title: Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.
abstract: Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.
authors.affiliation: Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, SP, 05508-000, Brazil. marcodms@iq.usp.br.|NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil. marcodms@iq.usp.br.
authors.forename: Marcos A
authors.initials: MA
authors.lastname: Demasi
authors.affiliation: Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, SP, 05508-000, Brazil.|NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil.
authors.forename: Erika
authors.initials: E
authors.lastname: de S Molina
authors.affiliation: NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil.
authors.forename: Christian
authors.initials: C
authors.lastname: Bowman-Colin
authors.affiliation: Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, SP, 05508-000, Brazil.|NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil.
authors.forename: Fernando H
authors.initials: FH
authors.lastname: Lojudice
authors.affiliation: Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, SP, 05508-000, Brazil.|NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil.
authors.forename: Angelita
authors.initials: A
authors.lastname: Muras
authors.affiliation: Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, SP, 05508-000, Brazil.|NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil.
authors.forename: Mari C
authors.initials: MC
authors.lastname: Sogayar
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045683:Furin / Q000378:metabolism*; D006801:Humans; D043484:Proprotein Convertases / Q000378:metabolism; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII; C080654:PCSK6 protein, human; C099101:PCSK7 protein, human; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C468731:FURIN protein, human; D045683:Furin
keywords: Coagulation factor VIII; PACE; PCSK5; PCSK6; PCSK7; Proteolytic processing