Welche Bedeutung hat die N-Glykosylierung in der rekombinanten Proteinproduktion? Was für 2 Typen der N-Glykosylierung gibt es und wie unterscheiden sie sich bezüglich des Aufbaus?

• Viele der rek. produzierten Therapeutika werden sezerniert, gehen durch den Sekretionsweg und sind daher oft glykosyliert.
• N-Glykosylierung häufigste PTM bei therapeutischen Proteinen, essentiell für Faltung und UE Assembly.
• N-Glykane sind Species spezifisch, daher problematisch bei rekombinanter Expression in anderen Host Zellen.
• Hyperglykosylierung (Mannosen) v.a. in Hefen. Glycoproteine aus S. cerevisiae werden im Blut schnell erkannt und abgebaut. Immunantwort!
• α-Galactosen kommen in N-Glykanen von anderen nicht-humanen Säugetierzelllinien vor (zB CHO).
• Pflanzen: β-1,2-Xylose und α-1,3-Fucose führt zu Immunantwort, (IgG Bildung), Diese IgG sind tw auch schon vorher vorhanden.
• Selbst in Säugetierzelllinien sind die N-Glykane der Proteine oft heterogen, auch Problem bei Organismen die humane Glykosylierung haben.

• Within the immune system the N-linked glycans on an immune cell's surface will help dictate that migration pattern of the cell, e.g. immune cells that migrate to the skin have specific glycosylations that favor homing to that site.[7] The glycosylation patterns on the various immunoglobulins including IgE, IgM, IgD, IgA, and IgG bestow them with unique effector functions by altering their affinities for Fc and other immune receptors.[7] Glycans may also be involved in "self" and "non self" discrimination, which may be relevant to the pathophysiology of various autoimmune diseases.[7]
• Functions of N-linked glycans
  • Intrinsic
    Provides structural components to the cell wall and extracellular matrix.
    Modify protein properties such as stability and solubility[8] (more stable to high temperature, pH, etc.).
  • Extrinsic
    Directs trafficking of glycoproteins.
    Mediates cell signalling (cell–cell and cell–matrix interactions).
• Many therapeutic proteins in the market are antibodies, which are N-linked glycoproteins. For example, Etanercept, Infliximab and Rituximab are N-glycosylated therapeutic proteins.
• The importance of N-linked glycosylation is becoming increasingly evident in the field of pharmaceuticals.[14] Although bacterial or yeast protein production systems have significant potential advantages such as high yield and low cost, problems arise when the protein of interest is a glycoprotein. Most prokaryotic expression systems such as E. coli cannot carry out post-translational modifications. On the other hand, eukaryotic expression hosts such as yeast and animal cells, have different glycosylation patterns. The proteins produced in these expression hosts are often not identical to human protein and thus, causes immunogenic reactions in patients. For example, S.cerevisiae (yeast) often produce high-mannose glycans which are immunogenic.
• Non-human mammalian expression systems such as CHO or NS0 cells have the machinery required to add complex, human-type glycans. However, glycans produced in these systems can differ from glycans produced in humans, as they can be capped with both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac), whereas human cells only produce glycoproteins containing N-acetylneuraminic acid. Furthermore, animal cells can also produce glycoproteins containing the galactose-alpha-1,3-galactose epitope, which can induce serious allergenic reactions, including anaphylactic shock, in people who have Alpha-gal allergy.

Viele der rek. produzierten Therapeutika werden sezerniert, gehen durch den Sekretionsweg und sind daher oft glykosyliert. N-Glykosylierung häufigste PTM bei therapeutischen Proteinen, essentiell für Faltung und UE Assembly. N-Glykane sind Species spezifisch, daher problematisch bei rekombinanter Expression in anderen Host Zellen. Hyperglykosylierung (Mannosen) v.a. in Hefen. Glycoproteine aus S. cerevisiae werden im Blut schnell erkannt und abgebaut. Immunantwort! α-Galactosen kommen in N-Glykanen von anderen nicht-humanen Säugetierzelllinien vor (zB CHO). Pflanzen: β-1,2-Xylose und α-1,3-Fucose führt zu Immunantwort, (IgG Bildung), Diese IgG sind tw auch schon vorher vorhanden. Selbst in Säugetierzelllinien sind die N-Glykane der Proteine oft heterogen, auch Problem bei Organismen die humane Glykosylierung haben. Ziel : die N-Glykosylierung ident der humanen Glykosylierung zu machen, damit sie z.B. vom Immunsystem nicht erkannt wird. 2 Typen : * Anmerkung: Der Komplexe Typ kann verschiedene Muster haben, hier nur 1 Beispiel Weitere Anmerkungen: Within the immune system the N-linked glycans on an immune cell's surface will help dictate that migration pattern of the cell, e.g. immune cells that migrate to the skin have specific glycosylations that favor homing to that site.[7] The glycosylation patterns on the various immunoglobulins including IgE, IgM, IgD, IgA, and IgG bestow them with unique effector functions by altering their affinities for Fc and other immune receptors.[7] Glycans may also be involved in "self" and "non self" discrimination, which may be relevant to the pathophysiology of various autoimmune diseases.[7] Functions of N-linked glycans Intrinsic Provides structural components to the cell wall and extracellular matrix. Modify protein properties such as stability and solubility[8] (more stable to high temperature, pH, etc.). Extrinsic Directs trafficking of glycoproteins. Mediates cell signalling (cell–cell and cell–matrix interactions). Many therapeutic proteins in the market are antibodies, which are N-linked glycoproteins. For example, Etanercept, Infliximab and Rituximab are N-glycosylated therapeutic proteins. The importance of N-linked glycosylation is becoming increasingly evident in the field of pharmaceuticals.[14] Although bacterial or yeast protein production systems have significant potential advantages such as high yield and low cost, problems arise when the protein of interest is a glycoprotein. Most prokaryotic expression systems such as E. coli cannot carry out post-translational modifications. On the other hand, eukaryotic expression hosts such as yeast and animal cells, have different glycosylation patterns. The proteins produced in these expression hosts are often not identical to human protein and thus, causes immunogenic reactions in patients. For example, S.cerevisiae (yeast) often produce high-mannose glycans which are immunogenic. Non-human mammalian expression systems such as CHO or NS0 cells have the machinery required to add complex, human-type glycans. However, glycans produced in these systems can differ from glycans produced in humans, as they can be capped with both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac), whereas human cells only produce glycoproteins containing N-acetylneuraminic acid. Furthermore, animal cells can also produce glycoproteins containing the galactose-alpha-1,3-galactose epitope, which can induce serious allergenic reactions, including anaphylactic shock, in people who have Alpha-gal allergy.