title: Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein.
abstract: 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.
authors: 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.
authors: M A
authors: MA
authors: Srour
authors: J
authors: J
authors: Grupp
authors: Z
authors: Z
authors: Aburubaiha
authors: T
authors: T
authors: Albert
authors: H
authors: H
authors: Brondke
authors: J
authors: J
authors: Oldenburg
authors: R
authors: R
authors: Schwaab
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D037281:Calnexin; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology
chemical_list: D037281:Calnexin; D005169:Factor VIII