title: Design and characterization of a photo-activatable hedgehog probe that mimics the natural lipidated form.
abstract: We have generated a photoactivatable form of sonic hedgehog protein by modifying the N-terminal cysteine with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (Bzm). The Bzm modification on ShhN imparted a significant increase in activity as assessed in the C3H10T1/2 functional assay with potency comparable to that of the endogenous dual-lipidated form of ShhN (ShhNp). Reversed-phase HPLC analysis indicated that the increase in activity compared to unmodified ShhN may be due in part to the hydrophobic nature of the benzophenone group. In contrast to the fully processed ShhNp, Bzm-ShhN is monomeric as assessed by analytical SEC and does not require detergent to be soluble. Further, we demonstrated that the Bzm-ShhN was able to crosslink in vitro in the presence of a known binding partner, heparin. We suggest that Bzm-ShhN can serve as a relatively facile and preferred source of ShhNp for in vitro assays and as a probe to identify novel Hh protein interactions.
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: House
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: Laura R
authors.initials: LR
authors.lastname: Daye
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Tarpley
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: Kezia
authors.initials: K
authors.lastname: Addo
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Lamson
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: Margie K
authors.initials: MK
authors.lastname: Parker
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA.
authors.forename: Warren E
authors.initials: WE
authors.lastname: Bealer
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA. Electronic address: kpwilliams@nccu.edu.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Williams
mesh_terms: D000818:Animals; D001577:Benzophenones / Q000737:chemistry; D040761:Biomimetic Materials / Q000737:chemistry*; D002460:Cell Line; D053823:Hedgehog Proteins / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry*; D055668:Photochemical Processes
chemical_list: D001577:Benzophenones; D053823:Hedgehog Proteins; D015335:Molecular Probes; C509239:SHH protein, human; C047723:benzophenone; D006493:Heparin
keywords: Benzophenone; Crosslinking; Heparin; Palmitoyl; Photoactivatable; Sonic hedgehog