Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.nanobody | CXCR4 antagonist | chemotaxis | HIV | stem cell mobilization

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TGF-␤-stimulated clone-22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved during evolution. Two homologues are known that share a similar leucine zipper domain and another conserved domain (designated the TSC box). Only limited data are available on the function of TSC-22 and its homologues. TSC-22 is transcriptionally up-regulated by many different stimuli, including anti-cancer drugs and growth inhibitors, and recent data suggest that TSC-22 may play a suppressive role in tumorigenesis. In this paper we show that TSC-22 forms homodimers via its conserved leucine zipper domain. Using a yeast two-hybrid screen, we identified a TSC-22 homologue (THG-1) as heterodimeric partner. Furthermore, we report the presence of two more mammalian family members with highly conserved leucine zippers and TSC boxes. Interestingly, both TSC-22 and THG-1 have transcriptional repressor activity when fused to a heterologous DNAbinding domain. The repressor activity of TSC-22 appears sensitive for promoter architecture, but not for the histone deacetylase inhibitor trichostatin A. Mutational analysis showed that this repressor activity resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain. Our results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors that can homodimerize and heterodimerize with other family members and that at least two TSC-22 family members may be repressors of transcription.

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPa prohibited intermolecular interactions. Interestingly, stress factors such as H 2 O 2 , UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPa and RPTPa-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPa-SpD2, Cys723, was required for the H 2 O 2 effect on RPTPa. H 2 O 2 induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by¯uorescence resonance energy transfer, with cyan¯uorescent protein (CFP) and yellow¯uorescent protein (YFP) anking RPTPa-SpD2 in a single chimeric protein. Importantly, H 2 O 2 treatment stabilized RPTPa dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPa-D2, leading to stabilization of RPTPa dimers, and thus to inhibition of RPTPa activity.