs) have 7 transmembrane domains and interact with G-proteins via intracellular loops to regulate their activity. Genetic variations in GPCRs and associated proteins can lead to diseases by altering receptor function, such as impaired or enhanced signaling. Single nucleotide polymorphisms in receptors like beta-adrenergic and chemokine receptors have been linked to conditions like asthma and HIV resistance.
The extracellular matrix (ECM) provides structural support and regulates cell behavior. It is composed of structural proteins like collagen and elastin, specialized proteins, and proteoglycans. Collagen makes up about 25% of body protein and contributes to tissue strength and integrity. It is synthesized through hydroxylation and glycosylation steps before assembling into fibrils outside the cell. Proteoglycans like hyaluronan and chondroitin sulfate provide compression resistance and regulate cell signaling. Bone matrix contains collagen and inorganic minerals like hydroxyapatite, providing rigidity. Osteoblasts deposit new bone matrix while osteoclasts resorb old bone, maintaining homeostasis through hormones.
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Indian dental academy has a unique training program & curriculum that provides students with exceptional clinical skills and enabling them to return to their office with high level confidence and start treating patients
State of the art comprehensive training-Faculty of world wide repute &Very affordable.
Similar to Lecture 7 glycosylation in cell culture (20)
1. Viral vaccines are produced using animal cell cultures which allow continuous availability of cell material for viral research and growth. Examples include vaccines for polio, rabies, foot-and-mouth disease, and influenza.
2. There are safety concerns around using animal cell lines for vaccine production including potential viral contamination from endogenous viruses in the cell lines and residual DNA contamination in the final vaccine product.
3. Extensive testing and characterization of cell lines and purification processes are used to ensure final vaccine products are free of contamination and safe.
This document discusses the production of recombinant proteins and glycoproteins from animal cells. It provides examples of several therapeutic proteins that were originally extracted from animal or human sources but are now produced recombinantly using cell culture, including insulin, interferons, erythropoietin, blood clotting factors, and tissue plasminogen activator. Recombinant production allows these proteins to be produced in larger quantities and avoids issues with variable supply or immunogenicity from animal sources. The proteins can also be engineered for improved properties when produced recombinantly compared to native forms.
Lecture 10 hybridomas and the production of antibodiesSarah Aira Santos
This document discusses hybridomas and the production of monoclonal antibodies. It describes how monoclonal antibodies are produced through cell fusion, where antibody-producing B-lymphocytes are fused with myeloma cells to create a hybridoma. This allows for the mass production of antibodies that target a single antigen. The document outlines the key steps of immunization, cell fusion, genetic selection of hybridomas, and clonal selection. It also discusses how mouse antibodies can be "humanized" for therapeutic use in humans to reduce immunogenicity. Major applications of monoclonal antibodies include diagnosis and treatment of diseases.
This document discusses strategies for scaling up animal cell culture production processes. It describes how pH and oxygen levels need to be carefully controlled as culture volumes increase. For larger cultures, pH can be automatically regulated by adding acid or base in response to probe readings. Oxygen supply also becomes an issue at larger volumes due to decreased surface area to volume ratio, so strategies like bubble prevention and alternate bioreactor designs are discussed. Control systems aim to keep the oxygen transfer rate higher than the uptake rate to prevent depletion.
This document discusses scaling up the production of animal cells for biotechnology. It describes various pieces of equipment used, including a motor driven magnetic stirrer to mix cultures, marine impellers to circulate liquid, and temperature control methods like electric heating jackets, heated water circulating around reactors, and probes to monitor temperature within cultures. Maintaining the proper temperature of around 37 degrees Celsius is important for cell growth.
Scaling up the production of animal cells requires moving to larger stirred tank bioreactors. These bioreactors provide a homogeneous and controlled environment for cell growth while allowing for easy sampling. However, scaling up also presents challenges like ensuring adequate oxygen supply and reducing shear forces that could damage delicate animal cells. Features like rounded bottoms, low stirring rates of 100-150 rpm, and additives like Pluronic F68 can help address these issues at larger scales. Axial flow impellers are also effective for lifting cells while exposing them to minimal shear during scaling up.
This document discusses scaling up animal cell and microbial production processes from small to large scale. Key points include:
1. Maintaining optimal physiological conditions as processes are scaled up from small to large bioreactors.
2. Factors that must be considered when scaling up include the fermentor/bioreactor system, agitation, pH, temperature, and dissolved oxygen levels.
3. Scaling up usually occurs gradually in 10x increments from 1L to 10,000L bioreactors. Limiting factors include oxygen supply, shear damage from mixing, and toxic metabolite build up.
The document discusses methods for genetically engineering animal cells, including transferring genes into cells using viral or non-viral methods, and selecting and amplifying transfected cells. Common methods to introduce genes include calcium phosphate transfection, lipofection, electroporation, and viral transduction. Genetic markers like DHFR or antibiotic resistance genes allow selection of cells containing the introduced gene.
1. Cell banks are used to store and maintain animal cell lines, with a master cell bank storing early passage cells and a working cell bank storing cells from the master bank for several passages.
2. Cells are cryopreserved in liquid nitrogen by slow freezing and fast thawing to maintain viability, with cryoprotectants like glycerol or DMSO added to minimize ice crystal formation.
3. Various methods are used to monitor and determine cell growth in culture, including counting cells with a hemocytometer, measuring protein, DNA, or glucose levels, or assessing metabolic activity or viability.
Here are the steps to solve this problem:
i) Number of generations = Log(final cell density/initial cell density) / Log(2)
= Log(2x106/3x105) / Log(2) = 1 generation
ii) Doubling time = Time for 1 generation x Log(2) / Specific growth rate
= 26 hours x Log(2) / 1 day = 26 hours
iii) Specific growth rate = Log(2) / Doubling time = Log(2) / 26 hours = 0.026 h-1
So in summary:
i) 1 generation
ii) Doubling time = 26 hours
iii) Specific growth rate = 0.026 h-1
The document discusses various components of typical cell culture media, including carbohydrates, amino acids, salts, buffers, vitamins and hormones, antibiotics, and serum. It describes the purposes and considerations for each component in maintaining optimal cell growth conditions and metabolism. Key factors include maintaining isotonicity, buffering pH, providing nutrients and growth factors, and preventing bacterial/fungal contamination.
The document discusses different biosafety levels for working with biohazardous materials from levels 1 to 4, with level 4 requiring the highest level of containment. It also describes various types of cell culture vessels and their characteristics such as multi-well plates, flasks, spinner bottles and roller bottles. Microcarriers that cells can grow on such as Cytodex and Cytopore beads are also discussed.
This lecture discusses characteristics of animal cell cultures. It begins by describing primary cultures established directly from tissue using mechanical and enzymatic methods. Specific cell types are then discussed, including fibroblasts, epithelial cells, muscle cells, neurons, and lymphocytes. The concepts of normal vs. transformed cells, anchorage dependence, passaging, contamination, cell differentiation, and stem cells are also covered. Common cell lines available from culture collections are listed at the end.
This lecture discusses animal cell biotechnology and cell culture. It begins by outlining some key applications of animal cell cultures, including for viral vaccines, monoclonal antibodies, recombinant proteins, and hormones. It then describes the basic concepts of cell culture, including how cells can continue growing when removed from tissue and supplied with nutrients. The lecture covers important historical figures like Harrison, Carrel, Hayflick and Moorhead and their key contributions to the field. It discusses the finite lifespan of cultured cells and how they can become immortal through transformation. The characteristics of normal versus transformed cells are also summarized.
Industrial microbiology involves using microorganisms to produce valuable products through fermentation. The lecture discusses fermentation processes and products. Key points:
1. Fermentation is used to produce foods, beverages, chemicals, fuels and more through microbial growth and product formation.
2. The fermentation process involves selecting microorganisms, culture media, growth conditions, and downstream processing to harvest and purify products.
3. Major fermentation products include antibiotics, vitamins, organic acids, alcohols and recombinant proteins. High value biopharmaceuticals produced in mammalian cell culture are a growing market.
1. Glycosylation of Proteins in Cell Culture
• Carbohydrates (glycans) are attached to proteins
as co-translational and post-translational
modifications (glycosylation)
2. Many biological molecules are Glycoproteins
Glycosylation affects the functional qualities of the protein. We must know how the
structure relates to the function to create effective biotherapeutics.
3. Overview
1. Why is glycosylation important in biotechnology.
2. Purpose of glycosylation in general.
3. Types of glycans: N-linked, O-linked.
4. Variation in glycosylation between cell types
5. Synthesis of N-linked glycans
6. Cell culture conditions that affect glycosylation
4. Understanding glycosylation in biological drugs is
important for two main reasons:
• Glycan can affect many of the protein properties:
pharmacokinetics (uptake and length of time in the body),
bioactivity, secretion, in vivo clearance, solubility, recognition,
and antigenicity
• Quantitative and qualitative aspects of glycosylation affected
by production process in culture, including cell line, method of
culture, extracellular environment, and protein itself
5. Why is the biotech industry concerned
about Glycosylation?
• Batch to batch variability of glycosylation patterns affect
product quality
• Too much variation in glycosylation leads to discarding of
product
• Regulatory agencies (eg FDA, Health Canada) have
regulations for amount of acceptable variability in
glycosylation –deviations can lead to redoing clinical trials
• Change in glycosylation can lead to another company
claiming a new patent
• Adverse reactions in patients to non-human glycosylation
9. The list of glycosylated
biopharmaceuticals in
rapidly growing.
Proper glycosylation is
essential for the
function of these
biotherapeutics.
Kawasaki et al 2008
10. FAQs about glycosylation
• 50% of eukaryotic proteins are glycosylated
• N-linked (Asn) and O-linked (Ser/Thr) glycosylation
• N-linked glycosylation is the more complex
• 65% of sequons (attachment sites) are occupied
• Macroheterogeneity = variation in occupancy of
sequons (eg. one site vs two site occupied)
• Microheterogeneity = variation in structures of
glycans (eg. Biantennary vs triantennary at site)
11. Why is Glycosylation Important in
Biotherapeutics?
Carbohydrate structures can affect the properties of the
glycoprotein, including:
→ pharmacokinetics
→ bioactivity
→ secretion
→ in vivo clearance
→ solubility
→ recognition
→ antigenicity
12. General Function of Glycosylation
• N-linked glycosylation prevalent in eukaryotes but not as
common in prokaryotes
• Function of glycans not well defined for many GP’s:
– May be to aid protein folding and transport process
– Prevent self adhesion of molecule (eg. beta-interferon)
– Oligosaccharide can limit the approach of other
macromolecules
• Eg. Inhibit digestion of glycoprotein by proteases (eg. high
concentration on cell surface)
– Regulatory roles
• Eg. Notch: cell surface signalling receptor – important for proper cell
fate determination (O-glycosylated)
13. Glycosylation is Important in Development:
Cell fate choices dependent on the Notch receptor require
appropriate glycan expression
A- Aberrant wing morphology results from mutation in glycosylation of the Notch receptor
B- normal neural development,
C-mutant with altered glycosylation
Essentials of Glycobiology
Second Edition Chapter 24, Figure 3
14. Purpose of Glycosylation (cont’d)
• Activation of secondary pathways:
– Eg. IgG (monoclonal antibodies)
• Differences in glycan structure can change the way the antibody
elicits a response when it binds to the antigen
Fv region
which
binds the
antigen
IgG oligosaccharide
affects the conformation Fab region which
of the Fab region and binds effector
affects how the antibody molecules and
binds to other molecules cells
which results in an
immunue response
16. Glycosylation of protein in cell culture
• Mammalian vs prokaryotes, lower eukaryotes
– Mammalian cells perform post-translational modifications and
achieve a product close to that produced in vivo
– Most Prokaryotes lack glycosylation machinery (exception:
Campylobacter, N-linked glycosylation)
– Yeast, insect, and plant cells produce different glycan structures
• glycan processing in golgi differs from mammalian cells
17. Organisms Differ in Glycosylation
• Bacteria are incapable of glycosylating recombinant
mammalian proteins
• Yeast have the tendency to hyper-mannosylate
• Plant and Insect produced glycoproteins tend to have α 1,3- Peptide
linked fucose and xylose residues N-acetylglucosamine
Mannose
Galactose
• CHO cells are most commonly used for recombinant protein Fucose
production N-glycolylneuraminic acid
– Close to human glycosylation N-acetylneuraminic acid
Xylose
• Important to use Mammalian cells for glycoprotein
production
Transgenic Transgenic
Bacteria Yeast Insect Plants Animal Cells Human
18. Two Primary types of glycosylation are
differentiated by the type of linkage to the
protein
Serine
Asparagine
19. Differences in Oligosacchride Structures in N-linked or O-linked
Glycans
N-acetylglucosamine
Mannose
Galactose
N-acetylneuraminic acid
Fucose
N-acetylgalactosamine
N-linked or O-linked oligosaccharide chains on proteins can have many
different patterns of sugar residues at the same sequon. This is called
Microheterogeneity.
20. O-glycans
• Glycan is bound via an O-glycosidic bond of GalNAc to a Ser/Thr (O-
glycosylation)
• Classified as one of 8 core structures
• Any Ser/Thr residue is a potential site for O-glycosylation, no
consensus sequence identified
• Addition of the glycan occurs on a fully folded protein
22. O-linked Glycans
• Huge variety of structures: from very short to very
long chains
• Are important in mucins (major component of
mucus), with very long chains
• Are often found altered in cancer cells
• Important in blood cell types (A, B, etc)
24. N-linked Glycosylation
• N-linked precursor added to most
proteins in RER membranes
• Only Asn in Asn-X-Ser/Thr become
glycosylated
• Core region survives extensive
oligosaccharide trimming in Golgi
Figure 12-51 (Alberts)
25. Presence of Sequon (Asn-X-Ser/Thr) does not
guarantee glycosylation
1. Spatial arrangement of the peptide during translation process may
expose or hide the tripeptide sequence
2. Glycosylation depends on X: (sequon Asn-X-Ser/Thr)
glycosylation high when X = Ser, Phe,
intermediate for Leu, Glu,
very low for Asp, Trp, and Pro
3. Availability and correct assembly of precursors (eg. nucleotide
sugars)
4. Level of expression of the oligosaccharyltransferase enzyme(s)
5. Disulfide bond formation within protein (makes site inaccessible to
precursor addition)
26. 3 Types of N-linked Glycans
Core region
Complex n-Linked Glycan:
Core with Terminal
Can be heterogeneous
-3 terminal branches
“Sequon” -2 or 4 also common
High Mannose N-linked Glycan:
•Not trimmed to core and more
mannose are added on
•2 to 6 Additional mannose added
onto core
Hybrid N-Linked Glycan:
Hybrid of high mannose and complex
One Mannose Branch
One GlcNAc and Gal branch
27. Protein Glycosylation in RER
Polypeptide
enters ER
lumen
Proteins and lipid-glycan are
generated separately then
glycan transferred on to the
protein structure from the
lipid.
9 Mannose
Oligosaccharyl
transferase enzyme
Man- Man
transfers precursor
Man
oligosaccharide from Man- Man
dolichol to Asn GlcNAc-GlcNAc-Man
Man-Man-Man-Glc-Glc-Glc
2 N- 3 glucose
Acetylglucosamine
Figure 12-52 (Alberts)
28. Production of the Lipid-Glycan
The molecule is flipped from the ER
membrane to the ER lumen.
Cytoplasm Lumen
Sugar residues are added sequentially to the Additional sugars are added via dolichol
lipid to give a Man5-Glc3 structure (using phosphate. Finally, the oligosaccharide
nucleotides sugars. (14 residues) is transferred to a specific
Asn in the lumen (Man9-Glc3)
29. Dolichol Cycle
-synthesis of the sugar chain on
the lipid, dolichol
FLIPPASE ENZYME
Flips oligosaccharide to
internal
lumen of ER membrane
Oligosaccharide is
transferred from dolichol-
phosphate to the protein at a
sequon (Asn-X-Thr/Ser)
30. The Processing Reactions: the introduction of structure variation in the glycan
Begins after the glycan is added to the protein.
l n
k
j m
jProcessing begins – removal of glucoses ER Lumen
kMannosidase I removes 1 mannose
lGolgi mannosidase I removes 3 mannose Golgi Lumen
mN-acetylglucosamine transferase I adds GlcNAc
nMannosidase II to removes 2 mannose
31. Role of N-linked Glycosylation in Protein folding
If the glycoprotein is not correctly folded, glucose will
be readded and sent back through the calnexin cycle
To the GOLGI for processing
and modification of the glycan
and protein
-Binds glycoprotein to help with folding
-Recognizes glucose residues and
glucosidase cleaves off
32. N-Linked Glycosylation PathwayOligomannose
Asn Xaa Ser/Thr
Type
Dol Endoplasmic
Reticulum
P
a-Glc I a-Glc II a-Glc II a-Man I
P NH2
Oligosaccharide
transferase
Man
Glc Golgi
Glc
Glc
SialT GalT GnTII Man II GnTI
FucT
Complex
type
Man Hybrid
Processing Reactions
type
33. Fig 11
Production of tri- and tetra-antennary structures
GnT IV GnT V
Asn
GnT V
Asn GnT IV Asn
M3Gn2 M3Gn4
Asn
M3Gn3
34. Fig 12
Reaction network for N-linked glycosylation
Leads to great diversity in structures
M9 (From Umana and Bailey, 1997)
4x ManI 1-4
M5
GTI 5
GalT13 M Gn GnTIII 20 27
M5 GnGn b GalT M
M5 GnG 5 5
GalT 6 GnTIII 28 GnGnbG
M4 GnG 14 M4 Gn 21 M4 GnGnb GalT M4
GalT 7 GnTIII GnGnbG
M3 GnG 29
22 M3 GnGnb GalT M3
15 M3 Gn
8 30 GnGnbG
16 GnTIII
M3 Gn2G GalT M Gn 23 M Gn Gnb GalT M3 Gn2GnbG
3 2 3 2
GnTIV10 9 GnTV
18GalT 17 GalT
M3 Gn3G M3 Gn3 M3 Gn3’ M3 Gn3’G
12 GnTIII
GnTIII GnTV
25 b GnTIV 24 GalT
M3 Gn3Gn 11 M3 Gn3 ’Gnb M3 Gn3’Gnb
M3 Gn4 31
GalT GalT GnTIII
32 19 26 GalT
M3 Gn3Gn bG M3 Gn4G M3 b
b
33 M3 Gn4Gn G
35. Cell-associated factors that affect product
glycosylation in cell culture
• host cell line
- complement of processing enzymes
• mode of culture
- suspension/ attached
- batch/ continuous
• specific protein productivity
- changes rate of transit through Golgi
• extracellular degradative enzymes
- release of sialidases by cells
36. Factors affecting protein
glycosylation (N-linked)
1. Host cell
• glycan structures on the same proteins can vary
between species and even different tissues
• due to:
– differences in relative activities of glycan processing
enzymes (glycosidases and glycosyltransferases)
– differences in the monosaccharide precursors
37. CHO and BHK
• Structure of sialic acid from CHO and BHK differ from human sialic acid
(also in rodents, pigs, sheep, cows, and new world monkeys)
– NGNA – N-glycoyl-neuraminic acid (humans don’t produce this)
– NANA – N-acetyl-neuraminic acid (most common sialic acid)
• Presence of a2,3 terminal sialic acid addition compared to a2,6 terminal
sialic acid (in humans)
• Absence of a functional a1,3 fucosyltransferase
• Absence of N-acetylglucosaminyltransferase III (Gn TIII)
– differences do not lead to immunogenic responses to glycoproteins
– no adverse physiological effect due to structural differences
38. Hamster vs Mouse cells
• Mouse cells express: a1,3 galactosyltransferase:
generating Gala1,3-Galb1,4-GlcNAc (not found in humans)
– gene is present in CHO and BHK but not expressed
• Limits use of murine cells in therapeutic glycoprotein production
39. 2. Culture environment
• Specific conditions of the culture can affect
glycosylation independently of the cell line
• During the process of a batch culture, nutrient
consumption and product accumulation can change
the culture environment
– gradually decreasing the extent of protein glycosylation
• may lead to variable glycoform heterogeneity and
batch-to-batch variation
40. Adherent Cells Suspension
3. Mode of culture
• adaptation from anchorage dependent growth to
suspension culture may also affect the
glycsosylation process
• presence or absence of serum also has a
significant affect on glycosylation
– presence of hormones and growth factors, high
activities of sialidase and fucosidase
41. 4. Protein productivity
• differences in growth rate, specific productivity,
and cell density among the bioreactors may
cause variation in the pattern of N-linked glycan
structures
• rate of protein expression may also affect
glycosylation
42. 5. Glucose availability
• glucose limitation results in incomplete protein glycosylation
– synthesis of abnormal dolichyl precursor oligosaccharides
– sequences that are normally glycosylated remain empty
6. Ammonia
• accumulated ammonia is inhibitory to cell growth and to protein
glycosylation
– increase in pH of the normally acidic distal golgi
– increase in the UDP-GNAc pool (reduces sialylation)
7. pH
• maximum glycosylation of a protein occurs between pH 6.9-8.2
43. 8. Oxygen limitations
• Limiting nutrient because of it’s low solubility in media
1. reduced dissolved oxygen (DO) may lead to reduction in
UDP-Gal
– reduced oxidative phosphorylation of UDP-Gal
– reduced UDP-Gal transport from the cytosol to the golgi
2. formation of premature disulfide bonds in the nascent protein
44. Effect of Dissolved Oxygen on
Sialylation of EPO
100
% sialylated structues
95
90
85
80
75
70
65
60
3% 10% 50% 100% 200%
DO concentration (% air saturation)
45. 9. Growth factors, vitamins and hormones
• up- and down-regulation of specific glycosyltransferases in
conjunction with hormonal induction of cell differentiation
• changes due to induction or repression or induction of the
enzymes involved in protein glycosylation
46. 10. Extracellular degradation of glycoproteins
• glycosidases may be released to the extracellular
environment by secretion or by cell lysis
• activity of glycosidases depends on medium
pH, temperature, residence time of glycoprotein, and
level of extracellular activity
Editor's Notes
The oligosaccharides have many diverse functions on glycoproteins, and defined functionality for some has only recently been determined. Much more research is required. Glycoproteins – associated with the cell membrane with the carbohydrate is sticking out – GlycoproteinFunction – interaction with other moleculesParetoif receptor that interacts with cell growth factorsAssists to cell-to-cell communicationModifies protein functionModulates antibody function- facilitates binding to viruses and bacteria
Recombinant glycoprotein – needs to be similar to the product in clinical trials – batch to batch variabilityPlatforms – consistent type of production systemEPO – needs to treat anemia and those that are in kidney dialysisAthletes who take EPO are caught because of the glycan structure differs in the drug compared to the natural one in the bodyDeviations – dramatic changes in glycosylation (percentage of difference) – can lead to redoing clinical trials (3rd stage) Clinical trials – w/ small group – later stages of a disease2nd stage – larger group than the first clinical trial – testing different dosage3rd stage – thousands of people – costs multimillion dollars needs to decide whether its worth to go into 3rd stage.Certain percentage of people have adverse reactions (antigenic reactions)
Recombinant glycoproteins have differences in the number of oligosacchrides attached. The site of attachment and type of oligosaccharide is specific for the glycoprotein.Glycosylation sites are protein specific Beta-interferon – one carbohydrate chain that is bound to the same siteEPO – have 3 glycosylation sitesMabs – glycosylation occurs between the two heavy chains in the Fc region of the antibody, and sometimes is also glycosylated in the tip of the Fab regionFab – antigen binding sitesFc – effector - binds to receptors – gets rid of antigen
Monoclonal antibodies (Mabs) are a large portion of the new biotherapeutics being developed. All monoclonal antibodies are glycoproteins. Mabs can be used in a wide variety of treatments, eg. cancer, asthma, inflammatory diseases.
Huge amount of research into generating monoclonals to treat cancer. Many are in use, but are not very good and are often used in combination with chemotherapy. Some are used to treat several types of cancer. Mab – aren’t good enough on their own, need to be combined with other treatments such as chemotherapy- due to glycosylation patterns
There are many other types of biotherapeutics which are glycoproteins, that treat many different types of diseases. They are a wide variety of applications, and they all work in different ways. For many of them, proper glycosylation is essential for their function.
N- linked seemed to be more important than O-linkedSequons – glycan attachment sitesMacrohetergeneity (larger picture) - # of glycosylation sites occupied by glycansMicroheterogeneity – variations of the glycan structure at a particular siteBoth are good to consider in quality of the proteinNeed to control both factors
Mucins have very long chains of glycans (oligosaccharides), which allows high hydration and the “slippery” character of the molecules.
Another series of enzymes leads to more branching and the production of triantennary and tetra-antennary structures.
This figure just shows the great amount of diversity that can be generated in the processing reactions.
The concentration of dissolved oxygen in the bioreactor has an effect on the amount of sialic acid that is added at the terminal end of the oligosaccharide. Sialic acid, because of its charge, can have a big effect on the function of the molecule.
Glycosidases secreted by the cell can affect glycosylation of the recombinant protein after it is secreted from the cell. Monitoring of glycosidase activity can be done to check for levels over the culture production.