We report the cloning of a novel human type I cell surface Ag mainly expressed by macrophages. The primary structure was established by molecular cloning, which yielded a 4579-bp cDNA sequence encoding a polypeptide chain of 1453 amino acid residues with 16 potential N-glycosylation sites. We designated this molecule M160. The domain organization features 12 scavenger receptor cysteine-rich domains followed by a transmembrane region and a cytoplasmic domain that occurs in two forms, a predominant form (M160-α) of 71 residues and an alternatively spliced form (M160-β) of 39 residues. M160-α contains three possible phosphorylation sites, which are lost in the alternatively spliced form. RT-PCR analyses showed M160 to be expressed by alveolar macrophages and by the monocyte cell lines HL60, U937, and THP1, but not by Jurkat or Raji cells. Stimulation of U937 cells with phorbol ester resulted in an increased expression of M160 from day 5 onward. RT-PCR analysis of 19 different human tissues showed signals for M160-α of varying intensity in all tissues, whereas M160-β was confined to the spleen. We conclude that M160 is a new member of the scavenger receptor cysteine-rich superfamily expressed by the monocyte/macrophage cell lineage.

In the process of cloning gp-340, a putative receptor for lung surfactant protein D (SP-D)3 and a member of the scavenger receptor cysteine-rich (SRCR) superfamily (1, 2), we isolated another gene for a member of this family of proteins. The sequence and the domain organization of this gene showed highest homology with CD163 (M130), a type 1 membrane protein exclusively expressed by cells of the monocyte/macrophage cell lineage (3).

The scavenger receptor superfamily was recognized during the analysis of the structure of the type I macrophage scavenger receptor (4, 5), a trimeric integral membrane protein found mainly on macrophages and involved in adhesion, host defense, and the pathogenesis of atherosclerosis (6, 7). As in other protein families such as the Ig family, the C-type lectin family and the epidermal growth factor family, the SRCR domains are found in secreted (8, 9, 10) and membrane-anchored proteins (11, 12) as single domains (4, 5), as part of multidomain mosaic proteins (1, 13), or as tandem repeats (3, 14, 15). Many SRCR domains are found on cells associated with the immune system (16), but SRCR domains are also synthesized by other cells, such as hepatocytes (9) and epithelial cells of the gastrointestinal tract (17, 18). Functionally, the SRCR domains are thought to mediate protein-protein interactions and ligand binding (6, 7).

The domain that defines the SRCR family of proteins consists of 100–110 amino acid residues (16). Molecules with SRCR domains are divided into two groups based on the localization and number of cysteine residues. Members of group A all have six cysteine residues and lack cysteine residues at positions 1 and 4. Members of group B have either eight or six cysteine residues, but the cysteine residues at positions 1 and 4 are always present (16). Another difference between group A and B SRCR proteins is seen at the genomic level, where group A SRCR domains are encoded by two different exons, whereas group B SRCR domains are encoded by a single exon. The structure of the group A SRCR domain from Mac-2 binding protein has recently been determined by x-ray crystallography (19). This revealed a compact fold consisting of a curved 6-stranded β-sheet cradling an α helix, and structure-based sequence alignment showed that this structure could be used as a template for the entire SRCR superfamily (19). Group B SRCR domains have so far only been found in vertebrates, whereas group A SRCR domains have been found in phyla ranging from vertebrates to the most primitive metazoa.

On the basis of their structure, sequence homologies, and domain organization, members of the group B SRCR family can be divided into three subgroups. The most extensively studied subgroup comprises CD5 (11), CD6 (12), and SPα (10). CD5 and CD6 have an extracellular region composed of three SRCR domains, a transmembrane domain, and a cytoplasmic region. SPα has three SRCR domains, showing high homology with CD5 and CD6, but lacks the transmembrane and cytoplasmic domains (10). CD5 and CD6 are predominantly expressed by thymocytes, mature T cells, and a subset of B cells involved in the regulation of T cell activation. Tyrosine residues in the cytoplasmic region of CD5 are transiently phosphorylated after activation of the T cells (20, 21, 22), and T cells in CD5-deficient mice are hyperresponsive to stimulation (23, 24), indicating that CD5 may act as a negative regulator of TCR-mediated signal transduction (23). Although CD5 and CD6 are closely related structurally, their ligands show no homology. CD5 has been shown to bind CD72 (25), and other candidate CD5 ligands have been described that are all compatible with a role in T cell/B cell interaction (26, 27, 28). CD6 binds to the activated leukocyte cell adhesion molecule ALCAM (29), the binding being mediated by the membrane-proximal SRCR domain of CD6 and the amino-terminal Ig domain of ALCAM (30). Transcripts encoding Spα are found in human bone marrow, spleen, lymph nodes, thymus, and fetal liver, but not in nonlymphoid tissues. Spα binds specifically to peripheral monocytes but not to T or B cells (10).

The second subgroup within the SRCR group B molecules includes gp-340 (1), DMBT1 (31), and their murine and rabbit counterparts CRP-Ductin (18), Ebnerin (17), and Hensin (32). Bovine gall-bladder mucin (33) and Pema SREG from the sea lamprey Petromyzon marinus (34) are also related to this subgroup. These molecules are synthesized by epithelial cells in the gastrointestinal tract and in ducts of exocrine glands. Gp-340 binds specifically to SP-D and is also synthesized by macrophages. Hensin is found in the proximal tubules of the kidney and has been suggested to induce the reversal of polarity of the intercalated cell, i.e., to shift the anion exchanger from the apical to the basolateral cell membrane (17).

The third subgroup of SRCR group B molecules is the WC1 family, comprising WC1 and CD163 (3). The WC1 gene is expressed by T cells of the cow (14), sheep (35), and pig (36). Bovine WC1 is composed of 11 SRCR domains, a transmembrane region, and a cytoplasmic domain (14). So far, four different forms of WC1 have been identified in pigs, representing both alternative spliced forms of the same gene and products of different genes (36). The largest of the pig WC1 genes encodes five SRCR domains. CD163 was originally defined by five different mAbs as a human monocyte/macrophage-associated Ag found on all circulating monocytes and on most tissue macrophages (37). Cloning of CD163 revealed that this protein is a membrane protein composed of nine type B SRCR domains followed by a transmembrane region and a cytoplasmic domain. No function has so far been described for the WC1 family of proteins.

We here report the primary structure of a new member of the WC1 family, which we have named M160 and characterized as a molecule primarily related to the monocyte/macrophage cell lineage.

Media and supplements were purchased from Life Technologies (Grand Island, NY). HL60, U937, Jurkat, and Raji cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 2 mM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. THP1 cells were grown in the same medium containing 0.05 mM 2-ME (M 7522; Sigma, St. Louis, MO). Cell viability was assessed by erythrosin B exclusion.

U937 cells in the logarithmic growth phase were incubated at 2 × 105 cells/ml for up to 9 days in the presence of 100 ng/ml PMA (Sigma P8139). The cells were removed from the culture flask with a rubber policeman and washed in PBS containing 0.1% human serum albumin (HSA). Peripheral blood was lysed by the ammonium chloride method, and peripheral blood leukocytes were washed three times in PBS. Bronchoalveolar washings were provided by Odense University Hospital, and cells from these were isolated by centrifugation and washed three times in PBS.

A previously isolated cDNA clone (gp-clone 7) spanning residues 3691–5499 of gp-340 cDNA was labeled with 32P and used to screen a human oligo(dT)-primed lung cDNA library (Stratagene no. 93210). Gp-clone 7 covers four and one-half SRCR domains, four SRCR-interspersed domains (SIDs), and one-half CUB domain (a domain first found in complement component C1r/C1s, Uegf, a sea urchin epidermal growth factor-containing protein, and Bmp1, bone morphogenetic protein 1). Approximately 2.4 × 105 individual clones were screened. Positive clones were replated, rescreened, subcloned, and sequenced.

Sequence analysis revealed that one clone (Z-2) included an SRCR sequence that was different from the SRCR sequences found in gp-340. Part of the sequence of clone Z-2 was identical with an expressed sequence taq clone (za25d06). This clone was obtained and sequenced. Clone Z-2 was used as probe to screen >5 × 106 individual clones from three different libraries. The libraries were a human lung 5′-stretch plus cDNA library (HL3004b; Clontech Laboratories, Palo Alto, CA), a human lung cDNA library in the Uni ZAP-XR vector (937210; Stratagene, La Jolla, CA), and a human alveolar macrophage cDNA library in the Lambda Zap Express vector (Stratagene), prepared as follows. Briefly, RNA was purified from bronchoalveolar lavage cells from 14 different individuals by means of the RNeasy Midi kit (Qiagen, Chatsworth, CA). Eighty micrograms of total RNA was obtained and used for the purification of messenger RNA using the Oligotex mRNA Mini kit (Qiagen). The yield of mRNA was 1.2 μg. This mRNA was then used to synthesize a cDNA library in strict accordance with the manufacturer’s protocols (Stratagene Lambda Zap Express cDNA synthesis kit and Lambda Zap Express Gigapack III gold cloning kit). No positive clones were obtained on screening these three cDNA libraries; therefore, another strategy was adopted.

By aligning the individual SRCR domains of M130 and the three known SRCR domains of Clone Z-2, two degenerated forward primers were designed (Deg forw SRCR1 9-26 and Deg forw SRCR1 125-148; Table I). These two degenerated primers were used as sense primers in all of the subsequent RT-PCR procedures. For each round of RT-PCR, a set of three M160-specific antisense primers was designed, one for first-strand cDNA synthesis (SP1), another for nested PCR (SP2), and a third for sequencing (SP3). Total RNA (2 μg) isolated from human spleen (Clontech) was used for first-strand synthesis using the antisense primers SP1 (a, b, c, d, and e). First-strand cDNA synthesis was performed with avian myeloblastosis virus reverse transcriptase supplied with the 5′/3′-rapid amplification of cDNA end (RACE) kit (Boehringer Mannheim, Indianapolis, IN) at 55°C according to the manufacturer’s protocol. The resulting single-stranded cDNA was purified with the High Pure PCR Product Purification kit (Boehringer Mannheim) and subjected to nested PCR. The reaction was performed in a final volume of 30 μl 50 mM Tris-HCl, pH 9.2, containing 16 mM (NH4)2SO4 and 2 mM MgCl2, with 3 μl cDNA, 25 pmol of each of the primers, 200 μM of each dNTP (Amersham Pharmacia Biotech, Piscataway, NJ), and 1 U Taq DNA polymerase (Amersham Pharmacia Biotech). After an initial denaturation for 2 min at 94°C, 40 cycles of 94°C for 30 s, 53°C for 30 s, 72°C for 2 min, and 72°C for 7 min were conducted with the specific primers SP2 (a, b, c, d, and e) in combination with Deg forw SRCR1 9-26 or Deg forw SRCR1 125-148. The resulting products were isolated by preparative agarose gel electrophoresis, purified with Sephaglas BrandPrep kit (Amersham Pharmacia Biotech) and sequenced using the SP3 (a, b, c, d, and e) primers in the DNA sequence reactions. A total of five rounds of “degenerated” RT-PCR were performed.

Table I.

Primers used for RT-PCR, 5′-RACE, and long-range PCR proceduresa

Degenerated sense primers used in all RT-PCR procedures  
Deg forw SRCR1 9–26 5′-NCTNAGNCTRGTRGATGG-3′ 
Deg forw SRCR1 125–148 5′-GTGRTNTGYMRNCARCTGGGMTGT-3′ 
Specific antisense primers for 1st RT-PCR  
SP1a 5′-CAGCCTTGAGGCTCTACAGTG-3′ 
SP2a 5′-CGCAGGGGTCATCTGCTCA-3′ 
SP3a 5′-CAGGAATGGAGTCCCACTTGTGG-3′ 
Specific antisense primers for 2nd RT-PCR  
SP1b 5′-CCAGAAGATGCCCGTGTTC-3′ 
SP2b 5′-CACTGGGACCCAGAAGATG-3′ 
SP3b 5′-CTCACTGTGTGACACC-3′ 
Specific antisense primers for 3rd RT-PCR  
SP1c 5′-CCAGAGAGCCTCATTTCA-3′ 
SP2c 5′-GGGTCTGCAATCAGGGTC-3′ 
SP3c 5′-GGCAACTTGAATGTGGGTCT-3′ 
Specific antisense primers for 4th RT-PCR  
SP1d 5′-GGACTGCACATATGATGGA-3′ 
SP2d 5′-CAGCTCTCTGGGACTGCAC-3′ 
SP3d 5′-CTGGGAATGAGTCAGCTC-3′ 
Specific antisense primers for 5th RT-PCR  
SP1e 5′-GGATGACATTTTATGCCAGG-3′ 
SP2e 5′-AGCCCTGCTGTATTGC-3′ 
SP3e 5′-GTTGTTAATAGCCCTGCTG-3′ 
Specific antisense primers used for 5′-RACE  
SP1f 5′-CCTGTTATGGAAATGAGTCA-3′ 
SP2f 5′-AAAATTTGGCTTGATGATGTTTCCTG-3′ 
SP3f 5′-ACATGGAAAAATTTGGCTTGATG-3′ 
Primers used for long-range PCR  
5′ primer 5′-CTCAGGAAGAGATAGACC-3′ 
3′ primer 5′-TCACCAGTCTTTATCAATTTAGC-3′ 
Primers used for RT-PCR  
1st strand:  
RT-PCR actin rev 1 5′-GTAACGCAACTAAGTCATAG-3′ 
RT-PCR M160 rev 1 5′-GCTGTTCTCCTATTGTTC-3′ 
RT-PCR CD163 rev 1 5′-TTTATAAATTCAGCAGCAGTC-3′ 
RT-PCR TNF-α rev 1 5′-GAAGGTTGGATGTTCGT-3′ 
PCR:  
RT-PCR M160 forw 5′-TGAATGCCTCCTCAGGT-3′ 
RT-PCR M160 rev2 5′-GGACAAGTTTTCCATAGGG-3′ 
RT-PCR actin forw 5′-ATGCAGAAGGAGATCACTGC-3′ 
RT-PCR actin rev2 5′-AGTCCGCCTAGAAGCATTTG-3′ 
RT-PCR CD163 forw 5′-ACAACAGGTCGCTCATCC-3′ 
RT-PCR CD163 rev2 5′-GCAGTCTTAGGAATCCTAG-3′ 
RT-PCR TNF-α forw 5′-GAGTGACAAGCCTGTAG-3′ 
RT-PCR TNF-α rev2 5′-CAATGATCCCAAAGTAGACC-3′ 
Degenerated sense primers used in all RT-PCR procedures  
Deg forw SRCR1 9–26 5′-NCTNAGNCTRGTRGATGG-3′ 
Deg forw SRCR1 125–148 5′-GTGRTNTGYMRNCARCTGGGMTGT-3′ 
Specific antisense primers for 1st RT-PCR  
SP1a 5′-CAGCCTTGAGGCTCTACAGTG-3′ 
SP2a 5′-CGCAGGGGTCATCTGCTCA-3′ 
SP3a 5′-CAGGAATGGAGTCCCACTTGTGG-3′ 
Specific antisense primers for 2nd RT-PCR  
SP1b 5′-CCAGAAGATGCCCGTGTTC-3′ 
SP2b 5′-CACTGGGACCCAGAAGATG-3′ 
SP3b 5′-CTCACTGTGTGACACC-3′ 
Specific antisense primers for 3rd RT-PCR  
SP1c 5′-CCAGAGAGCCTCATTTCA-3′ 
SP2c 5′-GGGTCTGCAATCAGGGTC-3′ 
SP3c 5′-GGCAACTTGAATGTGGGTCT-3′ 
Specific antisense primers for 4th RT-PCR  
SP1d 5′-GGACTGCACATATGATGGA-3′ 
SP2d 5′-CAGCTCTCTGGGACTGCAC-3′ 
SP3d 5′-CTGGGAATGAGTCAGCTC-3′ 
Specific antisense primers for 5th RT-PCR  
SP1e 5′-GGATGACATTTTATGCCAGG-3′ 
SP2e 5′-AGCCCTGCTGTATTGC-3′ 
SP3e 5′-GTTGTTAATAGCCCTGCTG-3′ 
Specific antisense primers used for 5′-RACE  
SP1f 5′-CCTGTTATGGAAATGAGTCA-3′ 
SP2f 5′-AAAATTTGGCTTGATGATGTTTCCTG-3′ 
SP3f 5′-ACATGGAAAAATTTGGCTTGATG-3′ 
Primers used for long-range PCR  
5′ primer 5′-CTCAGGAAGAGATAGACC-3′ 
3′ primer 5′-TCACCAGTCTTTATCAATTTAGC-3′ 
Primers used for RT-PCR  
1st strand:  
RT-PCR actin rev 1 5′-GTAACGCAACTAAGTCATAG-3′ 
RT-PCR M160 rev 1 5′-GCTGTTCTCCTATTGTTC-3′ 
RT-PCR CD163 rev 1 5′-TTTATAAATTCAGCAGCAGTC-3′ 
RT-PCR TNF-α rev 1 5′-GAAGGTTGGATGTTCGT-3′ 
PCR:  
RT-PCR M160 forw 5′-TGAATGCCTCCTCAGGT-3′ 
RT-PCR M160 rev2 5′-GGACAAGTTTTCCATAGGG-3′ 
RT-PCR actin forw 5′-ATGCAGAAGGAGATCACTGC-3′ 
RT-PCR actin rev2 5′-AGTCCGCCTAGAAGCATTTG-3′ 
RT-PCR CD163 forw 5′-ACAACAGGTCGCTCATCC-3′ 
RT-PCR CD163 rev2 5′-GCAGTCTTAGGAATCCTAG-3′ 
RT-PCR TNF-α forw 5′-GAGTGACAAGCCTGTAG-3′ 
RT-PCR TNF-α rev2 5′-CAATGATCCCAAAGTAGACC-3′ 
a

The codon usage was N = A+C+G+T; M = A+C; Y = C+T; R = A+G.

To reach the 5′ untranslated region, a last set of specific primers (SP1f, SP2f, and SP3f) were designed. The 5′-RACE reaction was performed with the 5′/3′-RACE kit (Boehringer Mannheim). First-strand synthesis was performed using 2 μg total RNA from the spleen as template and SP1f as primer. The resulting single-stranded cDNA was purified with the High Pure PCR Product Purification kit (Boehringer Mannheim) and poly(A) was added by means of the terminal transferase supplied with the kit. PCR was performed with the poly(T) primer and SP2f. The 5′-RACE products were then purified and sequenced.

From the sequences obtained from the 5′ and 3′ untranslated regions, sense (5′ primer) and antisense (3′ primer) primers were designed and used for long-range PCR to obtain full-length cDNA clones.

Poly(A)-RNA isolated from human spleen total mRNA (Clontech) was purified by means of the RNeasy Mini kit (Qiagen) and transcribed into cDNA by means of the Lambda ZAP Express Synthesis kit (Stratagene). Long-range PCR was conducted in a final volume of 30 μl by means of the Takara LA PCR kit (Biotechline, Copenhagen, Denmark). cDNA (0.5 μl) corresponding to 1 μg of cDNA was used as template, and 25 pmol of each primer (5′ primer, 3′ primer) were added. After denaturation for 2 min at 95°C, 30 cycles of 95°C for 30 s, 55°C for 45 s, and 68°C for 10 min were followed by a final extension step of 68°C for 15 min. The product was isolated by preparative agarose gel electrophoresis and purified with the Sephaglas BrandPrep kit (Amersham Pharmacia Biotech). Purified PCR products were ligated into the PCRIIvector and transformed into INVaF′ One Shot using the Original TA cloning kit (Invitrogen). Plasmids from four clones were purified by means of the Quantum Prep Plasmid Miniprep kit (Bio-Rad, Richmond, CA) and sequenced in both directions.

The DNA sequence reactions were performed with the Prism Ready Reaction BigDyeDeoxy Terminator sequencing kit (PE Applied Biosystems, Allerød, Denmark). Samples were subjected to electrophoresis on an ABI prism 310 Genetic Analyzer, read automatically, and recorded using ABI Prism Model Version 2.1.1. software (PE Applied Biosystems).

A tissue Northern blot was purchased from Clontech (no. PT1200-1) and hybridized in 5 ml ExpressHyb solution at 68°C according to the manufacturer’s instructions. The hybridization probe was a PCR fragment spanning base pairs 4080–4493 from the 3′ end of M160 cDNA. One hundred microliters of the PCR fragment was radiolabeled with [32P]dCTP (Amersham Pharmacia Biotech) by means of an oligolabeling kit (Amersham Pharmacia Biotech). The Northern blot was normalized with a β-actin probe supplied by the manufacturer. The blot was washed under high stringency conditions according to the protocol and used to expose x-ray film (BioMax MS; Kodak, Rochester, NY) with a double screen for 1 wk for the M160 probe and overnight without screen for the β-actin probe.

Samples of total RNA isolated from various human tissues were obtained from Clontech. Samples of total RNA from ∼2–5 × 106 cells of the cell lines HL60, U937, THP1, Raji, and Jurkat, and from alveolar macrophages, were purified by means of the RNeasy mini kit (Qiagen). RT-PCR was performed as duplex RT-PCR using β-actin as a standard. Total RNA was used for first-strand synthesis and primed by the two primers RT-PCR actin rev1 and RT-PCR M160 rev1. The reaction was conducted according to the protocol for SuperScript II (Life Technologies) in a volume of 19 μl with 1.5 μg of total RNA, 2 pmol of each primer, 4 μl of 5× first-strand buffer, 10 mM DTT, and 0.2 mM dNTP. The mixture was heated to 70°C for 10 min and then cooled to 42°C when 1 μl (200 U) SuperScript II (Life Technologies) was added. The mixture was incubated for 1 h at 42°C and finally heated to 70°C for 15 min.

Duplex PCR was performed in two rounds. A first round of 10 cycles using only the M160 specific primer RT-PCR M160 rev2 and RT-PCR M160 forw was conducted in 30 μl 50 mM Tris-HCl, pH 9.3, containing 16 mM (NH4)2SO4 and 2 mM MgCl2, with 3 μl of the first strand mixture, 25 pmol of each primer, 200 μM dNTP, and 1 U of Taq polymerase (Amersham Pharmacia Biotech). Cycling parameters were 94°C for 2 min followed by 10 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 45 s. The second round of 20 cycles was run with primers specific for both actin and M160. The PCR was conducted in 30 μl 50 mM Tris-HCl, pH 9.3, containing 16 mM (NH4)2SO4 and 2 mM MgCl2, with 5 μl of the product from the first round as template, 25 pmol of each primer, 200 μM dNTP, and 1 U of Taq polymerase (Amersham Pharmacia Biotech). After an initial denaturation for 2 min at 94°C, 20 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 45 s were performed. Ten microliters of each PCR product was run on a 2% agarose gel. To confirm the identity of the band obtained, bands of the relevant size were purified and sequenced.

Likewise, RT-PCR was performed with four different sets of primers specific for CD163, actin, TNF-α, and M160, respectively, on mRNA from U937 cells harvested on day 0 (before PMA stimulation) and on days 1, 3, 5, 7, and 9 after stimulation with PMA. RNA from the cells was purified by means of the RNeasy Mini kit (Qiagen). First-strand synthesis was performed as described for tissue RNA, but with a mixture of four different first-strand primers (RT-PCR TNF-α rev 1, RT-PCR actin rev1, RT-PCR CD163 rev1, and RT-PCR M160 rev1). PCR was performed in four different reactions with 2 μl of the first-strand reaction product as template and using 18 cycles for the actin reaction, 23 cycles for the M160 reaction, and 28 cycles for the TNF-α and CD163 reactions. The resulting products were analyzed on 2% agarose gels. The primers used were as follows: for actin, RT-PCR actin forw and RT-PCR actin rev2; for CD163, RT-PCR CD163 forw and RT-PCR CD163 rev2; for TNF-α, RT-PCR TNF-α forw and RT-PCR TNF-α rev2; and for M160, RT-PCR M160 forw and RT-PCR M160 rev2.

A novel member of the SRCR superfamily was identified in the process of cloning gp-340, a putative receptor for SP-D (1). Gp-340 is a mosaic molecule with thirteen SRCR group B domains separated by SIDs and followed by two CUB domains, which are themselves separated by a 14th SRCR group B domain and a zona pellucida domain. One cDNA clone isolated from a lung cDNA library by means of a gp-340-specific probe showed a high sequence homology with other SRCR group B domains, but the sequence was different from the SRCR sequences found in gp-340. The isolated cDNA clone of 1110 bp (clone Z-2) was sequenced in both directions and found to contain an open reading frame encoding a 370-aa polypeptide. The sequence included almost three SRCR domains and a cytoplasmic domain (Fig. 1 A). No SIDs were found between the SRCR domains, and although the repeated SRCR domains of gp-340 showed a high degree of domain-to-domain homology (88–100% identity at the protein level), the complete SRCR domains of the new polypeptide showed only 29% identity between themselves and an average identity with gp-340 SRCR domains of 32 and 56%, respectively. A database search showed a high degree of homology, but not identity, with human CD163 (M130) and bovine WC1, and the molecule was provisionally designated M160. No untranslated 3′ end or 5′ ends were found. By database screening, a human expressed sequence taq clone was identified (za25d06) that showed 100% identity with the 5′ end of clone Z-2 and that also included a 3′ end and a poly(A) tail.

FIGURE 1.

Cloning and structure of M160. A, cDNA clones and PCR used to establish the sequence of M160 are shown together with a schematic presentation of the domain structure of M160. The positions of the cDNA clones and long-range PCR products are indicated as solid lines. B, Leader sequence (signal peptide) and putative N-terminal sequence of M160 compared with the equivalent sequence of CD163. The experimentally determined N-terminal sequence of CD163 is underlined.

FIGURE 1.

Cloning and structure of M160. A, cDNA clones and PCR used to establish the sequence of M160 are shown together with a schematic presentation of the domain structure of M160. The positions of the cDNA clones and long-range PCR products are indicated as solid lines. B, Leader sequence (signal peptide) and putative N-terminal sequence of M160 compared with the equivalent sequence of CD163. The experimentally determined N-terminal sequence of CD163 is underlined.

Close modal

Clone Z-2 was used as a probe to screen both oligo(dT) and randomly primed human lung cDNA libraries, but this did not produce further cDNA clones. A strategy of using specific primers for first-strand synthesis following PCR with specific sense primers combined with degenerated antisense primers was then adopted. The degenerated primers were designed from a consensus sequence of the first three SRCR domains of M160 and the nine SRCR domains of CD163. A total of six rounds of degenerated RT-PCR were performed. The first four rounds gave products of ∼900 bp each, covering approximately three SRCR domains (Fig. 1,A). The fifth round gave a product of 350 bp covering one SRCR domain, and the sixth round gave no product. Subsequently, 5′-RACE PCR was performed on spleen RNA using specific sense primers and poly(T) primers. This reaction generated a 350-bp product. The sequence of this product showed homology with the sequence encoding the signal peptide of CD163 (Fig. 1,B). The translated sequence included a putative leader sequence of 42 aa followed by a sequence that showed high homology with the experimentally determined N-terminal sequence of CD163 (Fig. 1 B) (3).

Primers from the 5′ and 3′ untranslated ends were designed and used for long-range PCR with RNA purified from alveolar macrophages as template. A 4545-bp cDNA was obtained, subcloned, and sequenced in both directions. The open reading frame encodes a polypeptide of 1453 aa, including a possible hydrophobic signal sequence of 42 aa. The complete nucleotide sequence of M160 has been deposited at the GeneBank nucleotide sequence data base (accession no. AF264014) (Fig. 2).

FIGURE 2.

Nucleotide sequence and deduced amino acid sequence of M160. The initiation methionine is marked as +1. Nucleotides are numbered from the first nucleotide of the start codon. Potential glycosylation sites are shown in boxes. The positions of the primers used for long-range PCR are underlined. The polyadenylation sites are in bold.

FIGURE 2.

Nucleotide sequence and deduced amino acid sequence of M160. The initiation methionine is marked as +1. Nucleotides are numbered from the first nucleotide of the start codon. Potential glycosylation sites are shown in boxes. The positions of the primers used for long-range PCR are underlined. The polyadenylation sites are in bold.

Close modal

When the predicted signal peptide is omitted, the calculated molecular mass of the polypeptide chain is 154.5 kDa. The domain organization features 12 SRCR domains of 103–107 aa residues with a small interdomain segment of 31 aa residues between SRCR domains 9 and 10 (Fig. 2). All of the SRCR domains except SRCR 11 contain eight cysteine residues and belong to group B of the SRCR family. SRCR 11 is an atypical group B SRCR domain in that it lacks the Cys 2-Cys 7 bridge. A model of SRCR 11 of M160 based on the crystal structure of Mac-2 binding protein analyzed with the SwissPdbViewer suggests that the replacement of Cys 2 and Cys 7 by Gly and Ser, respectively, in SRCR 11 does not change the overall conformation of the domain (data not shown). An alignment of the SRCR domains of M160 compared with the SRCR domains of CD163 and WC1 in the cow and pig is shown in Fig. 3. SRCR 8 of CD163 and SRCR 1 in both bovine and porcine WC1 and the second SRCR domain of CD5 also lack the Cys 2-Cys 7 bridge.

FIGURE 3.

Alignment of the SRCR domains of M160 with the SRCR domains of CD163 and bovine and porcine WC1. The secondary structure elements as deduced from the Mac-2 binding protein SRCR structure are indicated below the alignment, and the disulfide bonding pattern is shown above the alignment. Potential N-linked glycosylation sites are shown in bold boxes, and the atypical Cys 6′ residues are shown in bold circles.

FIGURE 3.

Alignment of the SRCR domains of M160 with the SRCR domains of CD163 and bovine and porcine WC1. The secondary structure elements as deduced from the Mac-2 binding protein SRCR structure are indicated below the alignment, and the disulfide bonding pattern is shown above the alignment. Potential N-linked glycosylation sites are shown in bold boxes, and the atypical Cys 6′ residues are shown in bold circles.

Close modal

Interestingly, Cys 2 is also missing from SRCR 2 and SRCR 7 of bovine WC1 and SRCR 2 of porcine WC1 (Fig. 3). In these domains an extra Cys (Cys 6′) residue is found between Cys 6 and Cys 7. In all three domains Cys 6′ is flanked by proline residues and the 3′ proline is followed by two Gly residues. This sequence allows for a turn, and it is possible that Cys 6′-Cys 7 form a bridge stabilizing loop E-F.

The sequence contains 17 potential N-glycosylation sites, the first of which is located at the predicted position −1 and is therefore probably not used. A conserved glycosylation site is found in loop E-F between the Cys 5-Cys 6 disulfide bridge in six of the M160 SRCR domains, and the same glycosylation site is found in CD163 in five of nine SRCR domains. This glycosylation site is not found in any of the sixteen known WC1 SRCR domains and is therefore specific for the SRCR domains found on macrophages and may be related to macrophage function.

The homologies between the SRCR domains of M160 vary from 18 to 61% identity, domain 11 being the least well conserved. A long-range repeat of SRCR domains has previously been identified in bovine WC1 and in CD163 (3, 14) (Fig. 4). Multiple alignments and sequence homologies revealed that M160 has a similar repeating structure. This long-range repeating structure is composed of five consecutive SRCR domains with a small 31 amino acid residue linking domain between the second and third SRCR domains. This cassette has been designated a [b-c-d-e-d] cassette, where the WC1 domains 1–11 are arranged as a-[b-c-d-e-d] [b-c-d-e-d] and the CD163 domains are arranged as h-i-j-k-[b-c-d-e-d] (16). The M160 SRCR domains 7–12 can similarly be arranged as k-[b-c-d-e-d] with up to 75 and 77% identity between the SRCR domains of the M160 cassette and those of CD163 and WC1 cassettes, respectively. The “d” domains are the most highly conserved domains within and between the cassettes. The first six domains of M160 show identities between 40 and 61%, and these domains are similar to the h-i-j domains in CD163, with an overall identity of 52–57%. It is difficult to determine a precise hierarchy of relationships between these domains, but it is possible that CD163 emerged by a gene duplication of M160 and that three of the first six SRCR domains of M160 were lost in this process. The recent assignment of the gene encoding M160 to chromosome 12q13.3, close to the CD163 gene, supports this notion (38).

FIGURE 4.

The domain organization of M160 compared with other members of group B of the SRCR superfamily. Only the longest known translated transcripts are shown.

FIGURE 4.

The domain organization of M160 compared with other members of group B of the SRCR superfamily. Only the longest known translated transcripts are shown.

Close modal

The SRCR domains of M160 are followed by a small connecting peptide, a transmembrane region of ∼26 aa residues, and a cytoplasmic domain of 71 residues.

Northern blot analysis using M160 cDNA as probe revealed that mRNA encoding M160 is expressed in the spleen, lymph nodes, thymus, and fetal liver, whereas only weak expression was found in bone marrow and no expression was found in peripheral blood leukocytes (Fig. 5). The tissues expressing mRNA encoding M160 expressed only one transcript of 4.7 kb. The difference of only 96 bp between the M160-α and the M160-β variant forms described below is probably below the resolution limit of the Northern blot analysis.

FIGURE 5.

Tissue Northern blot showing the RNA message hybridizing with the M160 cDNA probe. PBL, peripheral blood leukocytes.

FIGURE 5.

Tissue Northern blot showing the RNA message hybridizing with the M160 cDNA probe. PBL, peripheral blood leukocytes.

Close modal

RT-PCR analysis using primers spanning the region from the untranslated 3′ end to SRCR 12 was performed on 19 different human tissues and showed that the principal site of M160 synthesis is the spleen (Fig. 6,A). When spleen mRNA was used as template, two clear bands were revealed (Fig. 6,A). The sequences of these two bands showed that the cytoplasmic domain of M160 occurs as two variant forms (Fig. 7), a predominant form (M160-α) of 213 nt, and an alternatively spliced form (M160-β) that lacks an in frame insertion of 96 nucleotides. Thus M160-α and M160-β have cytoplasmic domains of 71 and 39 aa residues, respectively. In this context it is interesting to note that two polyadenylation sites are found in the 3′ untranslated end of M160 cDNA. M160-α includes the consensus sequence for protein kinase C (R-[X]2-S/T-RR) (39), casein kinase II (S/T [X]2-D/E) (40), and cGMP-dependent kinase (R/K-R/K-X-S/T) (41), and these possible phosphorylation sites are all absent in M160-β. This suggests that M160-α may be involved in signal transduction through phosphorylation of the cytoplasmic domain and the relative expression level of the M160-α and M160-β forms may be involved in regulating this signal transduction.

FIGURE 6.

RT-PCR analysis of M160 expression in human tissues and cell lines. A, Analysis of 19 different human tissues. The alternative spliced form (M160-β) is only seen in spleen. B, Analysis of alveolar macrophages (from bronchoalveolar lavage, BAL), HL60, U937, THP1, Jurkat, and Raji cells. C, Analysis of M160, CD163, and TNF-α in unstimulated (day 0) and PMA-stimulated U937 cells (days 1, 3, 5, 7, and 9).

FIGURE 6.

RT-PCR analysis of M160 expression in human tissues and cell lines. A, Analysis of 19 different human tissues. The alternative spliced form (M160-β) is only seen in spleen. B, Analysis of alveolar macrophages (from bronchoalveolar lavage, BAL), HL60, U937, THP1, Jurkat, and Raji cells. C, Analysis of M160, CD163, and TNF-α in unstimulated (day 0) and PMA-stimulated U937 cells (days 1, 3, 5, 7, and 9).

Close modal
FIGURE 7.

Alignment of the C-terminal regions of the dominant (M160-α) and alternative (M160-β) form of M160. The 96-bp gap in M160-β excludes the potential phosphorylation sites found in M160-α, but induces neither a stop codon nor a frame shift. The potential phosphorylation site for cGMP-dependent kinase is indicated by ∗, the sites for casein kinase II by %, and the site for protein phosphokinase C by #.

FIGURE 7.

Alignment of the C-terminal regions of the dominant (M160-α) and alternative (M160-β) form of M160. The 96-bp gap in M160-β excludes the potential phosphorylation sites found in M160-α, but induces neither a stop codon nor a frame shift. The potential phosphorylation site for cGMP-dependent kinase is indicated by ∗, the sites for casein kinase II by %, and the site for protein phosphokinase C by #.

Close modal

Three cytoplasmic variants have also been described for CD163, and the genomic organization of the CD163 gene suggests that these variants arise from alternative splicing of intron 15 of the gene (42). The predominant form of CD163 has a cytoplasmic domain of 49 aa residues, whereas the two variant forms have cytoplasmic domains of 84 and 89 residues, respectively. Only weak consensus sequence patterns for potential phosphorylation sites are found in CD163, and these are not related to the different splicing forms of the molecule.

Different cell lines were then analyzed for the presence of M160 mRNA. RT-PCR analysis showed M160 mRNA expression in THP1 and U937 cell lines and low-level expression in HL60 cells and alveolar macrophages, but no expression in Jurkat or Raji cells (Fig. 6 B).

PMA stimulation of U937 cells provoked a small, delayed increase in M160 mRNA expression from day 5. In the same cells, transcripts of TNF-α appeared on day 0 and peaked on day 3 (Fig. 6,C). In contrast to M160, no expression of CD163 mRNA was found in resting U937 cells (Fig. 6 C), but PMA stimulation led to an increase in CD163 mRNA on day 3. These results indicate that the constitutive expression of CD163 and M160 differs and that these structurally closely related molecules are differentially regulated.

We conclude from the above results that M160 is a multidomain protein composed of twelve SRCR domains, a transmembrane region, and a cytoplasmic domain. The cytoplasmic domain is found in two alternatively spliced forms, one of which contains three potential phosphorylation sites. Therefore, it is possible that signaling through M160 could be regulated via alternative splicing of cytoplasmatic encoding exons. Our data suggest that M160 expression, like that of CD163, is restricted to the monocyte/macrophage lineage, and that its expression may be increased by stimulation with phorbol ester.

1

This work was supported by the Danish Medical Research Council, the Novo-Nordisk Foundation, Fonden til Lægevidenskabens Fremme, and the Benzon Foundation.

3

Abbreviations used in this paper: SP-D, lung surfactant protein D; SRCR, scavenger receptor cysteine-rich; SID, SRCR-interspersed domain; RACE, rapid amplification of cDNA end.

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