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A designer cell-based histamine-specific human allergy profiler.

ABSTRACT: Allergic disorders are markedly increasing in industrialized countries. The identification of compounds that trigger the immunoglobulin E-dependent allergic reaction remains the key to limit patients' exposure to critical allergens and improve their quality of life. Here we use synthetic biology principles to design a mammalian cell-based allergy profiler that scores the allergen-triggered release of histamine from whole-blood-derived human basophils. A synthetic signalling cascade engineered within the allergy profiler rewires histamine input to the production of reporter protein, thereby integrating histamine levels in whole-blood samples with remarkable sensitivity and a wide dynamic range, allowing for rapid results or long-term storage of output, respectively. This approach provides non-intrusive allergy profiles for the personalized medicine era.

SUBMITTER: Auslander D

PROVIDER: S-EPMC4143915 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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A designer cell-based histamine-specific human allergy profiler.

Ausländer David D Eggerschwiler Benjamin B Kemmer Christian C Geering Barbara B Ausländer Simon S Fussenegger Martin M

Nature communications 20140805

Allergic disorders are markedly increasing in industrialized countries. The identification of compounds that trigger the immunoglobulin E-dependent allergic reaction remains the key to limit patients' exposure to critical allergens and improve their quality of life. Here we use synthetic biology principles to design a mammalian cell-based allergy profiler that scores the allergen-triggered release of histamine from whole-blood-derived human basophils. A synthetic signalling cascade engineered wi ...[more]

PMID: 25093291

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Project description:A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [35S]GTP?S assays). The human H1R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH1R and insect cell G?q. By contrast, functional expression of the hH2R required the generation of an hH2R-Gs? fusion protein to ensure close proximity of G protein and receptor. Fusion of hH2R to the long (Gs?L) or short (Gs?S) splice variant of G?s resulted in comparable constitutive hH2R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH1R/hH2R and their guinea pig orthologues gpH1R/gpH2R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH3R with G?i1, G?i2, G?i3, and G?i/o in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li+, Na+, K+) and anions (Cl-, Br-, I-) revealed that anions with large radii most efficiently stabilize the inactive hH3R state. Potential sodium binding sites in the hH3R protein were analyzed by expressing specific hH3R mutants in Sf9 cells. In contrast to the hH3R, the hH4R preferentially couples to co-expressed G?i2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTP?S. The hH4R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH4R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH4R. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of HxR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

Dataset Information

A designer cell-based histamine-specific human allergy profiler.

Publications

A designer cell-based histamine-specific human allergy profiler.

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