Isolation, tissue distribution, and chromosomal localization of a novel testis-specific human four-transmembrane gene related to CD20 and FcepsilonRI-beta

CD20 and the beta subunit of the high affinity receptor for IgE (FcepsilonRIbeta) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcepsilonRIbeta. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcepsilonRIbeta respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcepsilonRIbeta have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcepsilonRIbeta family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex.     

A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23)

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.     

Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

Identification of CD16-2, a novel mouse receptor homologous to CD16/Fc gamma RIII

It is believed that mouse Fc gamma RIII arose by an evolutionarily recent recombination, which brought together the extracellular domains from Fc gamma RII with the transmembrane/cytoplasmic region from the ancestor Fc gamma RIII. Here, we report identification of a mouse gene encoding a transmembrane receptor that may be regarded as the true ortholog of nonrodent CD16/Fc gamma RIII. Designated CD16-2, the novel protein is highly similar to human Fc gamma RIIIA 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 Fc gamma RIIIA and mouse Fc gamma RIII (44%). However, the conserved transmembrane motif LFAVDTGL shared by rodent and human Fc gamma RIII and Fc epsilon RI has two replacements in CD16-2. The CD16-2 gene is tightly linked to the Fc gamma RIII and Fc gamma RII 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.     

Induction of Fc epsilon RII/CD23 on phytohemagglutinin-activated human peripheral blood T lymphocytes. I. Enhancement by IL-2 and IL-4.

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.     

Analysis of Fc gamma RIII (CD16) membrane expression and association with CD3 zeta and Fc epsilon RI-gamma by site-directed mutation.

Two genes encode Fc gamma RIII (CD16), a low affinity FcR for IgG. CD16-I is expressed as a phosphatidylinositol glycan-anchored membrane glycoprotein on neutrophils, whereas CD16-II is a transmembrane-linked glycoprotein on NK cells. Membrane anchoring is determined by codon 203. Site-directed mutation of codon 203 and transient expression of these cDNA in COS-7 cells indicated that Phe, Ile, Leu, and Val permit transmembrane expression, whereas Ser, Thr, Tyr, Asn, Gly, Ala, Asp and Lys enable phosphatidylinositol-glycan attachment. Thus, the involvement of amino acid 203 in membrane anchoring cannot be explained simply on the basis of size, charge, or polarity of the amino acid side groups at this site. Efficient expression of CD16-II in COS-7 cells requires co-transfection with either CD3 zeta or Fc epsilon RI-gamma. Truncation of the cytoplasmic segment of CD16 failed to affect association with CD3 zeta. CD3 zeta and Fc epsilon RI-gamma with truncated cytoplasmic segments were also able to facilitate membrane expression of CD16-II, implicating the transmembrane segments as the interaction site between CD16-II and CD3 zeta or Fc epsilon RI-gamma. Prior studies have suggested that the acidic residue in the CD3 zeta transmembrane segment may be important for the association of CD3 zeta complexes. Although site-directed mutation of CD3 zeta-Asp36 to Glu, Leu, or Val retained the ability to permit membrane expression of CD16-II, quantitatively the wild-type CD3 zeta-Asp36 provided optimal levels of expression, consistent with conservation of this amino acid in mouse and human CD3 zeta.     

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8beta and CD4-like genes

Partial cDNA sequences of both CD8beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8beta and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8beta is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8betas, carp CD8beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp.     

Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells

The high affinity receptor for IgE (Fc epsilon RI) found on mast cells and basophils is a tetrameric complex of a single alpha subunit, a single beta subunit, and two identical gamma subunits. The genes for the three subunits of mouse Fc epsilon RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat alpha is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the beta and gamma are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the alpha subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the alpha is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc epsilon RI and other mouse proteins reveal regions of high homology between the alpha subunit and Fc gamma RIIa and between the gamma subunit and the zeta chain of the T cell receptor. Cells transfected with the alpha gene express the alpha subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human alpha gene together with the rodent gamma without apparent need for beta. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Identification of a CD20-, FcepsilonRIbeta-, and HTm4-related gene family: sixteen new MS4A family members expressed in human and mouse

CD20, high-affinity IgE receptor beta chain (FcepsilonRIbeta), and HTm4 are structurally related cell-surface proteins expressed by hematopoietic cells. In the current study, 16 novel human and mouse genes that encode new members of this nascent protein family were identified. All family members had at least four potential membrane-spanning domains, with N- and C-terminal cytoplasmic domains. This family was therefore named the membrane-spanning 4A gene family, with at least 12 subgroups (MS4A1 through MS4A12) currently representing at least 21 distinct human and mouse proteins. Each family member had unique patterns of expression among hematopoietic cells and nonlymphoid tissues. Four of the 6 human MS4A genes identified in this study mapped to chromosome 11q12-q13.1 along with CD20, FcepsilonRIbeta, and HTm4. Thus, like CD20 and FcepsilonRIbeta, the other MS4A family members are likely to be components of oligomeric cell surface complexes that serve diverse signal transduction functions.     

Mouse macrophage β subunit (CD11b) cDNA for the CR3 complement receptor/Mac-1 antigen

The cDNA for the common \_Mac-1 subunit (CD11b) of the mouse LFA-1/Mac-1/p150,95 group of leukocyte cell adhesion receptors, formally designated integrin \₂, has been cloned and sequenced. Clones were isolated from cDNA libraries made from J774 macrophage and WEHI-3B myelomonocytic tumor cells which express this subunit as a component of the macrophage activation antigen 1 (Mac-1), also known as complement receptor type 3 (CR3). This subunit is expressed as a single, abundant mRNA species approximately 2.7 kilobase (kb) in size. The 2422 base pair (bp) cDNA sequence obtained codes for a 771 amino acid protein organized with leader, extracellular, transmembrane, and cytoplamic domains of 23, 680, 23, and 46 amino acids, respectively, yielding an 82700 mature protein of 747 amino acids. The mouse \_Mac-1 subunit is highly similar to its human counterpart with an overall sequence identity of 81% and identical positioning of 5 out of 6 potential N-linked glycosylation sites, as well as 56 Cys residues that are organized in repeating motifs characteristic of integrin \ subunits. The most highly conserved regions are the transmembrane and cytoplasmic domains where only 4 out of 69 amino acids differ, indicating that the functions associated with this domain in Mac-1-mediated processes, such as iC3b-triggered phagocytosis, have been evolutionarily conserved.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M31039. Offprint requests to: P. M. Hogarth.     

A B cell-specific differentiation antigen, CD23, is a receptor for IgE (Fc epsilon R) on lymphocytes

Two independent L cell transformants expressing human lymphocyte Fc epsilon R were established by using cellular DNA from RPMI 8866 cells. The surface expression of the receptor was confirmed on the basis of the binding of a panel of anti-Fc epsilon R antibodies and its ability to bind IgE. Anti-CD23 antibodies strongly stained the transformants, indicating possible identity or antigenic relationship between Fc epsilon R and CD23. This interesting observation warrants additional clarification as to the role of CD23 and Fc epsilon R in B cell differentiation.     

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.