title: Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABA(A) receptors.
abstract: This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the ρ1 and α1 subunits of GABA(A) receptors, for localizing reagents of interest to the target receptor. A cysteine residue was inserted at the C terminus of the cysteine-lacking B-domain (Bd) and used for conjugating maleimide-containing compounds. As determined by enzyme-linked immunosorbent assay (ELISA), binding of a Bd-cys-S-fluorescein conjugate to polyclonal guinea pig anti-GABA(A)-ρ1 and rabbit anti-GABA(A)-α1 IgG was similar to that exhibited by full-length protein A. Surface plasmon resonance analysis of the interaction of Bd-cys-S-PEG3400-biotin conjugate (where PEG is polyethylene glycol) with anti-GABA(A)-ρ1 and anti-GABA(A)-α1 yielded K(D) values of 6.4 ± 1.9 and 0.4 ± 0.1 nM, respectively. Fluorescence anisotropy analysis of the binding of Bd-cys-S-fluorescein to the two antibodies yielded EC50 values of 65 and 18 nM, respectively. As determined with biotin-reactive fluorescent reagents, Bd-cys-S-PEG3400-biotin specifically bound to the plasma membrane of Xenopus laevis oocytes that expressed α1β2γ2 or homomeric ρ1 GABA(A) receptors and were pretreated with the corresponding anti-GABA(A) IgG. The IgG-binding specificity and high affinity of Bd-cys conjugates illustrate the potential of these conjugates, in combination with a selected IgG, to localize compounds of interest at specific cell surface proteins.
authors.affiliation: Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Nasser M
authors.initials: NM
authors.lastname: Qtaishat
authors.forename: Hélène A
authors.initials: HA
authors.lastname: Gussin
authors.forename: David R
authors.initials: DR
authors.lastname: Pepperberg
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D016503:Drug Delivery Systems; D013058:Mass Spectrometry; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D011963:Receptors, GABA-A; D013205:Staphylococcal Protein A; D003545:Cysteine