RESUMEN

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galß1-3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the ß-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the ß-strands, requiring them to bend to match the concavity of the VLR solenoid.

Asunto(s)

Antígenos de Carbohidratos Asociados a Tumores/química , Proteínas de Peces/química , Lampreas , Receptores de Antígenos de Linfocitos T/química , Animales , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Unión Proteica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología

RESUMEN

Adaptive immunity in jawless vertebrates is mediated by leucine-rich repeat proteins called "variable lymphocyte receptors" (VLRs). Two types of VLR (A and B) are expressed by mutually exclusive lymphocyte populations in lamprey. VLRB lymphocytes resemble the B cells of jawed vertebrates; VLRA lymphocytes are similar to T cells. We determined the structure of a high-affinity VLRA isolated from lamprey immunized with hen egg white lysozyme (HEL) in unbound and antigen-bound forms. The VLRA-HEL complex demonstrates that certain VLRAs, like gammadelta T-cell receptors (TCRs) but unlike alphabeta TCRs, can recognize antigens directly, without a requirement for processing or antigen-presenting molecules. Thus, these VLRAs feature the nanomolar affinities of antibodies, the direct recognition of unprocessed antigens of both antibodies and gammadelta TCRs, and the exclusive expression on the lymphocyte surface that is unique to alphabeta and gammadelta TCRs.

Asunto(s)

Epítopos/inmunología , Linfocitos/inmunología , Petromyzon/inmunología , Proteínas/inmunología , Animales , Sitios de Unión , Pollos , Epítopos/química , Epítopos/metabolismo , Cinética , Proteínas Repetidas Ricas en Leucina , Linfocitos/metabolismo , Modelos Moleculares , Muramidasa/química , Muramidasa/inmunología , Muramidasa/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología

RESUMEN

Adaptive immunity in jawless vertebrates is mediated by antigen receptors that are fundamentally different from those of jawed vertebrates. Whereas antibodies and T cell receptors (TCRs) are composed of immunoglobulin (Ig) domains, the variable lymphocyte receptors (VLRs) of jawless fish consist of leucine-rich repeat (LRR) modules. As with antibodies and TCRs, VLRs are assembled by DNA recombination in a process that generates a vast repertoire of receptors. VLRs recognize as diverse an array of particulate and soluble antigens as Ig-based antibodies, and do so with similar affinity and specificity. X-ray crystallographic studies of VLRs in complex with protein and carbohydrate antigens have shown that these LRR-based receptors use nearly all their concave surface to bind ligands, in addition to a highly variable loop in their C-terminal LRR capping module. This structural information, combined with a comprehensive analysis of VLR sequences, has revealed an almost perfect match between antigen-contacting positions and positions with highest sequence diversity. The independent evolution approximately 500 million years ago of LRR-based and Ig-based receptors of comparable diversity and antigen-binding properties provides evidence for the survival value of adaptive immunity in vertebrates.

Asunto(s)

Inmunidad Adaptativa/inmunología , Evolución Molecular , Sistema Inmunológico/inmunología , Linfocitos/inmunología , Receptores Inmunológicos/inmunología , Animales , Maxilares , Conformación Proteica , Vertebrados

RESUMEN

Lamprey are members of the ancestral vertebrate taxon (jawless fish), which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of Ig superfamily domains, lamprey variable lymphocyte receptors (VLRs) consist of highly diverse leucine-rich repeats. Although VLRs represent the only known adaptive immune system not based on Ig, little is known about their antigen-binding properties. Here we report robust plasma VLRB responses of lamprey immunized with hen egg lysozyme and beta-galactosidase (beta-gal), demonstrating adaptive immune responses against soluble antigens. To isolate monoclonal VLRs, we constructed large VLR libraries from antigen-stimulated and naïve animals in a novel yeast surface-display vector, with the VLR C-terminally fused to the yeast Flo1p surface anchor. We cloned VLRB binders of lysozyme, beta-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide antigen, with dissociation constants up to the single-digit picomolar range, equivalent to those of high-affinity IgG antibodies. We also isolated from a single lamprey 13 anti-lysozyme VLRA clones with affinities ranging from low nanomolar to mid-picomolar. All of these VLRA clones were closely related in sequence, differing at only 15 variable codon positions along the 244-residue VLR diversity region, which augmented antigen-binding affinity up to 100-fold. Thus, VLRs can provide a protective humoral antipathogen shield. Furthermore, the broad range of nominal antigens that VLRs can specifically bind, and the affinities achieved, indicate a functional parallelism between LRR-based and Ig-based antibodies. VLRs may be useful natural single-chain alternatives to conventional antibodies for biotechnology applications.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Lampreas/inmunología , Proteínas/inmunología , Receptores de Antígenos/inmunología , Adaptación Fisiológica , Animales , Secuencia de Bases , Proteínas Repetidas Ricas en Leucina , Datos de Secuencia Molecular , Receptores de Antígenos/fisiología

RESUMEN

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs are fundamentally different from the antibodies of jawed vertebrates, which consist of immunoglobulin (Ig) domains. We determined the structure of an anti-hen egg white lysozyme (HEL) VLR, isolated by yeast display, bound to HEL. The VLR, whose affinity resembles that of IgM antibodies, uses nearly all its concave surface to bind the protein, in addition to a loop that penetrates into the enzyme active site. The VLR-HEL structure combined with sequence analysis revealed an almost perfect match between ligand-contacting positions and positions with highest sequence diversity. Thus, it is likely that we have defined the generalized antigen-binding site of VLRs. We further demonstrated that VLRs can be affinity-matured by 13-fold to affinities as high as those of IgG antibodies, making VLRs potential alternatives to antibodies for biotechnology applications.

Asunto(s)

Antígenos/química , Lampreas , Conformación Proteica , Proteínas/química , Proteínas/metabolismo , Receptores de Superficie Celular/química , Secuencia de Aminoácidos , Animales , Antígenos/genética , Antígenos/metabolismo , Sitios de Unión , Pollos , Proteínas Repetidas Ricas en Leucina , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Muramidasa/química , Muramidasa/genética , Muramidasa/inmunología , Proteínas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo

RESUMEN

2B4 belongs to the CD2 family of molecules and is expressed on all NK, gammadelta, and memory CD8(+) (alphabeta) T cells. The murine NK receptor 2B4 exhibits both inhibitory and activating functions, whereas human 2B4 has been reported to be an activating molecule. How murine 2B4 can act both as an activating and inhibitory receptor and what distinguishes its function from human 2B4 have remained largely unknown. We use here a model system that allows the study of human and murine 2B4 under identical and controlled conditions. These studies reveal that both human and mouse 2B4 can activate or inhibit NK cells. We show here that the level of 2B4 expression and the degree of 2B4 cross-linking play a significant role in the regulation of signaling lymphocyte activation molecule-associated protein-mediated activation by 2B4. A high level of 2B4 expression, heavy cross-linking, and relative paucity of signaling lymphocyte activation molecule-associated protein promote inhibitory function. Our studies demonstrate how a single receptor can have opposing function depending on the degree of receptor expression, extent of its ligation, and the relative abundance of certain adaptor molecules. Because the levels of 2B4 and CD48 are dynamically regulated, these findings have implications for the regulation of NK cell function.

Asunto(s)

Antígenos CD/fisiología , Receptores Inmunológicos/fisiología , Alelos , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno CD48 , Línea Celular , Humanos , Hibridomas , Inmunosupresores/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Activadas por Linfocinas/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ligandos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Familia de Moléculas Señalizadoras de la Activación Linfocitaria

RESUMEN

Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.

Asunto(s)

Antígenos CD/química , Células Asesinas Naturales/química , Activación de Linfocitos/fisiología , Glicoproteínas de Membrana/química , Modelos Inmunológicos , Receptores Inmunológicos/química , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno CD48 , Cristalografía por Rayos X , Humanos , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria

RESUMEN

The enzyme phospholipase Cgamma1 (PLCgamma1) is essential for T cell signaling and activation. Following T cell receptor ligation, PLCgamma1 interacts through its SH2 and SH3 domains with the adaptors LAT and SLP-76, respectively, to form a multiprotein signaling complex that leads to activation of PLCgamma1 by Syk tyrosine kinases. To identify the binding site for PLCgamma1 in SLP-76, we used isothermal titration calorimetry to measure affinities for the interaction of PLCgamma1-SH3 with a set of overlapping peptides spanning the central proline-rich region of SLP-76. PLCgamma1-SH3 bound with high specificity to the SLP-76 motif 186PPVPPQRP193, which represents the minimal binding site. To understand the basis for selective recognition, we determined the crystal structures of PLCgamma1-SH3 in free form, and bound to a 10-mer peptide containing this site, to resolutions of 1.60 A and 1.81 A, respectively. The structures reveal that several key contacting residues of the SH3 shift toward the SLP-76 peptide upon complex formation, optimizing the fit and strengthening hydrophobic interactions. Selectivity results mainly from strict shape complementarity between protein and peptide, rather than sequence-specific hydrogen bonding. In addition, Pro193 of SLP-76 assists in positioning Arg192 into the compass pocket of PLCgamma1-SH3, which coordinates the compass residue through an unusual aspartate. The PLCgamma1-SH3/SLP-76 structure provides insights into ligand binding by SH3 domains related to PLCgamma1-SH3, as well as into recognition by PLCgamma1 of signaling partners other than SLP-76.

Asunto(s)

Fosfoproteínas/química , Fosfolipasas de Tipo C/química , Dominios Homologos src , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sitios de Unión , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Fosfolipasa C gamma , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Fosfolipasas de Tipo C/metabolismo

RESUMEN

The transmembrane protein, linker for activation of T cells (LAT), is essential for T-cell activation and development. Phosphorylation of LAT at multiple tyrosines creates binding sites for the adaptors Gads and Grb2, leading to nucleation of multiprotein signaling complexes. Human LAT contains five potential binding sites for Gads, of which only those at Tyr171 and Tyr191 appear necessary for T-cell function. We asked whether Gads binds preferentially to these sites, as differential recognition could assist in assembling defined LAT-based complexes. Measured calorimetrically, Gads-SH2 binds LAT tyrosine phosphorylation sites 171 and 191 with higher affinities than the other sites, with the differences ranging from only several fold weaker binding to no detectable interaction. Crystal structures of Gads-SH2 complexed with phosphopeptides representing sites 171, 191 and 226 were determined to 1.8-1.9 A resolutions. The structures reveal the basis for preferential recognition of specific LAT sites by Gads, as well as for the relatively greater promiscuity of the related adaptor Grb2, whose binding also requires asparagine at position +2 C-terminal to the phosphorylated tyrosine.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformación Proteica , Tirosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sitios de Unión , Cristalografía por Rayos X , Proteína Adaptadora GRB2 , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Fosfopéptidos/genética , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Termodinámica , Dominios Homologos src