Molecular cloning and characterization of mouse CD97

Jörg Hamann, Chris van Zeventer, Astrid Bijl, Chris Molenaar, Kiki Tesselaar and René A. W. van Lier

Department of Immunobiology, CLB and Laboratory for Experimental and Clinical Immunology, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands

Correspondence to: J. Hamann


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The EGF-TM7 family (CD97 and EMR1) is a group of class II seven-span transmembrane receptors predominantly expressed by cells of the immune system. Recently, we have identified CD55, a regulatory molecule of the complement cascade, as a cellular ligand of human CD97 (hCD97). In this study, the molecular properties of mouse CD97 (mCD97) are described. Like hCD97, mCD97 has an extended extracellular region with several epidermal growth factor-like (EGF) domains. Due to alternative RNA splicing, isoforms with three and four EGF domains exist, designated mCD97(EGF1,2,4) and mCD97(EGF1,2,3,4) respectively. All EGF domains, except for the N-terminal one, possess a calcium-binding site. In a third isoform mCD97(EGF1,2,X,3,4), a sequence of 45 amino acids was found between the second and third EGF domain that does not correspond to any known protein module. Using newly generated mCD97 mAb, we show that analogous to the blood expression pattern of hCD97, mCD97 can be found on lymphoid and myeloid cells. Adhesion of mouse erythrocytes and splenocytes to COS cells expressing mCD97(EGF1,2,4) or mCD97(EGF1,2,3,4) could be blocked by mouse CD55 (mCD55) antibody, identifying mCD55 as a cellular ligand for mCD97. Consistent with the necessity of directly linked EGF domains for the integrity of the CD55-binding site on hCD97, no adhesion was detected to the largest mouse isoform mCD97(EGF1,2,X,3,4). Remarkably, we found that the interaction between CD97 and CD55 is phylogenetically restricted, as indicated by the selective adhesion of primate erythrocytes to hCD97 transfectants, and of mouse and rat erythrocytes to mCD97 transfectants respectively.

Keywords: adhesion, CD55, cDNA cloning, EGF-TM7 family, G protein-coupled receptors, phylogenetic restriction


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Seven-span transmembrane (7-TM) receptors form the largest superfamily of transmembrane proteins in nature with nearly 2000 members (1). A common feature of these receptors is their association with G proteins, which affect intracellular second messenger levels to regulate a variety of cellular processes (2). Based on the sequence of the membrane-spanning region, vertebrate 7-TM receptors can be divided into five classes. Class II has originally been described as a family of insect and mammalian peptide hormone receptors (3). In ancestral genes from this family, the structural variability of the extracellular region has increased during evolution by acquisition of new exons. In this way, the EGF-TM7 family evolved when exons encoding epidermal growth factor-like (EGF) domains accumulated (410). Similarly extended extracellular domains of lower and higher complexity have recently been identified in several orphan receptors (1121). In parallel with the structural diversification, the tissue specificity of receptor expression has markedly changed, suggesting that these class II 7-TM receptors are involved in different physiological processes. The members of the EGF-TM7 family, CD97 and EMR1, are predominantly expressed by cells of the immune system (10,2225).

The unusual molecular structure of human CD97 (hCD97) suggested that this molecule might be involved in cell–cell interactions (5,7). Indeed, adhesion studies demonstrated that both lymphocytes and erythrocytes specifically bind to hCD97 transfectants. Generating a ligand-specific mAb, the counterstructure was identified as human CD55 (hCD55) or decay accelerating factor (26), a glycosylphosphatidyl inositol-linked molecule involved in regulation of the complement cascade (27,28). The binding site for hCD55 is formed by the N-terminal EGF domain region, which consists of rod-like arranged classical and calcium-binding EGF domains (29). Affinity of the binding site is regulated by alternative RNA splicing. The smallest hCD97 isoform with three EGF domains binds hCD55 with significantly higher affinity than the larger isoforms possessing four and five EGF domains (29).

Despite accumulating data on the expression, structure and molecular characteristics of EGF-TM7 family members, little is known about their physiological function. Because mice allow the study of protein function in vivo, we cloned the mouse homologue of CD97 and generated mAb to the gene product. Here we report on the molecular structure and expression of mouse CD97 (mCD97) and describe its ability to bind mouse CD55 (mCD55). This study confirms and extends a recent report by Qian et al. (30).


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
cDNA cloning and sequence analysis
The hCD97 cDNA sequence (GenBank accession no. X84700) was used to search the expressed sequence tag (EST) database for homologous sequences. Seven partially overlapping mouse EST were identified. Alignment of these EST resulted in cDNA sequences of ~0.9 kb (GenBank accession nos AA118715, AA198691 and W90978) and 1.0 kb (GenBank accession nos W54498, AA080529, W20922 and AA204482).

mCD97 cDNA clones were isolated by a PCR-based strategy from a pCDM8 library of NOD mouse spleen, kindly provided by Dr David Simmons (Oxford, UK). Plasmid DNA was prepared from 12 pools of 30,000 clones each and screened by PCR (35 cycles, 30 s at 93°C, 30 s at 55°C, 45 s at 72°C) with the specific primers 5'-CTGTCCCTGATGGTGAAGGAG-3' [nucleotides 1104–1124 of the (+) strand] and 5'-TCCTGCACATGGTACTGAGCC-3' [nucleotides 1589–1609 of the (–) strand]. The primers had been designed using two overlapping EST. In three out of 12 pools a PCR product of 506 bp was amplified. Subdivision and PCR screening of the positive pools was followed by colony hybridization as described (5).

The isolated clones, with cDNA inserts of 2025 and 2335 bp, did not contain an open reading frame homologous to the full-length hCD97 cDNA. To amplify the complete 5' sequence by PCR, plasmid DNA was isolated from 12 library pools of 250,000 clones each. Using a standard T7 primer [(+) strand] and the specific primer 5'-CAGTGTCCAAGTTGG GAACGG-3' [nucleotides 822–842 of the (–) strand], a fragment of 560 bp was obtained. Although providing additional sequence information, the 5' end was not included in this sequence. Therefore, a pCDM8 library from concanavalin A (Con A)-stimulated splenocyte T cells of a BALB/c mouse (HGMP Resource Centre, Cambridge, UK) was tested. Using plasmid DNA from 12 pools of 24,000 clones each as template and the primers described, a fragment of 628 bp was amplified. This fragment contained the predicted start site of the open reading frame and 5' untranslated sequence.

Both strands of the isolated cDNA clones and PCR fragments were sequenced with the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) on an ABI 377 automated DNA sequencer (Applied Biosystems). PC Gene (IntelliGenetics, Mountain View, CA) was used for sequence analysis.

RT-PCR
RNAzol (Cinna/Biotecx, Friendswood, TX) was used to isolate total RNA from mouse splenocytes and from mouse lymph node cells, which had been stimulated for 3 h with 1 µg/ml phytohemagglutinin (PHA). First-strand cDNA was prepared from 1 µg of total RNA using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) and used for PCR amplification of the EGF domain region (35 cycles, 30 s at 95°C, 30 s at 54°C, 1 min at 72°C). PCR products, generated with a variety of flanking primers, were separated on a 1.5% agarose gel and sequenced.

Partial analysis of gene structure
Genomic clones were custom-isolated by Genome Systems (St Louis, MO) from a P1 genomic library from mouse ES-129 cells. Briefly, screening was based on a PCR strategy using the specific primers 5'-CACGTCACGAGCTGATATGTG-3' [nucleotides 1459–1479 of the (+) strand] and 5'-TCCTGCACATGGTACTGAGCC-3' [nucleotides 1589–1609 of the (–) strand]. DNA from one isolated clone (clone 16698, plate 247) was prepared according to manufacturer's recommendation. Using a series of specific primer pairs, intronic sequences were amplified by PCR (36 cycles, 30 s at 95°C, 30 s at 54–56°C, 1–3 min at 72°C). PCR products were separated on a 0.8% agarose gel to determine intron size and sequenced partially.

RNA blot analysis
A mouse multiple-tissue RNA blot (Clontech, Palo Alto, CA), loaded with 2 µg poly(A)+ RNA per lane, was hybridized with a 32P-labeled mCD97 PCR fragment and subsequently with a 32P-labeled human ß-actin cDNA fragment in ExpressHyb hybridization solution (Clontech) according to the instructions of the manufacturer.

Generation of expression constructs
To generate expression constructs encoding mCD97 isoforms, the N-terminal part of mCD97 was amplified by RT-PCR with the specific primers 5'-CCCAGACGCTGTCCGTTCCGTG-3' [nucleotides 1–22 of the (+) strand] and 5'-CAGTGTCCAAGTTGGGAACGG-3' [nucleotides 822–842 of the (–) strand]. The reaction was performed as described above using cDNA from lymph node cells as template and High-Fidelity polymerase (Boehringer Mannheim, Mannheim, Germany). After cloning the PCR product into pGEM-T Easy (Promega, Madison, WI), clones encoding three different isoforms were isolated. Expression constructs were obtained by replacing the N-terminal part of the above described cDNA of 2335 bp, which before had been recloned into the mammalian expression vector pcDNA3.1/Zeo(+) (Invitrogen, Leek, The Netherlands), with the PCR-amplified sequences. Accuracy of the constructs was verified by sequence analysis.

An expression construct encoding a chimeric receptor in which the first EGF domain of mCD97 has been replaced by the first EGF domain of hCD97 was generated by overlap extension-PCR as described (29) using High-Fidelity polymerase (Boehringer Mannheim). As a first step, two overlapping PCR fragments were generated in separate PCR reactions. The first EGF domain of hCD97 was amplified from hCD97 cDNA (5) using a standard T7 primer [(+) strand] and the specific primer 5'-GGGGCTGCCTCTGAACACTGCTGTAGTTGC-3' [nucleotides 240–260 of the (–) strand; the sequence overlapping with the second EGF domain of mCD97 is underlined]. EGF domain 2 and part of EGF domain 4 of mCD97 were amplified using the specific primers 5'- CTTGTGACGACATCAACGAGTGTTTACTAC-3' [nucleotides 238–258 of the (+) strand; the sequence overlapping with the first EGF domain of hCD97 is underlined] and 5'- CGGTGGAATTGTGACATTGATGC-3' [nucleotides 698–720 of the (–) strand]. The PCR fragments were purified and mixed together to serve as template for the second step of the overlap extension-PCR, performed with a standard T7 primer and the specific primer 5'-CGGTGGAATTGTGACATTGATGC-3'. The chimeric construct mCD97(EGF1h,2,4) was obtained by replacing the N-terminal part of the above described construct mCD97(EGF1,2,4) with the PCR amplicon. Accuracy of the construct was verified by sequence analysis.

Generation of mAb
Using Lipofectamine Plus reagent (Life Technologies), the Armenian hamster fibroblast line ARHO12 (31), kindly provided by Dr Jannie Borst (Amsterdam, The Netherlands), was transfected with pcDNA3.1/Zeo(+) containing the chimeric expression construct mCD97(EGF1h,2,4). Cells were selected with Zeocin (Invitrogen) at 500 mg/ml in culture medium. Resulting stably transfected clones were tested for mCD97(EGF1h,2,4) expression by flow cytometry with the mAb CLB-CD97/1 (26) that binds to the first EGF domain of hCD97 (29).

An Armenian hamster (Cricetulus migratorius; Cytogen, West Roxbury, MA) was injected i.p. with an ARHO12 clone stably expressing mCD97(EGF1h,2,4). Four injections with 107 irradiated cells (50 Gray) in PBS were given at weekly intervals. Three weeks after the fourth injection, the hamster was boosted i.p. with 107 cells. Three days later, hamster spleen cells were fused with mouse myeloma SP2/0 cells by standard hybridoma technology. Binding of hybridoma supernatants to the ARHO12 clone stably expressing mCD97(EGF1h,2,4) was tested by flow cytometry. Flow cytometry of COS cells expressing either hCD97 or mCD97 was used to identify hybridomas that recognize the mouse or human part of the chimera respectively. Selected hybridomas were subcloned until they were monoclonal and stable. The hybridoma 1A2 was grown in large amounts and Ig was purified using Protein A bound to Sepharose CL-4B (Sigma, St Louis, MO). Purified antibody was biotinylated using ImmunoPure NHS-LC-biotin (Pierce, Rockford, IL).

Flow cytometry
Flow cytometry was performed by standard procedures on a FACScan (Becton Dickinson, Mountain View, CA). Next to the mCD97 mAb 1A2 and the hCD97 mAb CLB-CD97/1 (26), the following FITC-conjugated anti-mouse mAb were used: 145-2C11 (CD3), 1D3 (CD19), DX5 (Pan-NK) and M1/70 (CD11b) (PharMingen, San Diego, CA). TER-119 (Ly-76) (PharMingen) was used as phycoerythrin (PE)-conjugated mAb. Prior to incubation with first-step mAb, Fc receptors on mouse cells were blocked with the mAb 2.4G2 (CD16/CD32; PharMingen). PE-conjugated streptavidin (Molecular Probes, Leiden, The Netherlands), goat anti-mouse Ig (Immunotech, Marseille, France) or goat anti-hamster Ig (Southern Biotechnology, Birmingham, AL) or Red670-conjugated streptavidin (Life Technologies) were used as second step reagents.

Expression of mCD97 in COS cells and adhesion studies
Using Lipofectamine Plus reagent (Life Technologies), COS cells were transfected with the expression constructs encoding mCD97 isoforms or hCD97(EGF1,2,5). Mock transfection was done without DNA. At day 1, cells were replated into 12-well cell culture plates. Expression of mCD97 and hCD97 was detected by flow cytometry with the mAb 1A2 and CLB-CD97/1 respectively. Adhesion studies were performed at day 3 by overlaying COS cells with 50x106 erythrocytes or 5x106 splenocytes for 30 min at 20°C as described (26). Non-adhering cells were removed by gentle washing with PBS prior to examination by microscopy and photography. Specificity of the interaction was tested by performing adhesion in the presence of mAb to mCD97 (1A2), hCD97 (CLB-CD97/1), hCD55 (CLB-CD97L/1) (26) or two independently raised polyclonal antibodies to mCD55 (final dilution 1:200), kindly provided by Dr Hidechika Okada and Dr Noriko Okada (Nagoya, Japan) (32) and Dr Yoshihiro Fukuoka (Cleveland, OH). Non-human primate erythrocytes were received from the Biomedical Primate Research Centre (Rijswijk, The Netherlands).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
cDNA cloning and sequence analysis of mCD97
A search of the EST database for homology with hCD97 identified seven mouse EST. Alignment of these EST revealed mCD97 cDNA sequences of together ~1.9 kb. Using EST-based primer pairs, two incomplete cDNAs, 2025 and 2335 bp in length, were isolated from a mouse spleen cDNA library. The 5' end of the mCD97 cDNA was PCR amplified from a cDNA library derived from Con A-stimulated T cells. Sequencing of the PCR products provided evidence for alternative RNA splicing. Therefore, RT-PCR was performed using RNA from non-stimulated splenocytes and PHA-stimulated lymph node cells. From both sources, three alternatively spliced mCD97 transcripts were amplified.

The nucleotide sequence and the deduced amino acid sequence of the mCD97 cDNA are shown in Fig. 1Go. A hydrophobic signal sequence with a potential cleavage site behind Ala21 suggests the existence of mature polypeptide isoforms of 797, 752 and 703 amino acids respectively, with a predicted molecular mass of 88, 83 and 78 kDa. Hydrophilicity analysis of the polypeptide sequence indicated that mCD97 has an extended extracellular region of 509, 464 or 415 amino acids respectively, followed by a transmembrane region of 243 amino acids, containing seven hydrophobic segments, and a cytoplasmic region of 45 amino acids.



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Fig. 1. Nucleotide and deduced amino acid sequence of the largest mCD97 isoform. The signal sequence and the seven hydrophobic transmembrane segments are underlined. The polyadenylation signal is underlined twice and potential N-glycosylation sites are boxed. EGF domains are shaded. Borders of exons 1–8 as determined by partial structural analysis of the CD97 gene (see Results and Fig. 2Go) are indicated. Alternative RNA splicing results in smaller isoforms. The mCD97 cDNA sequence has been submitted to EMBL database under accession no. Y18365.

 
Further sequence analysis revealed the presence of several EGF domains at the N-terminus of the extracellular region. As in hCD97, all EGF domains, except for the most N-terminal one, contain the consensus sequence for a calcium-binding site (33,34). The number of EGF domains appeared to be three in the smallest isoform and four in the middle isoform. In contrast to hCD97, in the largest isoform, in place of an additional EGF domain, a sequence of 45 amino acids was found between the second and third EGF domain that did not show homology with any known protein module (35). In analogy to hCD97, we designated the different isoforms mCD97(EGF1,2,4), mCD97(EGF1,2,3,4) and mCD97(EGF1,2,X,3,4). The schematic structure of mCD97 isoforms is presented in Fig. 2Go(A).



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Fig. 2. (A) Schematic structure of mCD97 isoforms possessing three EGF domains, four EGF domains or four EGF domains spaced by a sequence of 45 amino acids. (B) Genomic organization of the 5' structural regions of mCD97 and hCD97. The relation between exons and the molecular structure of CD97 are indicated: S, signal peptide; G, EGF domain; X, sequence of 45 amino acids; E, extracellular region. Exons that have been demonstrated to undergo alternative splicing are hatched. The structure of the hCD97 gene has been adapted from (7) by addition of the later identified exons G3 and G4 (8).

 
Comparison of the structure of mCD97 and hCD97
To understand the differences between mCD97 and hCD97 isoforms, we investigated the structure of the corresponding region of the mouse gene, which is located on central mouse chromosome 8, tightly linked to the gene encoding transcription factor Rfx 1 (36). A custom-isolated P1 clone containing the mCD97 gene was used to PCR amplify all introns present in the EGF domain region. The genomic organization of this region is delineated in Fig. 2Go(B). Sequence analysis showed that, like in hCD97 (7), all EGF domains are encoded by symmetrical class 1–1 exons. When sequencing intron 4, no homologue of human EGF domain 3 was found. The sequence of 45 amino acids, which was found instead in the largest mouse isoform, is encoded by a single exon ~0.5 kb upstream of the respective position of human exon 5.

An alignment of the amino acid sequences of mCD97 and hCD97 is shown in Fig. 3Go. The polypeptides exhibit an overall amino acid sequence identity of 60% (70% similarity). The identity with EMR1 (4) and its mouse homologue F4/80 (6,9) is 30% for both proteins. Directly upstream of the first membrane-spanning segment, four cysteines are present. This cysteine box is shared by the EGF-TM7 family (10) and related orphan receptors (1121), but absent from the secretin receptor family where a different cysteine box is conserved (3).



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Fig. 3. Amino acid sequence alignment of mCD97 and hCD97. Identical residues are joined by a dash and similar residues by a colon. The signal sequence and the seven hydrophobic transmembrane segments are underlined. EGF domains and the RGD motif in hCD97 are shaded. No RGD motif is found in mCD97. Asterisks indicate four cysteines upstream from the transmembrane region, which are conserved in the EGF-TM7 family and related orphan receptors.

 
Generation of mCD97-specific mAb
To characterize the mCD97 gene products, we set out to generate mCD97 mAb. An expression construct encoding a chimeric receptor in which the first EGF domain of mCD97 has been replaced by the first EGF domain of hCD97 (Fig. 4AGo) was generated and transfected into the Armenian hamster fibroblast line ARHO12 (31). Stably transfected clones were selected by flow cytometry with the mAb CLB-CD97/1 that binds to the first EGF domain of hCD97 (29). One clone was used to immunize an Armenian hamster. Screening of supernatants of the resulting hybridomas for specific binding to ARHO12 cells expressing mCD97(EGF1h,2,4) identified six positive clones (Fig. 4BGo). To evaluate whether hybridoma supernatants recognize mCD97 or the first EGF domain of hCD97, binding studies were performed with COS cells expressing either mCD97 or hCD97 respectively. Two hybridomas (1A2 and 1B1) were found to be specific for mCD97, whereas the other four recognized hCD97. As shown in Fig. 5Go, binding studies with 1A2 and 1B1 revealed that all three mCD97 isoforms are expressed on the surface of transfected COS cells.



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Fig. 4. (A) Schematic structure of the chimeric mCD97(EGF1h,2,4) receptor used for the immunization of an Armenian hamster. (B) Flow cytometric analysis of hybridoma supernatants. Untransfected ARHO12 cells and ARHO12 cells stably expressing mCD97(EGF1h,2,4) were stained with the supernatant of hybridomas 1A2 (solid line), 1B1 (dashed line) and an irrelevant control mAb (dotted line), using goat anti-hamster Ig–PE as second-step reagent. From six positive hybridomas, two (1A2 and 1B1) were found to be specific for mCD97 (see Results and Fig. 5Go), whereas the other four recognize the first EGF domain of hCD97.

 


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Fig. 5. Expression of mCD97 isoforms on COS cells transfectants. Flow cytometry analysis of COS cells expressing either hCD97(EGF1,2,5), mCD97(EGF1,2,4), mCD97(EGF1,2,3,4) or mCD97(EGF1,2,X,3,4) was performed with the mAb 1A2 (solid line), 1B1 (dashed line) and an irrelevant control mAb (dotted line). Goat anti-hamster Ig–PE was used as second step reagent. Both, 1A2 and 1B1 did not stain hCD97.

 
Expression of mCD97
In human tissues, CD97 is expressed on macrophages, dendritic cells and activated lymphocytes (L. Jaspars and J. Hamann, unpublished data). The analysis of a multiple-tissue RNA blot revealed the presence of mCD97 mRNA in various tissues (Fig. 6Go). In heart, brain, spleen, lung, liver, muscle, kidney and testis a diffuse signal, representing one or more splice variants of the mCD97 transcript, was detected. Consistent with the length of the cDNA, the transcript is ~3 kb in length. The strongest hybridization signal was found in the lung. The minor bands of larger size seen in this tissue are likely incompletely spliced pre-mRNAs.



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Fig. 6. Tissue distribution of mCD97 transcripts. A multiple-tissue RNA blot, loaded with 2 µg poly(A)+ RNA per lane, was hybridized with a 32P-labeled mCD97 PCR fragment. The position of RNA size markers in kb is shown on the right. The lower panel shows hybridization of the same blot with ß-actin probe as a control.

 
Next, we used the mAb 1A2 to document distribution of mCD97 on cells of the hematopoetic system. Representative profiles of two-parameter flow cytometry analysis are shown in Fig. 7Go. In the spleen, mCD97 expression was detected on most myeloid cells and variable portions of T cells, B cells and NK cells. In the bone marrow, where substantial populations of myeloid and erythroid cells persist, nearly all myeloid cells expressed mCD97, whereas little if any expression was found on erythroid cells.



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Fig. 7. mCD97 expression on mouse cells. Two-parameter flow cytometry of spleen-derived cells (A) or bone marrow-derived cells (B) was performed with markers for T cells (CD3), B cells (CD19), NK cells (DX5), myeloid cells (CD11b) or erythroid cells (Ly-76) and 1A2–biotin (anti-mCD97). Streptavidin–PE or streptavidin–Red670 was used as second step reagent. Markers were set using irrelevant control mAb.

 
Binding of mCD97 to mCD55
To evaluate ligand binding, COS cells expressing the three mCD97 isoforms were overlaid with mouse blood cells. As depicted in Fig. 8Go, erythrocytes efficiently adhered to mCD97(EGF1,2,4) and mCD97(EGF1,2,3,4) transfectants but not to COS cells expressing mCD97(EGF1,2,X,3,4). Similar adhesion was seen in binding studies with splenocytes (not shown). Polyclonal antibodies to mCD55 completely prevented cell adhesion, identifying mCD55 as a cellular ligand of mCD97. Cell adhesion was also blocked by the mAb 1A2 and 1B1, indicating that both mAb interfere with the CD55-binding site on mCD97.



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Fig. 8. mCD55 is a cellular ligand of mCD97. Mouse erythrocytes were overlaid on mock-transfected COS cells (A) or COS cells transfected with cDNAs encoding mCD97(EGF1,2,4) (B), mCD97(EGF1,2,3,4) (C) or mCD97(EGF1,2,X,3,4) (D). In the presence of a polyclonal antibody to mCD55, no binding to mCD97(EGF1,2,4) (E) or mCD97(EGF 1,2,3,4) (F) was detectable whereas adhesion was not effected by control antibodies.

 
In view of the sequence homology between mCD97 and hCD97, we expected that CD97–CD55 interactions would occur in a species-independent fashion. However, primate erythrocytes efficiently bound to COS cells expressing hCD97, but not to mCD97 transfectants (Table 1Go). Vice versa, adhesion of mouse and rat erythrocytes was restricted to mCD97 transfectants. Whereas hamster, rabbit, sheep and cattle erythrocytes did not bind to any of the transfectants, guinea pig erythrocytes, surprisingly, bound to COS cells expressing hCD97. Specificity of erythrocyte binding was demonstrated by performing adhesion assays in the presence of antibodies to hCD97, mCD97, hCD55 or mCD55 respectively.


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Table 1. Phylogenetic restriction of the CD97–CD55 interaction
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD97 is a member of the EGF-TM7 family of class II 7-TM receptors that is characterized by an extended extracellular region with several N-terminal EGF domains (410). In this study, we describe the cloning and characterization of mCD97, thereby confirming and extending a recent report by Qian et al. (30). The existence of three mCD97 transcripts initially suggested alternative RNA splicing similar to that occurring in hCD97, where isoforms possessing either three (EGF1,2,5), four (EGF1,2,3,5) or five EGF domains (EGF1,2,3,4,5) have been characterized (8). However, sequence analysis of RT-PCR products from the EGF domain region and examination of the corresponding region of the mCD97 gene revealed that mouse isoforms do not exclusively result from alternative splicing of exons encoding EGF domains. Only four EGF domain-encoding exons were identified in the mCD97 gene, which account for isoforms with three (EGF1,2,4) and four EGF domains (EGF1,2,3,4). In the largest isoform mCD97(EGF1,2,X,3,4), a sequence of 45 amino acids interrupts the EGF domain region that does not correspond with known protein modules.

The structural differences between mCD97 and hCD97 isoforms raised the question on the ligand-binding properties of the different mCD97 isoforms for CD55. Mouse erythrocytes and splenocytes, both expressing CD55 (32), strongly adhered to mCD97(EGF1,2,4) and mCD97(EGF1,2,3,4) transfectants. The specificity of the interaction was confirmed by polyclonal antibodies to mCD55, which completely blocked cell binding. In contrast, no cell adhesion to COS cells expressing the largest isoform mCD97(EGF1,2,X,3,4) was detectable. These findings are in agreement with a recent study in which we characterized the CD55-binding site on hCD97 (29). Three tandemly arranged EGF domains, the later two with a calcium-binding site, were found to be necessary for the interaction with hCD55. The calcium-binding EGF domains of CD97 belong to the fibrillin-like class I type (37). These calcium-binding EGF domains arrange in a near-linear order, stabilized by hydrophobic interactions between conserved amino acid residues of succeeding domains. Thus, in the largest isoform, mCD97(EGF1,2,X,3,4), the interfering sequence of 45 amino acids may disturb the structural integrity of the EGF domain region resulting in inactivation of the binding site for mCD55.

From their initial description, EGF-TM7 family members have been suggested as candidate receptors for cell–cell interactions (410). Next to the existence of EGF domains, which are known to mediate protein interactions (38), this assumption was based on the presence of the integrin-recognition motif Arg–Gly–Asp (RGD) (39) in the amino acid sequence of hCD97 and mouse EMR1 (F4/80). The finding that RGD motifs are absent in human EMR1 and, as shown here, in mCD97 questions whether interactions with integrins are used by members of the EGF-TM7 family to mediate adhesion.

Remarkably, only autologous erythrocytes or erythrocytes from closely related species can bind to transfectants expressing hCD97 or mCD97 respectively. Based on a similar phylogenetic selectivity, CD55 and other regulatory proteins protect cells against autologous complement activation (27,28). It is intriguing that two independent molecular interactions of CD55 are phylogenetically restricted. Hitherto, a functional link between CD97 and the complement system has not been demonstrated. A possible explanation might be that the necessity of selective interaction with autologous complement results in a similar selectivity of CD55 for other interactions that involve regions of the molecule close to the binding site for C3 convertases. The C3 convertase-regulatory function of hCD55 has been mapped to the short consensus repeats (SCR) 2–4 (4042), whereas blocking studies with hCD55 mAb indicated that the CD97-binding site resides within SCR 1–3 (26). The exact localization of the binding site for CD97 will show whether CD97 and C3 convertases interact with the same or overlapping regions on CD55.

The phylogenetic restriction in the CD97–CD55 interaction is not absolute as indicated by the adhesion of guinea pig erythrocytes to hCD97 but not to mCD97 transfectants. This finding is in line with the observation that soluble hCD55 inhibits the lysis of sensitized erythrocytes by guinea pig complement but not that by complement from mouse, rat or rabbit (43). The amino acid identity of guinea pig CD55 (gpCD55) is 58% to hCD55 but only 50% to mCD55 (4446). A higher conservation of protein sequences between guinea pig and man compared to mouse and man has repeatedly been reported (47,48), and reflects a current controversy about the phylogenetic position of the guinea pig (49). The observed cross-reactivity between hCD55 and gpCD55 in the interaction with both C3 convertases and CD97 seems to favor a close proximity of the respective binding sites.

The existence of a CD97–CD55 interaction in the mouse, without cross-reactivity to man, strengthens our earlier hypothesis that CD55 is a cellular ligand of CD97. The biological significance of this interaction is still unknown. The availability of mAb allows us to study the expression of the predicted mCD97 isoforms during physiological and pathological processes. They provide the opportunity to investigate the molecule structure and lineage restriction of mCD97 and to compare its tissue distribution with that of mouse EMR1, which has been described in detail by Gordon and coworkers, using the mAb F4/80 (10). More importantly, mAb allow interference with CD97 function in vivo. Since molecular structure, RNA processing, expression and ligand binding of mCD97 are largely similar to hCD97 the mouse can be used as a model system to investigate the physiological function of this molecule. Such studies might contribute to understanding the role of other recently discovered class II 7-TM receptors.


    Acknowledgments
 
This article is dedicated to the memory of Professor Michael Strauss. We thank Dr David Simmons for the NOD mouse spleen cDNA library, Dr Jannie Borst for the ARHO12 cell line, and Drs Hidechika Okada, Noriko Okada and Yoshihiro Fukuoka for polyclonal antibodies to mCD55. We are grateful to Drs Lucien Aarden, Peer Bork, Endre Kiss-Toth, Heleen Lie-Venema and Marilyn Moore for their comments and suggestions. This work was supported by grant no. 901-07-217 from the Netherlands Organization for Scientific Research (NWO). J. H. is a fellow of the Royal Netherlands Academy of Arts and Sciences.


    Abbreviations
 
7-TM seven-span transmembrane
Con A concanavalin A
EGF epidermal growth factor
EST expressed sequence tag
hCD55 human CD55
hCD97 human CD97
gpCD55 guinea pig CD55
mCD55 mouse CD55
mCD97 mouse CD97
PE phycoerythrin
PHA phytohemagglutinin
SCR short consensus repeat

    Notes
 
Transmitting editor: J. Borst

Received 13 September 1999, accepted 30 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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