From the
Secretory phospholipases A
Secretory phospholipases A
Snake venom PLA
In mammals, the prototype of group I
sPLA
Molecular mechanisms governing
the physiological effects mediated by these mammalian sPLA
Cloned 180-kDa sPLA
This paper reports the molecular cloning, expression, and
chromosomal localization of a membrane-bound and the existence of a
secreted soluble human sPLA
For internalization
experiments, 293 cells were transfected with the cDNA encoding for the
membrane bound human sPLA
Analysis of the
different clones isolated from the human kidney cDNA library shows the
presence of a second form of sPLA
Since the nucleotide
sequences of clones 2B11 and 2C11 are identical in a large overlapping
region, it is unlikely that the two forms of sPLA
We report here the molecular cloning of the human 180-kDa
sPLA
This work shows that, in addition to
the membrane-anchored sPLA
The mechanism of generation of this shorter
transcript of the sPLA
Quantitative PCR experiments show that
transcripts encoding for both forms of the human sPLA
Expression of the human
sPLA
A property of the human membrane-bound 180
kDa sPLA
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank/EMBL Data Bank with accession number(s)
U17033 and U17034.
We thank Dr. R. Kramer (Eli Lilly Co.) and Dr. H.
Verheij (Utrecht, The Netherlands) for the gift of human secretory type
II sPLA
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(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 sPLA
s. We report here the molecular cloning and
expression of one of these sPLA
receptors from human
kidney. Two transcripts were detected. One encodes for a transmembrane
form of the sPLA
receptor 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 sPLA
receptor 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 sPLA
receptors both bind sPLA
with high affinities.
However, the binding properties of the human receptors are different
from those obtained with the rabbit membrane-bound sPLA
receptor. The 180-kDa human sPLA
receptor gene has
been mapped in the q23-q24 bands of chromosome 2.
(sPLA
s,
(
)
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) .
s
(svPLA
s) 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 svPLA
s ( K
=
5-40 p
M), which have been characterized in rat brain
(8) as well as in other tissues
(9, 10, 11) using svPLA
s purified from the venom of the
Taipan snake Oxyuranus scutellatus scutellatus called OS
and OS
.
is 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 sPLA
has 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 sPLA
is 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 sPLA
is
considered to be a potent mediator of the inflammatory processes.
Several studies have shown that this group II sPLA
is
implicated in the production of potent lipid mediators of inflammation
(24, 25, 26) .
s
are not well understood. Some of these effects are thought to be linked
to the existence of high affinity sPLA
receptors 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
sPLA
receptor recognizes several svPLA
s,
including OS
and OS
(9) , with very high
affinities ( K
values = 5-50
p
M). It also tightly binds the porcine pancreatic group I and
the human inflammatory group II sPLA
s with
K
values of 1-10 n
M (28) .
receptors
(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) .
receptor. These sPLA
receptors have new binding properties as compared with the rabbit
sPLA
receptor.
Materials
OSand OS
were
purified as described previously
(8) . Porcine pancreatic
sPLA
(group I) was purchased from Boehringer Mannheim.
Human pancreatic sPLA
was 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
sPLA
A cDNA fragment corresponding to
nucleotides 2564-4687 of the rabbit sPLAReceptor
receptor
(28) was used to probe 10
phage 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 sPLA
receptor
(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 sPLA
receptor 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 sPLA
receptor, 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 sPLA
receptor. 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 sPLA
receptor
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 sPLA
receptor, 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 sPLA
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 Gene
receptor 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
10
disintegrations/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 sPLA
A human multiple tissue Northern blot (Clontech
Laboratories, Inc.) was hybridized with the Receptor
Expression
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 sPLA
The full-length cDNAs encoding for the
membrane-anchored and the soluble forms of the human sPLAReceptor into
Eukaryotic Cells
receptor 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/PO
procedure
(33) . 50% confluent cells were
transfected with 20 µg of DNA/75 cm
Petri dish on day
1. On day 2, the cells were trypsinized and replated. On day 3, cells
were scraped, and
I-OS
binding was performed
as described previously
(9) . For binding experiments to the
soluble human sPLA
receptor, the medium from transfected
cells was centrifuged at 10,000
g for 10 min. Aliquots
of the supernatant were incubated with
I-OS
as described for the membrane-bound sPLA
receptor
(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.
receptor 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-OS
in the presence or absence of
300 n
M unlabeled OS
in 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-OS
was 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-OS
was 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
.
Isolation of cDNA Clones Encoding for the Human Kidney
sPLA
Previous experiments have shown that
the mRNA encoding the rabbit sPLAReceptor
receptor is strongly
expressed in rabbit lung
(28) . A cDNA fragment corresponding to
nucleotides 2564-4687 of the rabbit sPLA
receptor
(28) was therefore used to screen a human lung cDNA library
constructed in the
gt11 vector. 10
clones 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 sPLA
receptor
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 sPLA
receptor mRNA is expressed at a high level in kidney (see below).
In order to obtain the full-length cDNA clone encoding the human
sPLA
receptor, 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 sPLA
receptor. 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 sPLA
receptor. 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 sPLA
receptor. Therefore, these
clones have been used to construct the full-length cDNA encoding for
the human membrane-anchored sPLA
receptor (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 sPLA
receptor
(29) , the rabbit sPLA
receptor 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 sPLA
receptor 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) .
receptor 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 sPLA
receptor-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 sPLA
receptor.
receptors
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 sPLA
receptor, 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 sPLA
receptor 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
sPLA
receptor (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 sPLA
receptor. 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 sPLA
receptor 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
sPLA
receptor.
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 sPLA
receptor, 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 P
is the quantity of product measured for a cycle n, eff.
is the efficiency of amplification between two consecutive cycles, and
P
is 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 sPLA
receptor 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
10
mol and 3.74
10
mol 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 sPLA
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
receptor 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 sPLA
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
receptor
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 sPLA
In order to examine the pharmacological
properties of both the membrane-anchored and the soluble human
sPLAReceptor
receptors, transient expression experiments have been
made in COS cells. Binding experiments with
I-OS
revealed that this svPLA
binds with a high affinity
( K
= 1.5 n
M) to the two
expressed forms of the human sPLA
receptor
( B
values 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 sPLA
s show
that both forms of human sPLA
receptors display the same
pharmacological profile. OS
and OS
inhibit
I-OS
binding with K
values of 2 and 4 n
M, respectively (Fig. 7). Both
porcine and human pancreatic sPLA
s inhibit
I-OS
binding with K
values of approximately 400 n
M, respectively
(Fig. 7). Conversely, the bee venom sPLA
and the
human inflammatory group II sPLA
are without effect on
I-OS
binding (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-OS
binding (Fig. 7). These results
are significantly different from those obtained with the rabbit 180-kDa
sPLA
receptor that we had previously characterized and
cloned
(28) . The rabbit sPLA
receptor binds
svPLA
s with higher affinities ( K
= 7 p
M for OS
and 40 p
M for
OS
) than those determined for the presently cloned human
sPLA
receptor. 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 sPLA
receptor 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-OS
as 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
sPLA
s or mannosylated-BSA on the membrane-bound ( panel
A) or the soluble ( panel B) human sPLA
receptors. Results are expressed as percentage of the maximal
specific binding measured in the absence of competitor. 100%
corresponded to 1 p
M of
I-OS
specifically bound. Nonspecific binding was measured in the
presence of 100 n
M unlabeled OS
and represented
20% of the total binding.
Internalization of the Human Membrane-bound sPLA
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 Receptor
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-OS
internalization by
the human 180-kDa sPLA
receptor 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-OS
in the absence (total binding) or the presence (nonspecific
binding) of 300 n
M unlabeled OS
for 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.
receptor. This receptor is strongly homologous to its
rabbit and bovine counterparts
(28, 29) . The human
membrane-anchored sPLA
receptor 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 sPLA
or 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
sPLA
binds to the sPLA
receptor via one of its
CRDs.
(
)
receptor transcripts, there
exists a second type of messengers for the sPLA
receptor 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 sPLA
receptor. Splicing of this intron is required
for the production of a longer transcript that directs the synthesis of
the membrane-anchored form of the sPLA
receptor. 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.
receptor 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) .
receptor are present in comparable amounts in human kidney. This
result suggests that the soluble human sPLA
receptor might
have an important physiological role.
receptors indicates that both membrane-bound and
soluble human sPLA
receptors display the same binding
properties. Therefore, the soluble human sPLA
receptor can
compete for binding of sPLA
s with the membrane-bound
receptor. The binding properties of the human sPLA
s
receptors are very different from those of their rabbit, bovine, and
rat counterparts. Indeed, rat, bovine, and rabbit receptors recognize
the porcine group I sPLA
with high affinities
( K
values = 0.5-10
n
M) (
(27, 28) . The human sPLA
receptor recognizes this sPLA
as well as human
pancreatic sPLA
with a relatively weak affinity
( K
≅ 400 n
M). Another intriguing
result is that the human sPLA
receptor does not recognize
the human inflammatory group II sPLA
as 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
sPLA
s but rather to associate with one of the newly
discovered mammalian sPLA
s such as the Enhancing Factor, a
novel group II sPLA
(51) , or two other human
sPLA
s of unknown function which have been recently cloned
(52, 53) .
receptor is its ability to promote sPLA
internalization. This finding suggests that one of the possible
functions of this receptor is a role in the clearance of circulating
sPLA
, thus inhibiting sPLA
action.
Alternatively, another possible role of these sPLA
receptors could be to direct the action of sPLA
into
its target cells, in order to obtain, before sPLA
degradation occurs, a transient extracellular production of
second messengers of the arachidonic acid cascade.
,
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).
and 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.