RESUMEN
Molecular interferences are an important challenge in biotechnologies based on antibody-antigen interactions, such as sandwich immunoassays. We report how a sandwich immunoassay with magnetic particles as label can be used to probe interference by surfactants. Surfactants are often used to improve the performance of immunoassays, however the surfactants can affect the involved proteins and the mechanism of action of surfactant molecules on the antibody-antigen system is mostly unknown. As an example, we investigated molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay with two monoclonal antibodies. The influence of the surfactant below the critical micelle concentration (0.00-0.04%) on dissociation properties was quantified in a magnetic tweezers setup, where a force is applied to the molecules via magnetic particle labels. The force-dependent dissociation curves revealed the existence of two distinct cTn-dependent bond types, namely a weak bond attributable to non-specific binding of cTn, and a strong bond attributable to the specific binding of cTn. The dissociation rate constant of the strong bonds increased with the surfactant concentration by about a factor of two. Circular dichroism spectroscopy data showed that the nonionic surfactant influences the conformation of cTn while not noticeably affecting the two monoclonal antibodies. This suggests that the surfactant-induced increase of the dissociation rate of the specific sandwich-type cTn binding may be related to a conformational change of the antigen molecule. The described methodology is an effective tool to study the influence of surfactants and other interferences on assays based on protein interactions.
Asunto(s)
Anticuerpos Monoclonales/química , Reacciones Antígeno-Anticuerpo , Inmunoensayo/métodos , Magnetismo , Anticuerpos Monoclonales/inmunología , Dicroismo Circular , Poloxámero/química , Tensoactivos/química , Troponina/inmunologíaRESUMEN
Tail-interacting 47-kDa protein (TIP47) binds the cytoplasmic domains of the cation-dependent (CD) and cation-independent (CI) mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the Golgi complex. TIP47 recognizes a phenylalanine-tryptophan signal in the CD-MPR. We show here that TIP47 interaction with the 163-residue CI-MPR cytoplasmic domain is highly conformation dependent and requires CI-MPR residues that are proximal to the membrane. CI-MPR cytoplasmic domain residues 1-47 are dispensable, whereas residues 48-74 are essential for high-affinity binding. However, residues 48-74 are not sufficient for high-affinity binding; residues 75-163 alone display weak affinity for TIP47, yet they contribute to the presentation of residues 48-74 in the intact protein. Independent competition binding experiments confirm that TIP47 interacts with the membrane-proximal portion of the CI-MPR cytoplasmic domain. TIP47 binding is competed by the binding of the AP-2 clathrin adaptor at (and near) residues 24-29 but not by AP-1 binding at (and near) residues 160-161. Finally, TIP47 appears to recognize a putative loop generated by the sequence PPAPRPG and other hydrophobic residues in the membrane-proximal domain. Although crystallography will be needed to define the precise interaction interface, these data provide an initial structural basis for TIP47-CI-MPR association.
Asunto(s)
Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Gestacionales , Receptor IGF Tipo 2/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Perilipina-3 , Unión Proteica , Receptor IGF Tipo 2/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Transporte VesicularRESUMEN
TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-independent and cation-dependent mannose 6-phosphate receptors and is required for their transport from late endosomes to the trans Golgi network in vitro and in vivo. We report here a quantitative analysis of the interaction of recombinant TIP47 with mannose 6-phosphate receptor cytoplasmic domains. Recombinant TIP47 binds more tightly to the cation-independent mannose 6-phosphate receptor (K(D) = 1 microm) than to the cation-dependent mannose 6-phosphate receptor (K(D) = 3 microm). In addition, TIP47 fails to interact with the cytoplasmic domains of the hormone-processing enzymes, furin, phosphorylated furin, and metallocarboxypeptidase D, as well as the cytoplasmic domain of TGN38, proteins that are also transported from endosomes to the trans Golgi network. Although these proteins failed to bind TIP47, furin and TGN38 were readily recognized by the clathrin adaptor, AP-2. These data suggest that TIP47 recognizes a very select set of cargo molecules. Moreover, our data suggest unexpectedly that furin, TGN38, and carboxypeptidase D may use a distinct vesicular carrier and perhaps a distinct route for transport between endosomes and the trans Golgi network.
Asunto(s)
Citoplasma/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endosomas/metabolismo , Glicoproteínas , Aparato de Golgi/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Gestacionales , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Transporte Biológico , Encéfalo/metabolismo , Carboxipeptidasas/metabolismo , Cationes , Bovinos , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Colorantes Fluorescentes/farmacología , Furina , Glutatión Transferasa/metabolismo , Immunoblotting , Cinética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Perilipina-3 , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Subtilisinas/metabolismo , Proteínas de Transporte Vesicular , Xantenos/farmacologíaRESUMEN
The endoplasmic reticulum (ER) is a highly dynamic organelle, continuously undergoing membrane fusion and fission. We have measured homotypic fusion between ER vesicles isolated from Chinese hamster ovary cells kinetically in vitro, using an assay based on the metabolic incorporation of pyrene-labeled fatty acids into the phospholipids of cellular membranes. An increase in pyrene-monomer fluorescence was observed after mixing labeled and unlabeled ER vesicles in the presence of ATP and GTP. The protein, temperature, and nucleotide dependence of the increase indicated that it was caused by membrane fusion rather than molecular transfer of labeled lipids to unlabeled membranes. This assay allowed the first kinetic measurements with virtually nonexchangeable probes of a homotypic membrane fusion event. At 37 degrees C, fusion started off immediately at a rate of 1.14 +/- 0.29%/min and reached a half-maximal level after 56 min. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), or after treatment of the membranes with N-ethylmaleimide, fusion was reduced but not completely inhibited. Addition of GTP during a fusion reaction immediately accelerated, and GTPgammaS immediately slowed down the fusion reaction. Thus, these kinetic measurements indicate that G-proteins might act to rapidly enhance fusion beyond a basic level.