Irene Tom

The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells

Here we have identified a surface protein, TIGIT, containing an immunoglobulin variable domain, a transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif that was expressed on regulatory, memory and activated T cells. Poliovirus receptor, which is expressed on dendritic cells, bound TIGIT with high affinity. A TIGIT-Fc fusion protein inhibited T cell activation in vitro, and this was dependent on the presence of dendritic cells. The binding of poliovirus receptor to TIGIT on human dendritic cells enhanced the production of interleukin 10 and diminished the production of interleukin 12p40. Knockdown of TIGIT with small interfering RNA in human memory T cells did not affect T cell responses. TIGIT-Fc inhibited delayed-type hypersensitivity reactions in wild-type but not interleukin 10–deficient mice. Our data suggest that TIGIT exerts immunosuppressive effects by binding to poliovirus receptor and modulating cytokine production by dendritic cells.

Attenuating Lymphocyte Activity: the crystal structure of the BTLA-HVEM complex

Five CD28-like proteins exert positive or negative effects on immune cells. Only four of these five receptors interact with members of the B7 family. The exception is BTLA (B and T lymphocyte attenuator), which instead interacts with the tumor necrosis factor receptor superfamily member HVEM (herpes virus entry mediator). To better understand this interaction, we determined the 2.8-Å crystal structure of the BTLA-HVEM complex. This structure shows that BTLA binds the N-terminal cysteine-rich domain of HVEM and employs a unique binding surface compared with other CD28-like receptors. Moreover, the structure shows that BTLA recognizes the same surface on HVEM as gD (herpes virus glycoprotein D) and utilizes a similar binding motif. Light scattering analysis demonstrates that the extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex. Alanine-scanning mutagenesis of HVEM was used to further define critical binding residues. Finally, BTLA adopts an immunoglobulin I-set fold. Despite structural similarities to other CD28-like members, BTLA represents a unique co-receptor.

Design of a long-acting peptide functioning as both a glucagon-like peptide-1 receptor agonist and a glucagon receptor antagonist

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.

A secreted protein microarray platform for extracellular protein interaction discovery

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.

Defining the Ligand Specificity of the Deleted in Colorectal Cancer (DCC) Receptor

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4–6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.

Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2

No going back The mammalian spinal cord coordinates neuronal systems across the body. Axons that cross the spinal cord midline during development first need permission to cross and then instruction not to keep crossing back and forth. Jaworski et al. studied the axonal guidance receptor ROBO3 and found a ligand NELL2 in mice that appears to help in this process. Science, this issue p. 961 Multiple fail-safe signaling pathways ensure that developing axons cross the spinal cord midline no matter what. Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2–mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)–mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

Background: PILRα is an inhibitory receptor predominantly expressed in myeloid cells. Results: NPDC1 and COLEC12 are novel PILRα ligands. PILRα arginine residues 133 (mouse) and 126 (human) are critical contact residues. Conclusion: PILRα/ligand interactions involve a conserved domain in PILRα and a sialylated protein domain in the ligand. Significance: PILRα interacts with various ligands to alter myeloid cell function. Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

Murine Insulin Growth Factor-like (IGFL) and Human IGFL1 Proteins Are Induced in Inflammatory Skin Conditions and Bind to a Novel Tumor Necrosis Factor Receptor Family Member, IGFLR1

Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions.

The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation.

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus–host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus–host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.

Protein Microarrays for Identification of Novel Extracellular Protein‐Protein Interactions

Functional protein microarrays offer the capability for high‐throughput protein interaction analysis and have long promised to be a powerful tool for understanding protein interactions at the proteome scale. Although popular techniques for protein‐protein interaction mapping like yeast‐two‐hybrid and affinity‐purification mass spectrometry have performed well for identifying intracellular protein‐protein interactions, the study of interactions between extracellular proteins has remained challenging for these methods. Instead, the use of protein microarrays appears to be a robust and efficient method for the identification of interactions among the members of this class of protein. This unit describes methods for extracellular protein microarray production, screening, and analysis. A protocol is described for enhanced detection of low‐affinity interactions by generating multivalent complexes using Fc‐fusion bait proteins and protein A microbeads, along with a statistical method for hit scoring and identification of nonspecific interactions. Curr. Protoc. Protein Sci. 72:27.3.1‐27.3.24.