Christopher S. Theile

Site-specific N-terminal labeling of proteins using sortase-mediated reactions

This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Staphylococcus aureus sortase A) or LPXTA (for Streptococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.

Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes

Significance Engineered human RBCs are attractive carriers for the introduction of novel therapeutics, immunomodulatory agents, and diagnostic imaging probes into the human body. Normal murine and human RBCs can be produced in tissue culture from progenitors; we have introduced genes into these cells encoding surface proteins that can be covalently and site-specifically modified on the cell surface. The engineering and labeling processes do not damage the cells or affect their survival in vivo. Most importantly, the engineered RBCs can be labeled with a wide array of functional probes, including small molecules, peptides, and proteins, and thus have the potential to be carriers of a variety of therapeutic substances into the bloodstream. We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes.

Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry.

Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3–4 d.

Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells

Monovalent engagement can trigger BCR signal transduction, and fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition.

A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short-term plasticity and vesicle number.

Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short‐term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short‐term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short‐term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates.

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca2+-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h−1, depending on the geometry of the reactor used.

Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments

The site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified fluorescent single-domain antibody fragment or an intact immunoglobulin produced in a sortase-catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single-domain and full-sized antibodies without deleterious effects on antigen binding.

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.

Graphene Oxide Nanosheets Modified with Single-Domain Antibodies for Rapid and Efficient Capture of Cells

Peripheral blood can provide valuable information on an individuals immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.1 Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single-domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (∼30 μL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Significance We used an unbiased screening strategy to capture deubiquitylases that participate in T cell receptor signaling in primary cells under physiological settings. We identified ubiquitin-specific peptidase (Usp) 12 as a crucial component of TCR expression at the cell surface, and found supporting evidence for its function by creating an inducible genetic knockout in Jurkat cells. Using proximity-based labeling, we identified LAT and Trat1 as substrates of Usp12. In Usp12-deficient cells, both LAT and Trat1 are ubiquitin-modified and lysosomally degraded, thus down-regulating TCR surface expression. Our data define a role of Usp12 in the TCR signaling pathway for the first time, to our knowledge. These results underscore the importance of deubiquitylases in fine-tuning signaling cascades and provide a basis for the screening of small molecules to identify potential inhibitors. Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.