Sequence and diversity of rat T-cell receptor Tcra V8 gene segments

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X80509 (8.2) - X80514 (8.7)     

Receptive range analysis of a mouse odorant receptor subfamily

Mammals deploy a large array of odorant receptors (ORs) to detect and distinguish a vast number of odorant molecules. ORs vary widely in the type of odorant structures recognized and in the breadth of molecular receptive range (MRR), with some ORs recognizing a small group of closely related molecules and other ORs recognizing a wide range of structures. While closely related ORs have been shown to have similar MRRs, the functional relationships among less closely related ORs are unclear. We screened a small group of ORs with a diverse odorant panel to identify a new odorant‐OR pairing (unsaturated aldehydes and MOR263‐3). We then extensively screened MOR263‐3 and a series of additional MORs related to MOR263‐3 in various ways. MORs related by phylogenetic analysis (several other members of the MOR263 subfamily) had MRRs that overlapped with the MRR of MOR263‐3, even with amino acid identity as low as 48% (MOR263‐2). MOR171‐17, predicted to be functionally related to MOR263‐3 by an alternative bioinformatic analysis, but with only 39% amino acid identity, had a distinct odorant specificity. Our results support the use of phylogenetic analysis to predict functional relationships among ORs with relatively low amino acid identity.

     

Structural studies on the alphabeta T-cell receptor

Alphabeta T-cell receptor (TcR) recognition of antigenic peptides bound to the major histocompatibility complex (pMHC), is integral to the cellular immune system. Crystallographic studies over the last decade have provided significant insight into this unique trimolecular recognition event. The TcR-pMHC structural information has been paralleled by biophysical studies that have further explored the emerging binding models in an attempt to answer fundamental immunological questions regarding MHC restriction, T-cell immunodominance and TcR cross-reactivity. However, despite the important data that has been generated regarding TcR-pMHC interactions, the scope of this information is still incomplete due to the limited range of TcRs that have been studied. These limitations are primarily due to difficulties in obtaining high yields of recombinant alphabeta TcR for crystallographic and biophysical analysis; here we will discuss some of the protein engineering strategies that have been employed to expand the pool of recombinant TcRs suitable for crystallographic studies and the subsequent studies that have utilized these proteins.     

A new method for quantitative analysis of the mouse T-cell receptor V region repertoires: comparison of repertoires among strains

We developed an adaptor ligation PCR-based microplate hybridization assay (MHA) to analyze the repertoires of mouse T-cell receptor (TCR) alpha- and beta-chain variable regions (TCRAV and TCRBV). RNA is transcribed to cDNA and an adaptor is ligated to the 5' end of the cDNA, which is then used as a template for PCR with an adaptor-specific 3' primer and a constant region-specific 5' primer. After hybridization of PCR products with TCRAV-and TCRBV-specific probes on the microplate, quantitative ELISA was carried out.The entire TCRAV or TCRBV repertoires could be analyzed using a single 96-well plate in triplicate and completed in less than 4 h. The assay results demonstrated the high level of specificity and reproducibility of this method. Furthermore, MHA results correlated well with those of fluorescence-activated cell sorting. This method may provide important information about various T-cell-associated diseases including autoimmune disease. The influence of the MHC on mouse TCR repertoires was next studied using the newly developed mouse TCRAV and TCRBV repertoire assay. The analysis in six strains showed no significant correlation between MHC haplotypes and TCRAV and TCRBV repertoires. However, large differences among strains was observed in TCRBV, but not in TCRAV repertoires. There were also large differences within same strain in TCRBV, but not in TCRAV repertoires, indicating differences in individuals independent of genetic factors. These data suggest that TCRBV repertoires are more susceptible than TCRAV repertoires not only to genetic factors but also some environmental factors.     

Organization, polymorphism, and expression of the human T-cell receptor AV1 subfamily

 At least 32 mostly single-member subfamilies of T-cell receptor alpha variable (TCRAV) genes have been described in humans. The AV1 subfamily is the largest, estimated by hybridization to contain as many as five members. However, a search of nucleotide sequence databases reveals a much greater number of unique sequences corresponding to this subfamily. In order to resolve this discrepancy between hybridization and nucleotide sequencing data, and to better understand the nature of variability among variable genes within a large subfamily, a genomic characterization of the AV1 subfamily in humans was carried out. Total genomic DNA, as well as isolated genomic clones spanning the TCRA region were screened for members of the AV1 subfamily by polymerase chain reaction (PCR) and nucleotide sequencing as well as by hybridization. A total of eight AV1 genes were identified and their nucleotide sequences were determined. Three of the sequences represent new genes. Based on structural features and the results of PCR screening of cDNA, none of these new genes appear to be functional. Several additional previously reported AV1 sequences were determined to represent alleles of AV1 genes, and simple PCR restriction digest assays were established for their detection. Use of each of the identified AV1 genes as hybridization probes failed to reveal any additional hybridizing bands. Thus the AV1genes represent the largest TCRAV subfamily with a maximum of eight members, several of which have common allelic forms. Received: 7 November 1996 / Revised: 5 December 1996     

Structural requirements for V2 vasopressin receptor proteolytic cleavage.

The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.     

Structural characteristics of the mouse transferrin receptor

Rat monoclonal antibodies against mouse transferrin receptor have been used to isolate and characterize the mouse receptor molecule. The molecule is a dimeric glycoprotein of Mr 200 000 resembling its human homolog of Mr 190 000. Receptor molecules prepared from different lymphoid cell populations show structural differences which can be explained by variations in the carbohydrate moiety of the molecule. Both the antibody-binding site and the transferrin-binding site are located on tryptic fragments of Mr 80 000 on the extracellular part of the molecule. After trypsin treatment, these fragments are partially retained at the cell surface, probably non-covalently bound to one intact receptor subunit, but they are released at higher trypsin concentrations. The soluble fragments retain their ability to bind transferrin and appear to exist as dimers. In this fragment, there are no disulfide bonds present. Disulfide bonds are located near the plasma membrane. Studies using a cleavable cross-linker indicated the presence of cross-linking sites at the intramembranous or the cytoplasmic part of the molecule.     

Functional analysis of a mammalian odorant receptor subfamily

Phylogenetic analysis groups mammalian odorant receptors into two broad classes and numerous subfamilies. These subfamilies are proposed to reflect functional organization. Testing this idea requires an assay allowing detailed functional characterization of odorant receptors. Here we show that a variety of Class I and Class II mouse odorant receptors can be functionally expressed in Xenopus laevis oocytes. Receptor constructs included the N-terminal 20 residues of human rhodopsin and were co-expressed with Galphaolf and the cystic fibrosis transmembrane regulator to allow electrophysiological measurement of receptor responses. For most mouse odorant receptors tested, these conditions were sufficient for functional expression. Co-expression of accessory proteins was required to allow functional surface expression of some mouse odorant receptors. We used this assay to examine the receptive ranges of all members of the mouse odorant receptor 42 (MOR42) subfamily. MOR42-1 responded to dicarboxylic acids, preferring a 10-12 carbon chain length. MOR42-2 responded to monocarboxylic acids (7-10 carbons). MOR42-3 responded to dicarboxylic acids (8-10 carbons) and monocarboxylic acids (10-12 carbons). Thus, the receptive range of each receptor was unique. However, overlap between the individual receptive ranges suggests that the members of this subfamily form one contiguous subfamily receptive range, suggesting that odorant receptor subfamilies do constitute functional units.     

Structural determinants of T-cell receptor bias in immunity

Antigen-specific T-cell responses induced by infection, transplantation, autoimmunity or hypersensitivity are characterized by cells expressing biased profiles of T-cell receptors (TCRs) that are selected from a diverse, naive repertoire. Here, we review the evidence for these TCR biases, focusing on crystallographic analysis of the structural constraints that determine the binding of a TCR to its ligand and the persistence of certain TCRs in an immune repertoire. We discuss the ways in which diversity in a selected TCR repertoire can contribute to protective immunity and the implications of this for vaccine design and immunotherapy.     

PCR-based analysis of the T-cell receptor V beta multigene family: experimental parameters affecting its validity.

The validity of semiquantitative, PCR-based analysis of gene expression within a multigene family, the human T-cell receptor (TCR) beta chain variable region family, was investigated. Primer comparability was addressed by grouping hybridization temperatures and limiting the size range of amplified fragments. Primers selected satisfied criteria for comprehensiveness, match to targets and discrimination of nontargets. Specificity was enhanced by maximizing mismatches with nontargets and using an elevated hybridization temperature. Reaction conditions are described that ensure specificity while maintaining sensitivity. Several results confirmed primer specificity. Limits on precision were documented: probable error was 3%-7% of mean value for target prevalences (% of all TCR mRNA represented by a particular V beta) in the 5%-40% range. Accuracy was limited by the nonlinear relationship between target prevalence and signal obtained. Because of this relationship, the effect of the observed limits of precision varied. Valid distinctions were possible between sufficiently separated prevalences, i.e., 0%-1%, 3%-5%, 10%, 30%, greater than 50%. Additional concerns addressed include: standardization of signals, coamplifiation and effects of primer artifacts, and the nature of the mRNA pool. Only when theoretical and practical limits in precision and accuracy are acknowledged can semiquantitative, PCR-based analysis be used with confidence to assess gene usage within a large, multigene family.     

Structural analysis of mouse S-antigen

Mouse S-antigen clones were isolated from a mouse retinal cDNA library using a bovine S-antigen cDNA probe. The largest clone (MSC-242) comprised 1532 bp and contained the entire coding sequence. The nucleotide sequence homology between the mouse and bovine coding regions was 84%, while non-coding regions appeared to be more divergent. The deduced amino acid sequence indicated that the mouse S-antigen had 403 residues and its molecular ratio was 44,930. An overall amino acid sequence similarity of 84% was observed between the mouse and bovine proteins. This degree of similarity dropped to 60% and 47% at the N and the C termini, respectively. The local homology with alpha-transducin observed in the bovine proteins, including the putative phosphoryl and rhodopsin binding sites, was conserved in the mouse as well. There was no overall sequence similarity with other proteins listed in the National Biomedical Research Foundation (NBRF) protein sequence database. Among the uveitopathogenic sites for experimental autoimmune uveitis (EAU), peptides N and M were identical to their bovine counterparts. Peptides 3 and K, however, were more divergent. The short repeats within these peptides were conserved.     

Restriction fragment length polymorphisms of the mouse T-cell receptor gene families

We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cell receptor genes of 25 inbred Mus musculus strains and 8 wild Mus species. Included in the inbred mice tested were several strains which spontaneously develop systemic autoimmune disease. Extensive polymorphism was evident for the variable (V) gene segments of the gene family for both the inbred strains and wild mouse species. Changes in the total number of bands hybridizing with probes for V gene segments suggest that members of a V gene segment subfamily are not closely linked, but are interspersed with members of other subfamilies; that expansion and contraction of the multimembered subfamilies may be an important diversifying factor. Our data obtained with gene probes revealed genomic diversity that is much more limited than that seen for the locus. Analysis of inbred mice with probes for the gene locus revealed some RFLPs, but little evidence of expansion or contraction in the numbers of gene segments. Among the autoimmune mice, NZW, NZB, and BXSB/MpJ all display distinctive differences with gene probes. NZW mice have a large deletion of the gene family, which has been reported previously. We found no differences to distinguish the MRL/MpJ lpr/lpr mice from non-autoimmune strains.