Keiko Tamura-Kawakami

Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin

Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years. DOI: http://dx.doi.org/10.7554/eLife.11621.001

Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti‐peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18‐residue affinity tag with ideal solution binding kinetics, providing a low‐cost purification means when combined with nondenaturing elution by water‐miscible organic solvents. Three‐dimensional information provides a firm structural basis for the antibody–peptide interaction, opening opportunities for further improvements/modifications.

A rapid screening method for cell lines producing singly-tagged recombinant proteins using the "TARGET tag" system.

Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 microl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, high producer cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol.

A Structure-Based Model of Substrate Discrimination by a Noncanonical PDZ Tandem in the Intramembrane-Cleaving Protease RseP

During the extracytoplasmic stress response in Escherichia coli, the intramembrane protease RseP cleaves the anti-σ(E) protein RseA only after the membrane-anchored protease DegS truncates the periplasmic part of RseA that suppresses the action of RseP. Here we analyzed the three-dimensional structure of the two tandemly arranged PSD-95/Dlg/ZO-1 (PDZ) domains (PDZ tandem) present in the periplasmic region of RseP and revealed that the two putative ligand-binding grooves constitute a single pocket-like structure that would lie just above the active center sequestrated within the membrane. Complete removal of the PDZ tandem from RseP led to the intramembrane cleavage of RseA without prior truncation by DegS. Furthermore, mutations expected to destabilize the tertiary structure of the PDZ tandem also caused the deregulation of the sequential cleavage. These observations suggest that the PDZ tandem serves as a size-exclusion filter to accommodate the truncated form of RseA into the active center.

Detection of endogenous LRP6 expressed on human cells by monoclonal antibodies specific for the native conformation

LRP6 is a cell surface molecule that plays a critical role in the Wnt signaling pathway, and is implicated in numerous human diseases. Studies of cellular signaling mediated by LRP6 have relied on overexpression experiments, due to the lack of good monoclonal antibodies (mAbs) reactive with native LRP6 ectodomain. By using native recombinant LRP6 ectodomain fragment produced in mammalian expression system, we succeeded in developing a panel of anti-human LRP6 mAbs. Selected mAbs were capable of staining endogenous LRP6 on cell surface by using flow cytometry and immunofluorescence microscopy, and enriching detergent-solubilized LRP6 from cell lysate by immunoprecipitation.

Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1.

LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules. Importantly, the extent of inter-domain motion is modulated by evolutionarily conserved N-glycan chains proximal to the joint. We also found that the LRP6 ectodomain becomes highly compact upon complexation with the Wnt antagonist Dkk1, suggesting a potential role for the ectodomain conformational change in the regulation of receptor oligomerization and signaling.

Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability

Antibody fragments are frequently used as a crystallization chaperone to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37xa0kDa, termed Fv-clasp, which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The clasp and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior chaperoning activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6β1.

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies

HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.

An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system

Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called eTev, which contains a recognition sequence for Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse Wise protein.

Substrate discrimination by size-exclusion in the intramembrane protease RseP

Regulated intramembrane proteolysis (RIP), wherein a target membrane protein is specifically cleaved within the transmembrane region, is now accepted as a form of cellular signaling. As a result of proteolysis, a soluble portion of the target membrane protein is liberated to act as a signaling molecule. RIP is catalyzed by intramembrane-cleaving proteases, which are now classified into site-2 protease, rhomboid and γ-secretase/SPP families based on the mechanism of catalysis. E. coli possesses a site-2 protease homolog RseP, which is implicated in the extracytoplasmic stress response. RseP cleaves a membrane-spanning anti-σE protein RseA to release σE from the membrane, where truncation of the C-terminal periplasmic part of RseA by a membrane-anchored protease DegS triggers the action of RseP. Hence, there must be some mechanism by which RseP senses the DegS-cleavage of RseA. RseP possesses two tandemly-arranged PDZ domains (PDZ tandem) in the periplasmic region, which have been suggested to be involved in the regulation of cleavage. Although PDZ domains generally recognize the C-terminal sequence of a ligand, most of the previous works suggested that the RseP PDZ domains are involved in the suppression of the intramembrane cleavage of RseA. In this study, we determined the 3D structure of the PDZ tandem by X-ray crystallography and SAXS and showed that the two PDZ domains are arranged in an overall “clam-like” configuration to constitute a “pocket-like” structure. Sequence analysis suggested that the PDZ tandem would lie just above the active center sequestrated within the membrane. Furthermore, chemical modification demonstrated that the interior of the pocket is inaccessible to a bulky reagent in the full-length RseP. Taken together, we have made a proposal that RseP accommodates the truncated RseA into the active center by a steric size-exclusion mechanism through the PDZ tandem, rather than by recognition of a specific sequence/motif of RseA.