title: Posttranslational modifications of recombinant myotube-synthesized human factor IX.
abstract: Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.
authors.affiliation: Department of Pediatrics and Pathology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA.
authors.forename: V R
authors.initials: VR
authors.lastname: Arruda
authors.forename: J N
authors.initials: JN
authors.lastname: Hagstrom
authors.forename: J
authors.initials: J
authors.lastname: Deitch
authors.forename: T
authors.initials: T
authors.lastname: Heiman-Patterson
authors.forename: R M
authors.initials: RM
authors.lastname: Camire
authors.forename: K
authors.initials: K
authors.lastname: Chu
authors.forename: P A
authors.initials: PA
authors.lastname: Fields
authors.forename: R W
authors.initials: RW
authors.lastname: Herzog
authors.forename: L B
authors.initials: LB
authors.lastname: Couto
authors.forename: P J
authors.initials: PJ
authors.lastname: Larson
authors.forename: K A
authors.initials: KA
authors.lastname: High
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000032:analysis; D000256:Adenoviridae / Q000235:genetics; D002241:Carbohydrates / Q000032:analysis; D018929:Cell Culture Techniques; D017077:Culture Media, Conditioned / Q000737:chemistry; D005164:Factor IX / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000378:metabolism; D010314:Partial Thromboplastin Time; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein; D012694:Serine / Q000378:metabolism; D013431:Sulfates; D014161:Transduction, Genetic; D014443:Tyrosine / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D013431:Sulfates; D014443:Tyrosine; D012694:Serine; D015055:1-Carboxyglutamic Acid; D005164:Factor IX