title: The disulfide bond of an RGD4C motif inserted within the Hi loop of the adenovirus type 5 fiber protein is critical for retargeting to αv -integrins.
abstract: BACKGROUND
  The α(v) -integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie and adenovirus receptor. However, until now, it has been unclear whether disulfide bonds in the RGD4C motif influence the retargeting potential of RGD4C-modified Ad5.
  
  
  METHODS
  Replication deficient Ad5 bearing wild-type fiber (Ad5wt) or RGD4G, RGD4C and RGD2C2G insertions within the HI loop of the fiber protein (Ad5RGD4G, Ad5RGD4C and Ad5RGD2C2G, respectively) were used to transduce a panel of cancer cell lines, with or without previous treatment of these Ad5s with the reducing agent dithiothreitol (DTT). In parallel, native and DTT-treated fiber proteins isolated from purified Ad5RGD4C were compared by mass spectrometry.
  
  
  RESULTS
  Ad5RGD4C transduced all studied cell lines much more efficiently than Ad5wt, whereas Ad5RGD4G transduced cells only slightly more efficiently than Ad5wt. DTT treatment had no effect on cell transduction by wild-type Ad5wt and Ad5RGD4G but abolished the increased transduction efficacy of Ad5RGD4C in a dose-dependent manner. The mass spectra of native and DTT-reduced tryptic digests of the Ad5RGD4C fiber protein are consistent with the presence of a C(547) -C(549) linkage in the C(547) DC(549) RGDC(553) FC(555) motif. Finally, the high transduction efficacy of Ad5RGD4C is conserved in Ad5RGD2C2G.
  
  
  CONCLUSIONS
  We provide genetic and biochemical data strongly suggesting that cysteines C(547) and C(549) from the C(547) DC(549) RGDC(553) FC(555) motif inserted in the HI loop of the Ad5 fiber form a single disulfide bond, with this disulfide bond being crucial for Ad5RGD4C retargeting to av-integrins.
authors.affiliation: Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Dragomira
authors.initials: D
authors.lastname: Majhen
authors.forename: Jennifer
authors.initials: J
authors.lastname: Richardson
authors.forename: Bojana
authors.initials: B
authors.lastname: Vukelić
authors.forename: Ivana
authors.initials: I
authors.lastname: Dodig
authors.forename: Mario
authors.initials: M
authors.lastname: Cindrić
authors.forename: Karim
authors.initials: K
authors.lastname: Benihoud
authors.forename: Andreja
authors.initials: A
authors.lastname: Ambriović-Ristov
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D019138:Antibodies, Blocking / Q000276:immunology / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004220:Disulfides; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D039564:Integrin alphaV / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D000906:Antibodies; D019138:Antibodies, Blocking; D036022:Capsid Proteins; D004220:Disulfides; D039564:Integrin alphaV; C018315:hexon capsid protein, Adenovirus