©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human 180-kDa Receptor for Secretory Phospholipases A
MOLECULAR CLONING, IDENTIFICATION OF A SECRETED SOLUBLE FORM, EXPRESSION, AND CHROMOSOMAL LOCALIZATION (*)

Philippe Ancian , Gérard Lambeau , Marie-Geneviève Mattéi (1), Michel Lazdunski (§)

From the (1) Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France and the Unité de Génétique Médicale et Développement, Inserm U406, Faculté de Médecine, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 05, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Secretory phospholipases A(sPLA) are structurally related enzymes found in mammals as well as in insect and snake venoms. They have been associated with several physiological, pathological, and toxic processes. Some of these effects are apparently linked to the existence of specific receptors for both venom and mammalian sPLAs. We report here the molecular cloning and expression of one of these sPLAreceptors from human kidney. Two transcripts were detected. One encodes for a transmembrane form of the sPLAreceptor and the other one is an alternatively processed transcript, caused by polyadenylation occurring at a site within an intron in the C terminus part of the transcriptional unit. This transcript encodes for a shortened secreted soluble sPLAreceptor lacking the coding region for the transmembrane segment. Quantitative polymerase chain reaction experiments indicate a 1.6:1 ratio between the levels of transcripts encoding for the membrane-bound and soluble forms of the receptor, respectively. Soluble and membrane-bound human sPLAreceptors both bind sPLAwith high affinities. However, the binding properties of the human receptors are different from those obtained with the rabbit membrane-bound sPLAreceptor. The 180-kDa human sPLAreceptor gene has been mapped in the q23-q24 bands of chromosome 2.


INTRODUCTION

Secretory phospholipases A(sPLAs,() 14 kDa) have been purified from a variety of sources including mammalian pancreas, spleen, lung, and serum, as well as from insect and snake venoms (1, 2, 3) . They have been classified into different groups according to their primary structures (4, 5) .

Snake venom PLAs (svPLAs) display different types of toxic effects including neurotoxicity, myotoxicity, anticoagulant, and proinflammatory activity (6, 7) . Some of these effects are apparently linked to the existence of a variety of very high affinity receptors for these toxic svPLAs ( K= 5-40 p M), which have been characterized in rat brain (8) as well as in other tissues (9, 10, 11) using svPLAs purified from the venom of the Taipan snake Oxyuranus scutellatus scutellatus called OSand OS.

In mammals, the prototype of group I sPLAis the pancreatic sPLA. It is secreted from pancreas during digestion and has long been thought to only act as a digestive enzyme (12) . More recently, in relation with its localization in several tissues of non-digestive origin (13, 14, 15) , this pancreatic-type sPLAhas been suggested to play a role in airway (16) and vascular smooth muscle contraction (17) , as well as in cell proliferation (18) . The prototype of group II sPLAis the inflammatory-type sPLA. Because of its presence in large amounts in plasma and synovial fluids of patients with inflammatory diseases such as rheumatoid arthritis, acute pancreatitis, and endotoxic shock and its up-regulation by proinflammatory cytokines like tumor necrosis factor and interleukin-1 (reviewed in Refs. 19-23), this type II sPLAis considered to be a potent mediator of the inflammatory processes. Several studies have shown that this group II sPLAis implicated in the production of potent lipid mediators of inflammation (24, 25, 26) .

Molecular mechanisms governing the physiological effects mediated by these mammalian sPLAs are not well understood. Some of these effects are thought to be linked to the existence of high affinity sPLAreceptors with molecular masses of 180-200 kDa which have been identified in rabbit, rat, and bovine tissues (9, 10, 11, 27) . The rabbit 180-kDa sPLAreceptor recognizes several svPLAs, including OSand OS(9) , with very high affinities ( Kvalues = 5-50 p M). It also tightly binds the porcine pancreatic group I and the human inflammatory group II sPLAs with Kvalues of 1-10 n M (28) .

Cloned 180-kDa sPLAreceptors (28, 29) have the same structural organization as the macrophage mannose receptor, a membrane protein involved in the endocytosis of glycoproteins, and the phagocytosis of microorganisms bearing mannose residues on their surface (30, 31) .

This paper reports the molecular cloning, expression, and chromosomal localization of a membrane-bound and the existence of a secreted soluble human sPLAreceptor. These sPLAreceptors have new binding properties as compared with the rabbit sPLAreceptor.


EXPERIMENTAL PROCEDURES

Materials

OSand OSwere purified as described previously (8) . Porcine pancreatic sPLA(group I) was purchased from Boehringer Mannheim. Human pancreatic sPLAwas kindly provided by Dr. Hubertus Verheij (Department of Enzymology and Protein Engineering, 3508 TB, Utrecht, The Netherlands). Human non-pancreatic sPLA(group II) was a generous gift from Dr. Ruth Kramer (Eli Lilly). It is a recombinant protein expressed in Syrian Hamster AV 12 cells. p-Aminophenyl-- Dmannopyranoside-BSA (mannose-BSA, catalog no. A4664) was purchased from Sigma.

Isolation of cDNA Clones Encoding for the Human sPLAReceptor

A cDNA fragment corresponding to nucleotides 2564-4687 of the rabbit sPLAreceptor (28) was used to probe 10phage plaques of a dT-primed human lung cDNA library in gt11 vector (Clontech Laboratories, Inc.) at moderate stringency (hybridization for 16 h at 45 °C in 5 SSC and 30% formamide followed by four washes of the hybridization membranes in 2 SSC at 45 °C, where 1 SSC = 0.15 M NaCl, 15 m M trisodium citrate, pH 7). Two positive clones were isolated. The longest one, 2.3 kilobase pairs (kbp) in length (HdT319) was subcloned into the pBluescript II plasmid vector (Stratagene Cloning Systems) and sequenced in both strands using the dye-terminator kit and automatic sequencing (Applied Biosystems model 373 A sequencer). DNA for sequencing was prepared with the Erase-A-base system (Promega Corp.) using the manufacturer's instructions. The cDNA sequence included only a 1.2-kbp open reading frame, which was 84% homologous to the C-terminal part of the rabbit sPLAreceptor (28) . This P-labeled clone was used to probe 500,000 phage plaques of a homemade oligo(dT)-primed human kidney cDNA library in ZAPII (Stratagene Cloning Systems) at high stringency (hybridization for 16 h at 50 °C in 5 SSC, 50% formamide followed by two washes of the membranes in 2 SSC at 50 °C and two final washes in 0.2 SSC at 60 °C). Four positive clones were isolated. The 4.6-kbp clone 2C11 contained the N terminus part of the receptor. The 3.8-kbp clone 2B11 contained the C terminus part of the receptor, and the two other 3.5-kbp partial clones 3B11 and 3B12 contained the sequence corresponding to nucleotides 525-4067 of the rabbit receptor (28) . 2C11 and 2B11 were sequenced on both strands as described above. The full-length cDNA clone encoding the membrane-anchored human sPLAreceptor was obtained by ligation at the SphI site (located at nucleotide 3171) of these two latter human kidney partial clones.

PCR Experiments

RT-PCR experiments have been performed on human kidney poly(A) mRNA reverse transcribed with the Moloney murine leukemia virus reverse transcriptase (Promega Corp.). The specific amplification of fragments corresponding to the membrane-anchored and the soluble form of the sPLAreceptor cDNAs was achieved with a sense oligonucleotide called A (5`-CAGATGGTCTGGTTGAATGC-3`, nucleotides 4142-4162) common to both forms of the receptor, in combination with either the antisense oligonucleotide B (5`-ACAAAGTTCCAGCCAAGCAT-3`, nucleotides 4866-4886) or C (5`-GTTTTCAATAGGGAGTTTCT-3`, nucleotides 4450-4470), specific for the membrane-anchored and the soluble form of the sPLAreceptor, respectively (see Fig. 2). PCR conditions were: denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 1.5 min. The same combinations of oligonucleotides have been used on 500 ng of human genomic DNA as template, following a 5-min period of denaturation at 98 °C (35 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 2 min, and extension at 72 °C for 3 min).


Figure 2: Nucleotide and amino acid sequences of the human sPLA 180- kDa receptor. Panel A, potential Asn-glycosylation sites are marked with asterisks. The predicted signal peptide is boxed and has been determined by sequence comparison with the rabbit sPLAreceptor. The putative transmembrane segment is heavily underlined. The putative phosphorylation site by casein-kinase II is marked with an open circle. The open triangle shows the beginning of the diverging sequence of clone 2C11. Panel B, the nucleotide and deduced amino acid sequences of clone 2C11, which occurs following the nucleotide 4179 of the membrane-anchored sPLAreceptor cDNA ( open triangle). The splice donor sequence is doubly underlined. The two different polyadenylation signals are underlined. A-D show the positions and orientations of the oligonucleotides used for the PCR experiments (see text).



Quantitative PCR Experiments

Random-primed human kidney first strand cDNA were synthetized using the Moloney murine leukemia virus reverse transcriptase with 1 µg of human kidney poly(A) mRNA as template. For the negative control, the reverse transcriptase was omitted in the reaction mixture. The specific amplification of fragments corresponding to the membrane-anchored and the soluble forms of the sPLAreceptor cDNAs was achieved with a sense oligonucleotide called Qc (5`-CCAATGCCCAATACCTTAGAA-3`, nucleotides 3552-3571) common to both forms of the receptor, in combination with either the antisense oligonucleotide Qm (5`-TGATGGGGCTTGAAGTAGTC-3`, nucleotides 4251-4270) or Qs (5`-CAAGGAGAGTTTTCTGGGTC-3`, nucleotides 4252-4271), specific for the membrane-anchored and the soluble forms of the sPLAreceptor, respectively. The assay mixture (100 µl) contained 20 m M Tris-HCl, pH 8.4, 50 m M KCl, 1.5 m M MgCl, 0.15 µ M of primers Qc and Qm (or Qc and Qs), 100 µ M each of dATP, dGTP, dTTP, 20 µ M dCTP, 12.5 µCi of [P]dCTP and 25 ng of template. The mixture was heated at 94 °C during 2 min after which 2.5 units of Taq polymerase (Life Technologies, Inc.) were added. Amplification was performed in sequential cycles at 94 °C, 1 min; 56 °C, 1 min; and 72 °C, 2 min. From 17 to 30 cycles, 5 µl of the reaction mixture were removed at each cycle and incubated for an additional 10 min at 72 °C. The aliquots were electrophoresed on 6% acrylamide gels. Amplified DNA was fixed in the gel using 7% acetic acid and the gels were stained with 2 µg/ml ethidium bromide, photographed, and exposed 2 h for autoradiography. Bands of amplified DNA were cut from the gels and counted for the determination of incorporated radioactivity.

Chromosomal Mapping of the Human sPLAReceptor Gene

In situ hybridization was carried out on chromosome preparations obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 h. 5-Bromodeoxyuridine was added for the final 7 h of culture (60 µg/ml of medium) to ensure a posthybridization chromosomal banding of good quality. The sPLAreceptor clone HdT319 containing an insert of 2300 base pairs (bp) corresponding to the C-terminal region of the receptor in the plasmid vector pBluescript II (stratagene) was tritium labeled by nick translation to a specific activity of 2 10disintegrations/min/µg. The radiolabeled probe was hybridized to metaphase spreads at a final concentration of 100 ng/ml of hybridization solution as described previously (32) . After coating with nuclear track emulsion (Kodak NTB), the slides were exposed for 15 days at +4 °C, then developed. To avoid any slipping of silver grains during the banding procedure, chromosome spreads were first stained with buffered Giemsa solution and metaphase photographed. R-banding was then performed by the fluorochrome-photolysis-Giemsa (FPG) method and metaphases rephotographed before analysis.

Northern Blot Analysis of Human sPLAReceptor Expression

A human multiple tissue Northern blot (Clontech Laboratories, Inc.) was hybridized with the P-labeled HdT319 cDNA at high stringency (hybridization for 16 h at 55 °C in 5 SSC, 50% formamide followed by two washes of the membranes in 2 SSC at 50 °C, and two final washes in 0.2 SSC at 65 °C). As this commercial Northern blot did not show a good resolution in the labeled region of interest, we performed a new Northern blot with 2 µg of human kidney poly(A) mRNA (Clontech Laboratories, Inc.) separated in 1% agarose gel, transferred to a nylon membrane, and hybridized as described above.

Expression of the Human sPLAReceptor into Eukaryotic Cells

The full-length cDNAs encoding for the membrane-anchored and the soluble forms of the human sPLAreceptor were subcloned into the efficient expression vector pcDNA I (Invitrogen Corp.) and transfected into Chinese hamster ovary, 293, or COS cell lines (American Type Cell Collection) by the Ca/POprocedure (33) . 50% confluent cells were transfected with 20 µg of DNA/75 cmPetri dish on day 1. On day 2, the cells were trypsinized and replated. On day 3, cells were scraped, and I-OSbinding was performed as described previously (9) . For binding experiments to the soluble human sPLAreceptor, the medium from transfected cells was centrifuged at 10,000 g for 10 min. Aliquots of the supernatant were incubated with I-OSas described for the membrane-bound sPLAreceptor (9) except that reaction mixtures were filtered through Whatman GF/F glass-fiber filters. Mock-transfected cells were obtained by transfection with the parent vector pcDNA I.

For internalization experiments, 293 cells were transfected with the cDNA encoding for the membrane bound human sPLAreceptor and plated onto 24-well plates precoated with 10 µg/ml of polylysine. The next day, confluent cells were incubated for 3 h at 4 °C with 800 p M of I-OSin the presence or absence of 300 n M unlabeled OSin 0.25 ml of incubation buffer (Dulbecco's modified Eagle's medium/Ham's F-12 (50/50) supplemented with 0.1% of BSA and 20 m M HEPES, pH 7.4). The cells were rinsed twice with cold medium and then incubated for the kinetic experiment at 37 °C. The incubation medium (0.5 ml) was removed at indicated times, and cells were rinsed once with the above buffer. Cell-surface-associated I-OSwas removed by incubating the cells for 5 min in 0.5 ml of a dissociating buffer (50 m M sodium acetate, pH 3.5, 500 m M NaCl, 0.1% BSA, and 0.1 m M CaCl). Wells were rinsed once with this acidic buffer, and internalized I-OSwas counted after cell solubilization in 1 ml of NaOH 0.1 N. The degradation of the ligand was measured by precipitation of the incubation medium with 5% trichloroacetic acid. The radioactivity associated with the trichloroacetic acid-insoluble fraction is referred to as the intact ligand, as confirmed by gel filtration chromatography on Sephadex G-25. The radioactivity associated with the trichloroacetic acid-soluble fraction is refered to as degradation products of I-OS.


RESULTS

Isolation of cDNA Clones Encoding for the Human Kidney sPLAReceptor

Previous experiments have shown that the mRNA encoding the rabbit sPLAreceptor is strongly expressed in rabbit lung (28) . A cDNA fragment corresponding to nucleotides 2564-4687 of the rabbit sPLAreceptor (28) was therefore used to screen a human lung cDNA library constructed in the gt11 vector. 10clones were screened at moderate stringency, and two positive clones were isolated. The longest clone called HdT319 has a significant homology (85%) with the 1.2-kbp C-terminal region of the rabbit sPLAreceptor and contains 1.1 kbp of 3` non-coding sequence as well as a poly(A) stretch consistent with a poly(A) tail (Fig. 1). Northern blot analysis with the clone HdT319 as a probe reveals that the human sPLAreceptor mRNA is expressed at a high level in kidney (see below). In order to obtain the full-length cDNA clone encoding the human sPLAreceptor, 500,000 clones of a oligo(dT)-primed human kidney ZAPII cDNA library were screened with the above P-labeled clone. Four positive overlapping clones were isolated (Fig. 1). Sequence analysis revealed that the clone 2C11 of 4.6 kbp contained a 5`-non-coding region of 207 bp, a large open reading frame encoding for the amino acids 1-1325 of the receptor as well as a 3`-non-coding sequence of 466 bp, terminated by a poly(A) tail. The clone 2B11 of 3.8 kbp contained an open reading frame encoding for the amino acid sequence 536-1465 of the receptor followed by 1.1 kbp of 3`-non-coding sequence identical to that of HdT319. The two other clones 3B11 and 3B12 of 3.5 kbp contained a large open reading frame encoding for the amino acid sequence 149-1330 of the receptor.


Figure 1: Schematic representation of the human sPLA receptor cDNA clones. These clones were obtained by screening of a dT-primed human lung cDNA library ( HdT319) and of a dT-primed human kidney cDNA library (others). Open bars, coding regions of the membrane-anchored ( 2B11-3B11-3B12) or the soluble form ( 2C11) of the human sPLAreceptor. The black and hatched black boxes represent the 3`-non-coding region of the soluble and membrane-anchored form of the receptor, respectively. The white hatched box shows the 5`-non-coding region of the sPLAreceptor. 5` end, 3` end, and nucleotide positions of stop codons ( S) are indicated. SphI restriction site (nucleotide 3171) was used to construct the full-length cDNA clone encoding for the membrane-anchored form of the receptor (named BC11).



None of these cDNA clones contained the expected complete coding region when compared with the rabbit sPLAreceptor. When sequences of clones 2B11 and 2C11 were overlapped, the combined sequence displayed a significant homology with the previously cloned membrane-anchored rabbit sPLAreceptor. Therefore, these clones have been used to construct the full-length cDNA encoding for the human membrane-anchored sPLAreceptor (named BC11), using the SphI restriction site shown in Fig. 1. The open reading frame of this cDNA sequence encodes for a protein of 1465 amino acids with a predicted molecular mass of 168 kDa (Fig. 2). It has a striking homology with its rabbit (28) and bovine (29) counterparts (84 and 85% of homology at the amino acid level, respectively). The first 25 amino acids following the initiator methionine have the features of a signal sequence (34) . An alanine located 26 amino acids downstream of this methionine was deduced to be the N-terminal residue of the mature protein, when compared to the rabbit receptor (28) . The sequence has 15 potential N-glycosylation sites (Fig. 2) while the rabbit equivalent has 16 (28) . Like the rabbit receptor, the human protein displays a N-terminal cysteine-rich domain (residues 26-165), a fibronectin-like type II domain (residues 166-222), eight carbohydrate recognition domains (CRDs) in tandem (residues 223-1381), a unique transmembrane segment (residues 1398-1423), and a short C-terminal cytoplasmic tail (residues 1424-1465). This cytoplasmic tail of the receptor contains the typical consensus sequence NP XY, which is presumably involved in the internalization of the ligand-receptor complex (35) . This consensus sequence is also found in the bovine corpus luteum 180-kDa sPLAreceptor (29) , the rabbit sPLAreceptor having the sequence NSYY. It also contains a consensus sequence site for casein-kinase II phosphorylation on Ser-1460, suggesting a possible role of this kinase in the regulation of the receptor activity (Fig. 2). Comparison of the protein sequence with data bases revealed that this human sPLAreceptor has a 29% sequence homology with the human macrophage mannose receptor, a protein involved in the endocytosis of glycoproteins bearing terminal carbohydrates residues and in the phagocytosis of yeast and other pathogenic microorganisms (30, 31) .

Analysis of the different clones isolated from the human kidney cDNA library shows the presence of a second form of sPLAreceptor transcript in this tissue. The cDNA corresponding to this second form (clone 2C11) has a diverging sequence after the nucleotide 4179 with a 5` potential consensus splice donor site (36) at this nucleotide position (Fig. 2). Open reading frame analysis shows the presence of a stop codon 5 nucleotides downstream of the diverging sequence, thus changing the amino acids Asn-Glu-Thr (residues 1327-1329 of the membrane-anchored form of the receptor) into the sequence Ser-Lys-stop. This stop codon is followed by an untranslated region of 466 nucleotides which contains a canonical polyadenylation signal AATAAA located 19 bases upstream from a poly(A) tail. This alternatively processed transcript encodes for a sPLAreceptor-related protein, lacking a part of the eighth CRD, the transmembrane segment and the cytoplasmic tail, suggesting the existence of a soluble secreted form of the sPLAreceptor.

Since the nucleotide sequences of clones 2B11 and 2C11 are identical in a large overlapping region, it is unlikely that the two forms of sPLAreceptors are generated from two different genes. In agreement with that view, a single labeling has been observed for the chromosomal localization using a probe common to both transcripts (clone HdT319). Taken together, these results show that these two mRNA forms are transcribed from the same gene. To obtain further information relative to the mechanism of generation of the shortened transcript (clone 2C11), PCR experiments have been performed on both human kidney first strand cDNA and human genomic DNA as templates, with a sense oligonucleotide common to both forms (oligonucleotide A), and two antisense oligonucleotides, B and C, specific for the membrane-anchored and the soluble sPLAreceptor, respectively (Figs. 2 and 3 B). As expected, a fragment of 0.75 kbp has been amplified on human kidney first strand cDNA using oligonucleotides A and B, but no amplified product was seen on human genomic DNA (Fig. 3 A), suggesting that the genomic DNA fragment of the sPLAreceptor between these two oligonucleotides is too large to allow the amplification of this gene fragment. Using oligonucleotides A and C, the same 0.31-kbp fragment of DNA has been amplified on both human kidney first strand cDNA and human genomic DNA (Fig. 3 A). When transferred to Nylon membrane and probed with the corresponding fragment of clone 2C11, these two 0.31-kbp DNA fragments were strongly labeled (data not shown). Moreover, the diverging sequence of this transcript begins with a 5` consensus splice donor site and displays a polyadenylation signal 19 bp upstream from a poly(A) tail (Fig. 2 B). Another PCR experiment on human genomic DNA has been performed using the sense oligonucleotide A and an antisense oligonucleotide D corresponding to nucleotides 4192-4212 of the membrane-anchored form of the sPLAreceptor (Figs. 2 and 3 B). This oligonucleotide is located 29 bp downstream from oligonucleotide A in the cDNA encoding for the membrane-anchored form of the sPLAreceptor. A DNA fragment of 1.65 kbp has been amplified, subcloned, and sequenced at both ends. The 5` region of this amplified intron displays the same sequence as the diverging region of the shorter transcript (data not shown). Moreover, the 3` region of this intron displays a 3` splice acceptor consensus sequence (5`-TTTTCTCACAG-3`) (36) . Taken together, these results lead to the conclusion that the shortened transcript would be generated from an alternative processing of the sPLAreceptor mRNA precursor by truncation at an alternative polyadenylation site located within an intron (Fig. 3 B). When this intron is spliced, the resulting transcript encodes for the membrane-anchored form of the sPLAreceptor.


Figure 3: PCR experiments on human kidney first strand cDNA and on human genomic DNA. Panel A, cDNA: RT-PCR experiments on human kidney first strand cDNA. A-C shows the amplification products obtained with oligonucleotides A and C, specific for both transcripts and for the soluble form of the receptor, respectively. +, experiment done with 20 ng of cDNA as template. For the negative control (-), the reverse transcriptase has been omitted for the synthesis of the first strand cDNA. A and B show the amplification products obtained with oligonucleotides A and B, specific for both transcripts and for the membrane-anchored form of the receptor, respectively. Genomic DNA, PCR experiments on human genomic DNA. The (+) experiment has been done with 500 ng of human genomic DNA. The negative control (-) has been done without genomic DNA. Panel B, proposed model for the alternative polyadenylation and RNA splicing in the sPLAreceptor transcriptional unit: AATAAA shows the consensus site for polyadenylation. Open arrow shows the 5` splice consensus donor site. Gray arrow shows the 3` splice consensus acceptor site. A-D show the positions and orientations of oligonucleotides used for the PCR experiments (see text). The gray box represents the end of the amino acid sequence encoded by the first exon. The black box shows the amino acid sequence encoded by the beginning of the unspliced intron, and the white box represents the beginning of the amino acid sequence encoded by the second exon. Hatched box represents the coding region of the transmembrane segment.



To determine the relative amounts of both transcripts in human kidney, quantitative PCR experiments have been performed using random-primed human kidney first strand cDNA as template. The amplification of products specific for both forms of the sPLAreceptor was achieved using an oligonucleotide (Qc), common to both transcripts, in combination with oligonucleotides (Qm or Qs), specific for the transmembrane and soluble forms, respectively (see ``Experimental Procedures''). The concentration of products accumulating in consecutive cycles is presented in Fig. 4. In this experiment, incorporation of [P]dCTP was measurable after cycles 21 and 23 for the membrane-bound and the soluble forms of the sPLAreceptor, respectively. The plateau phases were reached at cycle 26 and 30, respectively. Between these cycles, the amplification of products was exponential. The equation describing product accumulation at a cycle n is: Log( P) = nLog(eff.) + Log( P) where Pis the quantity of product measured for a cycle n, eff. is the efficiency of amplification between two consecutive cycles, and Pis the initial amount of the amplified product (37) . This equation has been analyzed by linear regression (Fig. 4). The efficiencies of amplification were 1.64 and 1.59 for the membrane-bound and the soluble forms, respectively. The ratio between the amounts of the transcripts encoding for the membrane-bound and the soluble sPLAreceptor at 0 cycle ( P) was found to be 1.6. The negative control for this experiment gave no signal after 30 cycles.


Figure 4: Quantitative PCR of fragments specific for both forms of human sPLA receptor. Results were obtained with 25 ng of first strand human kidney cDNA as template (37). Results were fitted with the following equations: Log( P) = 0.2149 n - 19.2191 ( r = 0.998) and Log( P) = 0.2037 n - 19.4265 ( r = 0.998) for the membrane-bound and the soluble form, respectively. Initial quantities of template ( P, see ``Results'') were found to be 6.03 10mol and 3.74 10mol for 5 µl of reaction mixture. Points on the plateau ( hatched curves) have not been taken for the linear regression. Sizes of amplified fragments were 719 and 720 bp for the membrane-bound and the soluble form, respectively.



Gene Mapping of the Human sPLAReceptor

In the 120 metaphase cells examined after in situ hybridization, there were 214 silver grains associated with chromosomes, and 54 of these (25.2%) were located on chromosome 2; the distribution of grains on this chromosome was not random: 35/54 (64.8%) of them mapped to the q23-q24 region of chromosome 2 long arm with a maximum in the q24 band (Fig. 5). These results allowed us to map the sPLAreceptor gene to the 2q23-2q24 bands of the human genome. Until now, no genetic disorder has been associated within this chromosomal region.

Northern Blot Analysis of the Human 180-kDa sPLAReceptor

When the clone HdT 319 was used as a probe on a human multiple tissue Northern blot, a strong labeling was observed in human kidney but also in placenta, lung, and skeletal muscle (Fig. 6). The tissue distribution pattern of the human sPLAreceptor mRNA is significantly different from that obtained for the rabbit (28) . The labeling obtained with this commercial Northern blot did not provide the precise size of the labeled mRNAs. A new Northern blot has then been prepared with 2 µg of human kidney mRNA and hybridized with the same probe which indicated that two mRNAs of 6.5 and 5.4 kilobases were labeled.

Expression of the Human sPLAReceptor

In order to examine the pharmacological properties of both the membrane-anchored and the soluble human sPLAreceptors, transient expression experiments have been made in COS cells. Binding experiments with I-OSrevealed that this svPLAbinds with a high affinity ( K= 1.5 n M) to the two expressed forms of the human sPLAreceptor ( Bvalues were 440 fmol/mg of protein and 260 fmol/ml for the membrane-bound and the soluble forms, respectively) (data not shown). No specific binding was observed on cell membranes or medium prepared from mock-transfected COS cells (data not shown). Competition experiments with several unlabeled sPLAs show that both forms of human sPLAreceptors display the same pharmacological profile. OSand OSinhibit I-OSbinding with Kvalues of 2 and 4 n M, respectively (Fig. 7). Both porcine and human pancreatic sPLAs inhibit I-OSbinding with Kvalues of approximately 400 n M, respectively (Fig. 7). Conversely, the bee venom sPLAand the human inflammatory group II sPLAare without effect on I-OSbinding (data not shown and Fig. 7 , respectively). Moreover, mannosylated-BSA, a neoglycoprotein which is known to bind to the mannose receptor with a high affinity of 20 n M (38) has no effect on I-OSbinding (Fig. 7). These results are significantly different from those obtained with the rabbit 180-kDa sPLAreceptor that we had previously characterized and cloned (28) . The rabbit sPLAreceptor binds svPLAs with higher affinities ( K= 7 p M for OSand 40 p M for OS) than those determined for the presently cloned human sPLAreceptor. This rabbit receptor also tightly binds the porcine pancreatic sPLA( K= 10 n M) and the human group II sPLA( K= 1 n M) and mannosylated-BSA ( K= 5 n M). Transient expression experiments have also been made into 293 and Chinese hamster ovary cells, two other cell lines widely used for heterologous expression of proteins. An identical pharmacological profile was obtained after transfection of the human sPLAreceptor into these cell lines (data not shown). Binding experiments on human kidney membranes (expressing 15 fmol of receptor/mg of protein as determined with I-OSas ligand) revealed the same binding properties as those obtained on membranes prepared from cells used for heterologous expression, thus indicating that the newly cloned receptor is functionally expressed.


Figure 7: Transient expression of the human 180-kDa receptor cDNA into eukaryotic cells. Competition experiments between I-OS(10 p M) and other sPLAs or mannosylated-BSA on the membrane-bound ( panel A) or the soluble ( panel B) human sPLAreceptors. Results are expressed as percentage of the maximal specific binding measured in the absence of competitor. 100% corresponded to 1 p M of I-OSspecifically bound. Nonspecific binding was measured in the presence of 100 n M unlabeled OSand represented 20% of the total binding.



Internalization of the Human Membrane-bound sPLAReceptor

The human 180-kDa receptor, as the macrophage mannose receptor, displays in its cytoplasmic domain a consensus sequence for the internalization of the ligand-receptor complex (39) . This finding prompted us to verify if the expressed receptor is able to internalize I-OS. Thus, transfected cells were incubated with the ligand at 4 °C for 3 h, then the medium containing free ligand was removed, and the temperature raised to 37 °C. At various times, we then determined the radioactivity released in the medium during the warming period, the cell-surface ligand radioactivity (released from the cells by acid treatment), and the intracellular-associated ligand radioactivity (resistant to acid treatment). Fig. 8 A shows that the prebound ligand rapidly disappears from the cell surface upon warming while the intracellular-associated radioactivity rises concomitantly, reaching a plateau after 10 min. Fig. 8 A also shows that the radioactivity rapidly appears in the incubation medium. This latter has been subjected to trichloroacetic acid precipitation to determine the amount of native (trichloroacetic acid-insoluble) and degraded (trichloroacetic acid-soluble) ligand. Fig. 8 B shows that the radioactivity released in the incubation medium is composed of two distinct components: during the first 15 min, most of the released radioactivity is composed of intact ligand which probably results from cell surface dissociation. After 15 min of warming, the degraded ligand begins to appear in the medium. This experiment clearly shows that the expressed human receptor is able to internalize and promote the degradation of I-OS. It is interesting to note that the rate of I-OSinternalization by the human 180-kDa sPLAreceptor is very similar to the rate of mannosylated-BSA internalization by the human macrophage mannose receptor (40) .


Figure 8: Time course of internalization ( A) and degradation ( B) of I-OS by 293 cells transfected with the human sPLA receptor. Panel A, confluent transfected cells were incubated with 800 p M I-OSin the absence (total binding) or the presence (nonspecific binding) of 300 n M unlabeled OSfor 3 h at 4 °C. The unbound ligand was removed and the cells were warmed to 37 °C (time 0). All results are expressed as specifically associated radioactivity, calculated by substraction of the nonspecific binding from the total binding. Mock-transfected cells did not show binding or internalization of I-OS. Panel B, trichloroacetic ( TCA) precipitation of the warmed incubation medium. The trichloroacetic acid-insoluble and -soluble fractions are referred to as intact and degraded ligands, respectively.




DISCUSSION

We report here the molecular cloning of the human 180-kDa sPLAreceptor. This receptor is strongly homologous to its rabbit and bovine counterparts (28, 29) . The human membrane-anchored sPLAreceptor shares the same structural organization as the macrophage mannose receptor (30, 31) . This structural organization is composed of several distinct domains. The N-terminal cysteine-rich domain shares no significant homology with other known protein sequences. Its role remains unclear, even in the case of the mannose receptor. The fibronectin-like type II domain is found in several other proteins including fibronectin (41) , type IV collagenase (42) , and the cation-independent mannose 6-phosphate receptor (43) . The functional role of this domain in sPLAor mannose receptors is unknown. CRDs are found in numerous C-type lectins and are known to bind carbohydrate residues of glycoproteins or microorganisms (44) . Site-directed mutagenesis experiments have shown that the sPLAbinds to the sPLAreceptor via one of its CRDs.()

This work shows that, in addition to the membrane-anchored sPLAreceptor transcripts, there exists a second type of messengers for the sPLAreceptor in human kidney. The use of two different processing events at an intron within the region encoding for the eighth CRD appears to govern the production of these two types of messengers. Polyadenylation within this intron results in a shorter transcript which encodes for a secreted sPLAreceptor. Splicing of this intron is required for the production of a longer transcript that directs the synthesis of the membrane-anchored form of the sPLAreceptor. In general, it appears that the addition of a poly(A) tail to 3` ends of nuclear RNA occurs more rapidly than RNA splicing (45, 46, 47) , so that it is the control of polyadenylation at this site that probably determines the processing pathway that is used.

The mechanism of generation of this shorter transcript of the sPLAreceptor resembles the alternative polyadenylation within an intron which is used for the generation of soluble form of the receptor for the complement C3b/C4b called CD35 (48) . Cytokine receptors like the receptors for interleukin-4 and for interleukin-7 exist both in membrane-anchored or soluble forms, but these latter are obtained by alternative splicing. In both cases, an exon is added in the 3` region of the transcript and introduces a stop codon before the exon encoding for the transmembrane segment (49, 50) .

Quantitative PCR experiments show that transcripts encoding for both forms of the human sPLAreceptor are present in comparable amounts in human kidney. This result suggests that the soluble human sPLAreceptor might have an important physiological role.

Expression of the human sPLAreceptors indicates that both membrane-bound and soluble human sPLAreceptors display the same binding properties. Therefore, the soluble human sPLAreceptor can compete for binding of sPLAs with the membrane-bound receptor. The binding properties of the human sPLAs receptors are very different from those of their rabbit, bovine, and rat counterparts. Indeed, rat, bovine, and rabbit receptors recognize the porcine group I sPLAwith high affinities ( Kvalues = 0.5-10 n M) ( (27, 28) . The human sPLAreceptor recognizes this sPLAas well as human pancreatic sPLAwith a relatively weak affinity ( K≅ 400 n M). Another intriguing result is that the human sPLAreceptor does not recognize the human inflammatory group II sPLAas the rabbit receptor does. One possibility is that the functional role of the presently cloned receptor is not to bind the pancreatic and/or the inflammatory sPLAs but rather to associate with one of the newly discovered mammalian sPLAs such as the Enhancing Factor, a novel group II sPLA(51) , or two other human sPLAs of unknown function which have been recently cloned (52, 53) .

A property of the human membrane-bound 180 kDa sPLAreceptor is its ability to promote sPLAinternalization. This finding suggests that one of the possible functions of this receptor is a role in the clearance of circulating sPLA, thus inhibiting sPLAaction. Alternatively, another possible role of these sPLAreceptors could be to direct the action of sPLAinto its target cells, in order to obtain, before sPLAdegradation occurs, a transient extracellular production of second messengers of the arachidonic acid cascade.


FOOTNOTES

*
This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Association pour la Recherche sur le Cancer (ARC), and Ministère de la Défense Nationale Grant DRET 93/122. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U17033 and U17034.

§
To whom correspondence should be addressed: Institut de Pharmacologie Moléculaire et Cellulaire, UPR 411 du CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. Tel.: 33-93957700/02; Fax: 33-93957704.

The abbreviations used are: sPLA, secretory phospholipase A; svPLA, snake venom phospholipase A; OS, Oxyuranus scutellatus scutellatus toxin 1; OS, Oxyuranus scutellatus scutellatus toxin 2; CRD, carbohydrate recognition domain; BSA, bovine serum albumin; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s).

J. P. Nicolas, P. Ancian, E. Zvaritch, G. Lambeau, and M. Lazdunski, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. R. Kramer (Eli Lilly Co.) and Dr. H. Verheij (Utrecht, The Netherlands) for the gift of human secretory type II sPLAand of human pancreatic sPLA, respectively, and Dr. R. Waldman for providing us with the human kidney cDNA library. We are grateful to M.-M. Larroque and R. Pichot for their excellent collaboration, to J.-L. Nahon and J.-P. Hugnot for very helpful discussions, and to F. Aguila and C. Roulinat for their most skillful assistance.


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