Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils

The epidermal growth factor (EGF)-TM7 subgroup of G-protein-coupled receptors is composed predominantly of leukocyte-restricted glycoproteins defined by their unique hybrid structure, in which extracellular EGF-like domains are coupled to a seven-span transmembrane moiety via a mucin-like stalk. The EGF-TM7 group comprises mouse F4/80, human EGF module-containing mucin-like hormone receptor (EMR) 1, human EMR2, and human and mouse CD97, the genes for which map to human chromosome 19p13 and the syntenic regions of the mouse genome. In this study we describe the cloning and characterization of EMR3, a novel human EGF-TM7 molecule, and show the existence of its cellular ligand. The EMR3 gene maps closely to the existing members of the EGF-TM7 family on human chromosome 19p13.1 and, in common with other EGF-TM7 genes, is capable of generating different protein isoforms through alternative splicing. Two alternative splice forms have been isolated: one encoding a 652-amino acid cell surface protein consisting of two EGF-like domains, a mucin stalk, and a putative G-protein-coupled receptor domain and the other encoding a truncated soluble form containing only two EGF-like domains. As with other members of the EGF-TM7 family, EMR3 mRNA displays a predominantly leukocyte-restricted expression pattern, with highest levels in neutrophils, monocytes, and macrophages. Through the use of soluble EMR3 multivalent probes we have shown the presence of a ligand at the surface of monocyte-derived macrophages and activated human neutrophils. These interactions suggest a potential role for EMR3 in myeloid-myeloid interactions during immune and inflammatory responses.     

The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense

The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors predominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. Together, these findings indicate an essential role for CD97 in the migration of neutrophils.     

EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages,binds to a putative cellular ligand on B lymphoma cell line A20

A novel member of the EGF-TM7 family, mEMR4, was identified and characterized. The full-length mouse EMR4 cDNA encodes a predicted 689-amino acid protein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localized in the distal region of mouse chromosome 17 in close proximity to another EGF-TM7 gene, F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in Biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-alpha-treated resident peritoneal macrophages, whereas interleukin-4 and -10 dramatically reduce the expression. mEMR4 was found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated noncovalently as a heterodimer. The proteolytic cleavage site was identified by N-terminal amino acid sequencing and located at the conserved GPCR (G protein-coupled receptor) proteolytic site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.     

MUC17, a novel membrane-tethered mucin

Membrane mucins have several functions in epithelial cells including cytoprotection, extravasation during metastases, maintenance of luminal structure, and signal transduction. In this paper we describe a large membrane mucin expressed in the normal intestine. This novel mucin, designated MUC17, contains an extended, repetitive extracellular glycosylation domain and a carboxyl terminus with two EGF-like domains, a SEA module domain, a transmembrane domain, and a cytoplasmic domain with potential serine and tyrosine phosphorylation sites. RNA blot analysis and in situ hybridization indicates that MUC17 is expressed in select pancreatic and colon cancer cell lines and in intestinal absorptive cells. Radiation hybrid mapping localized MUC17 to chromosome 7q22 where it resides in close proximity with three other membrane mucin genes, MUC3A, MUC3B, and MUC12. Thus, these membrane mucins reside together in a gene cluster, but are expressed in different tissues and are likely to have different functions as well.     

Structural and functional characterization of a novel T cell receptor co-regulatory protein complex, CD97-CD55

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.     

Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles.

MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidin-like domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluorescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergent-sensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG-E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

CD312, the human adhesion-GPCR EMR2, is differentially expressed during differentiation, maturation, and activation of myeloid cells

EMR2/CD312 is a member of the adhesion-GPCR family that contains extracellular EGF-like domains. Previously it has been shown to interact with chondroitin sulphate glycosaminoglycans in an isoform-specific manner. Although EMR2 expression has been found to be restricted to human myeloid cells, its expression profile has not yet been systemically characterized. In this report, we show that EMR2 receptor expression is up-regulated during differentiation and maturation of macrophages, and is conversely down-regulated during dendritic cell maturation. We also demonstrate that EMR2 receptor alternative splicing and glycosylation is regulated during myeloid differentiation. In monocytes and macrophages, EMR2 can be specifically up-regulated by LPS and IL-10 via an IL-10-mediated pathway. In inflamed tissues, EMR2 is detected in subpopulations of myeloid cells including macrophages and neutrophils. The results presented here further support the idea that EMR2 plays a role in the migration and adhesion of myeloid cells during cell differentiation, maturation, and activation.     

Distribution of bacteriophage phi 3T homologous deoxyribonucleic acid sequences in Bacillus subtilis 168, related bacteriophages, and other Bacillus species.

The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage phi 3T. At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to phi 3T deoxyribonucleic acid (DNA). The thymidylate synthetase gene, thyA, of B. subtilis and the sequences adjacent to it were shown to be homologous to the region in the phi 3T DNA containing the phage-encoded thymidylate synthetase gene, thyP3. SP beta, a temperate bacteriophage known to be integrated into the B. subtilis 168 chromosome, was demonstrated to be closely related to phi 3T. Other regions of the bacterial genome were also found to hybridize to the phi 3T probe. The nature and location of these sequences in the bacterial and phage chromosomes were not identified. It was shown however, that they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene. The chromosomes of other Bacillus species were also screened for the presence of phi 3T homologous sequences, and the thyP3 gene was localized in the linear genomes of phages phi 3T and rho 11 by heteroduplex mapping. It is suggested that the presence of sequences of phage origin in the B. subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs.     

Analysis of CD97 expression and manipulation: antibody treatment but not gene targeting curtails granulocyte migration

The heptahelical receptor CD97 is a defining member of the EGF-TM7 family of adhesion class receptors. In both humans and mice, CD97 isoforms are expressed with variable numbers of tandemly arranged N-terminal epidermal growth factor-like domains that facilitate interactions with distinct cellular ligands. Results from treatment of mice with mAbs in various disease models have suggested a role for CD97 in leukocyte trafficking. Here, we aimed to thoroughly characterize the expression profile of CD97, and delineate its biological function. To this end, we applied a novel polyclonal Ab, which is the first antiserum suitable for immunohistochemistry, and combined this analysis with the study of Cd97-lacZ knock-in mice. We show that similar to the situation in humans, hematopoietic, epithelial, endothelial, muscle, and fat cells expressed CD97. Despite this broad expression pattern, the Cd97(-/-) mouse that we created had no overt phenotype, except for a mild granulocytosis. Furthermore, granulocyte accumulation at sites of inflammation was normal in the absence of CD97. Interestingly, application of CD97 mAbs blocked granulocyte trafficking after thioglycollate-induced peritonitis in wild-type but not in knock-out mice. Hence, we conclude that CD97 mAbs actively induce an inhibitory effect that disturbs normal granulocyte trafficking, which is not perturbed by the absence of the molecule.     

The EGF-TM7 family: a postgenomic view

With the human and mouse genome projects now completed, the receptor repertoire of mammalian cells has finally been elucidated. The EGF-TM7 receptors are a family of class B seven-span transmembrane (TM7) receptors predominantly expressed by cells of the immune system. Within the large TM7 superfamily, the molecular structure and ligand-binding properties of EGF-TM7 receptors are unique. Derived from the processing of a single polypeptide, they are expressed at the cell surface as heterodimers consisting of a large extracellular region associated with a TM7 moiety. Through a variable number of N-terminal epidermal growth factor (EGF)-like domains, EGF-TM7 receptors interact with cellular ligands such as CD55 and chondroitin sulfate. Recent in vivo studies demonstrate a role of the EGF-TM7 receptor CD97 in leukocyte migration. The different number of EGF-TM7 genes in man compared with mice, the chimeric nature of EMR2 and the inactivation of human EMR4 point toward a still-evolving receptor family. Here we discuss the currently available information on this intriguing receptor family.     

Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4.

We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124-126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of approximately 52 kb. The first intron is approximately 16 kb in length and is followed by an unusually large exon (approximately 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract.     

Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: dissection of the CD97-CD55 complex

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events. Unlike the immunoglobulin (Ig) superfamily, very little is known about the general properties of intermolecular interactions encoded by these common modules, and in particular, how specificity of binding is achieved. We have dissected the binding of CD97 (a member of the EGF-TM7 family) to the complement regulator CD55, two cell surface modular proteins that contain EGF and SCR domains, respectively. We demonstrate that the interaction is mediated solely by these domains and is characterized by a low affinity (86 microm) and rapid off-rate (at least 0.6 s(-1)). The interaction is Ca(2+) -dependent but is unaffected by glycosylation of the EGF domains. Using biotinylated multimerized peptides in cell binding assays and surface plasmon resonance, we show that a CD97-related EGF-TM7 molecule (termed EMR2), differing by only three amino acids within the EGF domains, binds CD55 with a K(D) at least an order of magnitude weaker than that of CD97. These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCR-EGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface.     

The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor

Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.     

The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.

Epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues and implicated in epithelial cell protection, adhesion modulation and signaling. The gene encoding human MUC3 (hMUC3), localised to chromosome 7q22, is most highly expressed in the small intestine. It has previously been reported to be a non-transmembrane mucin with minimal homology to its suggested orthologues from rat (rMuc3) and mouse (mMuc3). RT-PCR was performed to investigate the carboxyl terminus of the published sequence of hMUC3 from normal colon and small intestine tissues and also from a series of 10 colorectal cancer cell lines. Two distinct PCR products were identified. In contrast to the previously published hMUC3 sequence, which terminates shortly after a single cysteine-rich EGF-like domain, conceptual protein translation of the dominant and largest PCR product identified two extracellular cysteine-rich EGF-like domains separated by an N-glycosylation-rich domain and a potential coiled-coil region, followed by a putative transmembrane region and a 75 amino acid cytoplasmic tail. The smaller of the two PCR products was found to be an alternative splice variant of MUC3 including the first EGF-like domain but lacking part of the second EGF-like domain and the transmembrane region. Nine out of 10 colorectal cancer cell lines were found to express MUC3. Interestingly, one of the cell lines, LoVo, expressed predominantly the alternative splice form lacking a transmembrane domain. Structural homology of the new protein sequence of hMUC3 with rMuc3 and mMuc3 indicates it is closely related to the rodent proteins and is likely to be involved in ligand-binding and intracellular signaling. The new finding that MUC3 encodes a transmembrane molecule presents a new paradigm for the structure of this mucin and the manner in which it may function.     

CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.     

Characterization of a mouse Scube3 reporter line

The SCUBE gene family encode secreted, extracellular proteins that share a distinct domain organization of at least five recognizable motifs, including an amino-terminal signal peptide sequence, multiple EGF-like domains, a large spacer region containing multiple N-linked glycosylation sites, three repeated stretches of six-cysteine residues and a carboxy-terminal CUB domain. We describe a Scube3(tm1Dge/H) targeted allele, which replaces the entire coding region for Exons 2 and 3 with a neomycin-lacZ selectable marker cassette predicted to delete the first two EGF-like domains of the transcribed protein. Scube3(+/tm1Dge/H) embryos demonstrate strong β-galactosidase activity in the early facial epithelium, including the branchial arches and facial processes, the otic vesicle, limb buds, and neural tube. In addition, strong reporter activity was identified in the epithelial compartments of developing teeth and hair follicles. However, analysis of the Scube3(tm1Dge/H) allele revealed that it encodes a truncated protein, which contains part of the spacer region and CUB domain. It is likely that this protein retains functionality because our analysis reveals that Scube3(tm1Dge/H; tm1Dge/H) mice are phenotypically normal. Whilst acting as a useful reporter, these mice do not provide any insight into the potential role of Scube3 during embryonic development.