Ludmila V. Mechetina

IDENTIFICATION OF AN IL-8 HOMOLOG IN LAMPREY (LAMPETRA FLUVIATILIS) : EARLY EVOLUTIONARY DIVERGENCE OF CHEMOKINES

Subtractive hybridization was used to study river lamprey ( Lampetra fluviatilis ) leukocyte‐specific cDNA. A clone representing the most abundant component (12 %) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3 ′ untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA‐1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA‐1 has the highest similarity to the chicken EMF‐1 (40 %) and to the mammalian IL‐8 (32 – 33 %). However, it lacks the ELR motif essential for the function of the mammalian IL‐8‐related chemokines. Based on the phylogenetic analysis of the LFCA‐1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.

Identification of CD16-2, a novel mouse receptor homologous to CD16/FcγRIII

Abstract. It is believed that mouse FcγRIII arose by an evolutionarily recent recombination, which brought together the extracellular domains from FcγRII with the transmembrane/cytoplasmic region from the ancestor FcγRIII. Here, we report identification of a mouse gene encoding a transmembrane receptor that may be regarded as the true ortholog of nonrodent CD16/FcγRIII. Designated CD16-2, the novel protein is highly similar to human FcγRIIIA in the signal peptide (60% identical residues), and in the extracellular domains (65%). Although the similarity between the two proteins is less conspicuous in the transmembrane/cytoplasmic region (54%), it is higher than between human FcγRIIIA and mouse FcγRIII (44%). However, the conserved transmembrane motif LFAVDTGL shared by rodent and human FcγRIII and FcεRI has two replacements in CD16-2. The CD16-2 gene is tightly linked to the FcγRIII and FcγRII genes and consists of five exons. Northern blot analysis revealed that CD16-2 is expressed in peripheral blood leukocytes, as well as in spleen, thymus, colon and intestine. RT-PCR showed prominent expression in macrophage cell line J774. Based on sequence comparisons, it is suggested that the modern repertoire of the mammalian low affinity Fc receptors has resulted from repetitive duplications and/or recombinations of three ancestral genes.

A family of highly diverse human and mouse genes structurally links leukocyte FcR, gp42 and PECAM-1.

Abstract. A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger receptor superfamily-related domain at the C-terminus. The other cDNAs code for the type I transmembrane proteins with the extracellular parts comprised of one to six Ig-like domains. Five homologous types of the Ig-like domains were defined and each protein was found to have a unique combination of the domain types. The cytoplasmic tails of the transmembrane proteins show different patterns of the tyrosine-based signal motifs. While the human IFGP members appear to be B-cell antigens, the mouse genes have a broader tissue distribution with predominant expression in brain. Sequence comparisons revealed that the IFGP family may be regarded as a phylogenetic link joining the leukocyte FcRs with the rat NK cell-specific gp42 antigen and platelet endothelial cell adhesion molecule-1 (PECAM-1), two mammalian leukocyte receptors whose close relatives were not found previously. It is suggested that FcRs, the IFGP proteins and gp42 have arisen by a series of duplications from a common ancestor receptor comprised of five Ig-like domains. The organization of the human genes shows that the IFGP family evolved through differential gain and loss of exons due to recombination and/or mutation accumulation in the duplicated copies.

CD3ε homologues in the chondrostean fish Acipenser ruthenus

Abstract. CD3ε is an essential component of the T-cell receptor (TCR) complex for antigen. We report here molecular cloning and characterization of cDNAs encoding the CD3ε homologues in sterlet (Acipenser ruthenus), a representative of primitive chondrostean fishes. Sequence analysis of the cDNA clones demonstrated unexpectedly high CD3ε gene heterogeneity in this species. While some cDNAs encoded proteins with the structure typical of mammalian CD3ε, others coded for proteins lacking the membrane-proximal half of the extracellular domain. Two cDNAs contained in-frame stop codons in the region encoding the cytoplasmic domain. Based on genomic blot analysis and RT-PCR typing of individual spleen RNAs, we suggest that sterlet may possess two highly polymorphic CD3ε loci, of which one can produce alternatively spliced transcripts. The structural elements shown to be functionally important in the mammalian CD3ε are strongly conserved in the sterlet CD3ε. The cytoplasmic region contains an immunoreceptor tyrosine-based activation motif (ITAM) with YEPI and YSGL tyrosine-containing sequences that are characteristic of only this TCR subunit. The pattern of sequence conservation indicates also that strong selection pressure was imposed on a motif VYYW at the C-end of the transmembrane domain and on a CD3ε-specific proline-rich motif RXPPVP juxtaposed to the N-terminus of the ITAM. Weak similarity of the sterlet CD3ε with the chicken and Xenopus CD3γ/δ indicates that these two TCR subunits diverged before radiation of bony fishes and tetrapods. While the role of CD3ε heterogeneity in sterlet remains to be elucidated, the data obtained show that the basic mechanisms of TCR signaling have ancient evolutionary origin.

The Xenopus FcR family demonstrates continually high diversification of paired receptors in vertebrate evolution

BackgroundRecent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representatives Xenopus tropicalis and Xenopus laevis.ResultsThe diploid genome of X. tropicalis contains at least 75 genes encoding paired FcR-related receptors designated XFLs. The allotetraploid X. laevis displays many similar genes primarily expressed in lymphoid tissues. Up to 35 domain architectures generated by combinatorial joining of six Ig-domain subtypes and two subtypes of the transmembrane regions were found in XFLs. None of these variants are shared by FcR-related proteins from other studied species. Putative activating XFLs associate with the FcRγ subunit, and their transmembrane domains are highly similar to those of activating mammalian KIR-related receptors. This argues in favor of a common origin for the FcR and the KIR families. Phylogenetic analysis shows that the entire repertoires of the Xenopus and mammalian FcR-related proteins have emerged after the amphibian-amniotes split.ConclusionFcR- and KIR-related receptors evolved through continual species-specific diversification, most likely by extensive domain shuffling and birth-and-death processes. This mode of evolution raises the possibility that the ancestral function of these paired receptors was a direct interaction with pathogens and that many physiological functions found in the mammalian receptors were secondary acquisitions or specializations.

Expression pattern of FCRL (FREB, FcRX) in normal and neoplastic human B cells

FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B‐cell lymphoma, diffuse large B‐cell lymphoma and lymphocyte predominance Hodgkins disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut‐off that distinguished subsets of diffuse large B‐cell lymphoma. Our results also showed that FCRL tended to be negative in T‐cell‐rich B‐cell lymphoma and in classical Hodgkins disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B‐cell neoplasms.

Cloning of a CXCR4 homolog in chondrostean fish and characterization of the CXCR4-specific structural features.

The chemokine receptor CXCR4 and its ligand SDF-1 are essential components of hematopoiesis, organogenesis and immunomodulation in mammalian species. We cloned a cDNA encoding CXCR4 homolog of sterlet (Acipenser ruthenus), a representative of chondrostean fishes. The deduced amino acid sequence of sterlet CXCR4 is almost equally distant from mammalian and teleost CXCR4 (66-68% identical residues). Percent identity with the other chemokine receptors varies in the 30-35% range. The CXCR4 sequences from the three phylogenetically diverged lineages were compared with the sequences of the other chemokine receptors to determine the CXCR4-specific structural elements that were conserved during vertebrate evolution. The characteristic residues and/or motifs are located predominantly in the intracellular and extracellular regions and in the third, fourth and fifth transmembrane domains. The data presented may be helpful for structure-function analysis of the CXCR4 ligand binding and signal transduction.

FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.

The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like Receptors

In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3-4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRgamma subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

FCRL6 receptor: Expression and associated proteins

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.