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Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein. 2008

M A Srour, and J Grupp, and Z Aburubaiha, and T Albert, and H Brondke, and J Oldenburg, and R Schwaab
Institute for Experimental Hematology and Transfusion Medicine, University Clinics Bonn, University of Bonn, Sigmund-Freud-Str. 25, 53105, Bonn, Germany, rainer.schwaab@ukb.uni-bonn.de.

Recently, it was shown that glycoproteins with N-glycans close to the NH(2) terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH(2) terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII. To improve FVIII secretion, a 14-amino-acid-long polypeptide with (G3) or without (G0; control) three N-linked glycosylation consensus sites was inserted upstream of the NH(2) terminus of a B-domain deleted FVIII protein. Expression of G3- and G0-constructs in three different cell lines resulted in the same or even higher expression rate of protein as found for the B-domain deleted FVIII. However, as demonstrated by Western blot analysis, the G3- as well as the G0-protein variants were mainly retained inside the cells in similar amounts. Thus, glycosylation alone does not automatically lead to higher secretion rates, but must be in context to the normal structure of the FVIII protein.

UI MeSH Term Description Entries
D007668 Kidney Body organ that filters blood for the secretion of URINE and that regulates ion concentrations. Kidneys
D002522 Chlorocebus aethiops A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research. African Green Monkey,Cercopithecus aethiops,Cercopithecus griseoviridis,Cercopithecus griseus,Cercopithecus pygerythrus,Cercopithecus sabeus,Cercopithecus tantalus,Chlorocebus cynosuros,Chlorocebus cynosurus,Chlorocebus pygerythrus,Green Monkey,Grivet Monkey,Lasiopyga weidholzi,Malbrouck,Malbrouck Monkey,Monkey, African Green,Monkey, Green,Monkey, Grivet,Monkey, Vervet,Savanah Monkey,Vervet Monkey,Savannah Monkey,African Green Monkey,Chlorocebus cynosuro,Green Monkey, African,Green Monkeys,Grivet Monkeys,Malbrouck Monkeys,Malbroucks,Monkey, Malbrouck,Monkey, Savanah,Monkey, Savannah,Savannah Monkeys,Vervet Monkeys
D003412 Cricetulus A genus of the family Muridae consisting of eleven species. C. migratorius, the grey or Armenian hamster, and C. griseus, the Chinese hamster, are the two species used in biomedical research. Hamsters, Armenian,Hamsters, Chinese,Hamsters, Grey,Armenian Hamster,Armenian Hamsters,Chinese Hamster,Chinese Hamsters,Grey Hamster,Grey Hamsters,Hamster, Armenian,Hamster, Chinese,Hamster, Grey
D005169 Factor VIII Factor VIII of blood coagulation. Antihemophilic factor that is part of the factor VIII/von Willebrand factor complex. Factor VIII is produced in the liver and acts in the intrinsic pathway of blood coagulation. It serves as a cofactor in factor X activation and this action is markedly enhanced by small amounts of thrombin. Coagulation Factor VIII,Factor VIII Clotting Antigen,Factor VIII Coagulant Antigen,Factor VIII Procoagulant Activity,Thromboplastinogen,Blood Coagulation Factor VIII,F VIII-C,Factor 8,Factor 8 C,Factor Eight,Factor VIIIC,Hyate-C,Hyatt-C,F VIII C,Hyate C,HyateC,Hyatt C,HyattC
D006031 Glycosylation The synthetic chemistry reaction or enzymatic reaction of adding carbohydrate or glycosyl groups. GLYCOSYLTRANSFERASES carry out the enzymatic glycosylation reactions. The spontaneous, non-enzymatic attachment of reducing sugars to free amino groups in proteins, lipids, or nucleic acids is called GLYCATION (see MAILLARD REACTION). Protein Glycosylation,Glycosylation, Protein
D006224 Cricetinae A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS. Cricetus,Hamsters,Hamster
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D016466 CHO Cells CELL LINE derived from the ovary of the Chinese hamster, Cricetulus griseus (CRICETULUS). The species is a favorite for cytogenetic studies because of its small chromosome number. The cell line has provided model systems for the study of genetic alterations in cultured mammalian cells. CHO Cell,Cell, CHO,Cells, CHO
D019556 COS Cells CELL LINES derived from the CV-1 cell line by transformation with a replication origin defective mutant of SV40 VIRUS, which codes for wild type large T antigen (ANTIGENS, POLYOMAVIRUS TRANSFORMING). They are used for transfection and cloning. (The CV-1 cell line was derived from the kidney of an adult male African green monkey (CHLOROCEBUS AETHIOPS).) COS-1 Cells,COS-7 Cells,COS 1 Cells,COS 7 Cells,COS Cell,COS-1 Cell,COS-7 Cell,Cell, COS,Cell, COS-1,Cell, COS-7,Cells, COS,Cells, COS-1,Cells, COS-7

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