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Complement Biology Group, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analogues of DAF have been isolated from erythrocytes of guinea pig (9), rabbit (10), mouse (11), and orangutan (12). Although DAF in each of these species was similar in many respects to the human protein, some interesting differences emerged. Guinea pig, rabbit, and orangutan DAF were all GPI anchored, whereas mouse DAF was not released by PIPLC in all tissues, indicating that it was in part transmembrane anchored (11). Guinea pig erythrocyte DAF was shown to comprise three distinct species with Mr of 55, 70, and 88 kDa, all of which had identical amino-terminal sequences (13). Clarification of these differences emerged from the cloning of DAF analogues from guinea pig (14), mouse (15, 16), and rat (17). In each species, several isoforms were identified, all of which contained the four-SCR structure that typifies human DAF, but with substantial variability in the ST region and the membrane anchor within and between species. In guinea pig and mouse, transmembrane and GPI-anchored isoforms were present, and in rat, guinea pig, and mouse, putative secreted forms were found. In most species these multiple isoforms have arisen by alternative splicing in a single gene, but in the mouse there is an added complication, in that there are two DAF genes, each alternatively spliced to generate several isoforms (15, 18). An alternative splice product has also been described for human DAF, a frame shift downstream of the region encoding the SCRs and ST resulting in a hydrophilic carboxyl terminus that is predicted to encode a secreted form of the protein (19). Expression in vivo of this alternatively spliced form of human DAF has not been demonstrated.
Regulation of C in the pig has become a subject of interest because of the planned use of pig organs for transplantation to man (20). To circumvent C-mediated hyperacute rejection, an inevitable consequence of pig-human transplants, pigs are now bred that express human C regulators on endothelium (21, 22). However, the contribution of the endogenous pig inhibitors to C regulation remains to be assessed. We have undertaken characterization of membrane regulators of C in the pig and have recently described the purification and molecular analysis of the pig analogues of human MCP and CD59 (23, 24). We here set out to identify and clone the pig analogue of human DAF. Cloning from a cDNA library and sequencing from tissue mRNA identified multiple isoforms of pig DAF, including transmembrane-anchored and GPI-anchored forms. Intriguingly, pig DAF differed substantially from human DAF and all other species analogues of DAF heretofore described in that all isoforms found contained only three SCR domains. We present a comprehensive analysis of this unique form of DAF and comment on the implications of these findings to current efforts in xenotransplantation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All general reagents were obtained from Fisher Scientific
(Loughborough, U.K.) or Sigma Aldrich (Poole, U.K.) unless otherwise
stated. The porcine muscle Uni-ZAP XR cDNA library was obtained from
Stratagene (La Jolla, CA). UltraSpec RNA isolation medium was purchased
from Biotecx (Houston, TX). RNase H– Superscript
reverse transcriptase, RNase H, terminal deoxynucleotide transferase,
and all oligonucleotides were obtained from Life Technologies (Paisley,
U.K.). Nick columns for radioactive probe purification, Taq polymerase,
restriction enzymes and buffers, Hybond-N nylon membranes, Rapid-Hyb
buffer, RediPrime DNA labeling system and
[
-32P]dCTP were purchased from Amersham
Pharmacia (Milton Keynes, U.K.). Deoxy-NTPs were purchased from Bioline
(London, U.K.). RNase inhibitor rRnasin and pGEM-T vector kit were
obtained from Promega (Southampton, U.K.). The GeneClean II DNA
purification kit was obtained from Anachem (Luton, U.K.). Plasmid and
mRNA purification kits were purchased from Qiagen (Dorking, U.K.). The
expression vector pDR2
EF1 (25) was a gift from Dr.
I. Anegon (Institut National de la Santé et de la Recherche
Médicale, Unité 437, Nantes, France) and contains the
hygromycin resistance gene, allowing the selection of stable colonies,
and the powerful chain elongation factor 1 promoter.
Tissues, cells, and sera
Pig blood was obtained fresh from the animal facility of the University of Wales College of Medicine or from a local abattoir, either without anticoagulant for collection of serum or with EDTA (20 mM final concentration) for cells and plasma. Serum was harvested and stored in aliquots at -70°C. Human serum was obtained from normal donors. Pig erythrocytes were separated from EDTA blood by centrifugation, careful removal of buffy coat, and washing in PBS as described previously (23). Pig leukocytes (total and separated granulocytes and mononuclear cells) were obtained by centrifugation of EDTA blood over Ficoll by standard methods as previously described (23). Pig tissues were obtained fresh from the facilities noted above and processed immediately for mRNA isolation. The pig cell lines IB-RS-2 (kidney) and ST (testis) were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were propagated in DMEM supplemented with 10% FCS, glutamine, pyruvate, essential amino acids, and penicillin/streptomycin.
Abs and proteins
Mouse mAb anti-human C3 (C3/30) was a gift from Dr. Peter Taylor (Novartis, Horsham, U.K.). Rabbit polyclonal anti-pig C3 antiserum was generated in-house by immunization with purified pig C3 in adjuvant. Rabbit and guinea pig anti-Chinese hamster ovary (CHO) cell antisera were generated by repeated immunization with cells in pertussis vaccine. FITC- and HRP-conjugated secondary Abs against mouse and rabbit Ig were obtained from Bio-Rad (Hemel Hempstead, U.K.). PE-conjugated secondary Abs against mouse and rabbit Ig were obtained from Dako (High Wycombe, U.K.) and Sigma Aldrich, respectively. Mouse mAb anti-pig CD59 (MEL-2 and MEL-3) and anti-human DAF (MBC1) were raised in this laboratory (24, 26). Mouse mAb anti-human DAF (BRIC216) was obtained from International Blood Group Reference Laboratories (Oxford, U.K.). Mouse mAb anti-pig MCP (6C5) was raised as previously described (23).
Screening of a pig cDNA library
A DNA fragment encompassing nucleotides +26 to +1131 (aa
residues -26 to +345) of the human DAF cDNA sequence (GenBank
accession no. M15799) was generated by a
SstII-NcoI digest of the expression vector
pDR2EF1-DAF (25). The fragment was isolated from an
agarose gel and labeled with [-32P]dCTP
using the RediPrime kit (Amersham) according to the manufacturer’s
protocol. The labeled probe was purified and used to screen 600,000
plaque colonies of the porcine aortic smooth muscle cDNA library plated
at 50,000 PFU/plate and grown on a lawn of XL1-Blue Escherichia
coli for 8 h. Duplicate lifts were taken onto Hybond-N nylon
membranes, denatured, UV cross-linked, and incubated with
32P-labeled probe (18 h at 45°C). Membranes
were washed and exposed to x-ray film.
Plaques positive on both duplicate membranes were identified, isolated
in Agar plugs, eluted in 1 ml of SM buffer (100 mM NaCl, 8 mM
MgSO4, 50 mM Tris-HCl, 0.01% eluate in pH 7.5) for 24
h, and replated. The above screening protocol was then repeated.
Individual positive plaques from the secondary screening were isolated
in agar plugs and eluted in SM buffer. Putative positive clones were
confirmed by PCR and Southern blotting. PCR was achieved using M13
forward (TGA CCG GCA GCA AAA TG) and reverse (AAG AGC TAT GAC GAT G)
phage primers at 94°C for 30 s, 56°C for 1 min (ramp 1.0),
and 72°C for 3 min for 25 cycles. The PCR products were resolved on
agarose gels, transferred to Hybond-N, and probed using the human DAF
probe described above. The cDNA inserts were recovered from PCR
screen-positive colonies using the Exassist/SOLR system (Stratagene, La
Jolla, CA). Individual bacterial colonies containing recombinant
phagemid were grown up in Luria Bertani broth containing 50 µg/ml
kanamycin, and phagemid DNA was purified using a QIAprep spin plasmid
mini-prep kit (Qiagen, Valencia, CA). Automated sequencing was
conducted in-house using an ABI model 377 DNA sequencer (Applied
Biosystems, Warrington, U.K.). Sequences were analyzed using PSORT-II
software (http://psort.ims.u-tokyo.ac.jp/).
Northern blotting, RT-PCR analysis, and Southern blotting
Total RNA was isolated from fresh pig tissues and cells using UltraSpec RNA isolation reagents (Biotecx) following the manufacturer’s instructions. mRNA was isolated from pig cell lines using the Oligotex mRNA purification kit (Qiagen). A pig DAF-specific cDNA probe encompassing SCR1–3 of pig DAF was generated by PCR amplification from pBluescript vector containing pig DAF sequence. The PCR used primers N5 (GAC TGC AGC CTT CCA CCC GA) and N7AS (ATC ACT CCA CTC AAC ATC ATT T) with the following reaction conditions: 94°C for 30 s, 61°C for 30 s (ramp 1.0), and 72°C for 40 s for 25 cycles. The probe was purified from an agarose gel and 32P-labeled as described above. A 32P-labeled cDNA probe specific for GAPDH was a gift from Dr. D. Llewellyn (University of Wales College of Medicine, Cardiff, U.K.). For Northern analysis, total RNA (15–20 µg) was separated on a 1% formaldehyde-agarose gel, transferred to Hybond-N membranes, and UV cross-linked. Membranes were probed for 18 h at 50°C with each of the probes, washed, and exposed to x-ray film.
For RT-PCR analysis, RNA was reverse transcribed using poly(dT) DNA primers. The presence of cDNA encoding the transmembrane and GPI isoforms of pig DAF was determined by PCR. The reaction used primer N1 (GAC TTG TTA TTT GGC GCA TCC) located in SCR3 with either primer pDAF-AS (GGT GGA TCC CTT TGG CTA AGT CAG CTA GCC) to amplify all isoforms of pig DAF or primer TM-AS (ATC GCC AGT CGC AGG TAA AT) chosen to specifically amplify only transmembrane-linked isoforms. Cycling conditions were 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 25 cycles. The PCR products obtained were resolved on a 1% agarose gel, blotted onto Hybond-N membranes, and subjected to denaturation and UV cross-linking as described above.
To generate a probe for Southern blotting of PCR products, a 459-bp DNA
fragment was PCR amplified using primers N1 and TM-AS as described
above and was purified by agarose gel electrophoresis. The purified DNA
fragment was labeled with alkaline phosphatase (AlkPhos Direct,
Amersham Pharmacia), following the manufacturer’s protocol. Membranes
were hybridized with the probe and washed, and the signal was detected
using CDP star detection reagent (Amersham Pharmacia). The positions of
all primers are shown in Fig. 1
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Total RNA was isolated as described above. The poly(dT) adaptor
primer QT (28 pmol) was used to reverse
transcribe mRNA from 2–5 µg of total RNA. The 3' ends of the cDNAs
so obtained were amplified using a modification of the RACE method as
previously described (24, 27). Nested PCR was performed
using vector primers Q0 and
Q1 derived from the QT
poly(dT) primer (28) and pig DAF-specific primers N1
(defined above) and N2 (CTT TTT CTC ATG TAA CGC AGG) designed from the
sequence obtained from clones isolated from the cDNA library and
located in SCR3. In the first amplification, 7% of the
QT-primed cDNA was amplified using 25 pmol of
primers Q0 and N1 with the following reaction
conditions: 94°C for 30 s, 64°C for 30 s (ramp 1.0), and
72°C for 3 min for 25 cycles. In the second amplification a 3-µl
aliquot of a 1/20 dilution of the first reaction was amplified using 25
pmol of Q1 and 25 pmol of N2 with the following
reaction conditions: 94°C for 30 s, 61°C for 30 s (ramp
1.0), and 72°C for 2 min for 25 cycles. The PCR products were
resolved on agarose gels, purified, and sequenced as described above.
The positions of all primers are shown in Fig. 1
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Generation of a pig DAF-Ig fusion protein and production of mAbs
cDNA encoding the three SCRs of pig DAF was cloned into the
expression vector Signal pIg Plus (R&D Systems, Abingdon, U.K.)
according to the manufacturer’s instructions. The cloning site was
located just upstream from DNA encoding the hinge and Fc regions of
human IgG1. DNA encoding DAF, Ab hinge, and Fc was amplified by PCR
using purified vector as template and was sequenced to confirm
fidelity. Primers incorporated restriction sites enabling subsequent
ligation into the expression vector pDR2EF1. CHO cells were
transfected with vector using Lipofectamine (Life Technologies,
Gaithersburg, MD) as described previously (24). A
stable line was established by selection with hygromycin B, and
culture supernatant containing pig DAF-Ig fusion protein (pDAF-Ig) was
collected. The fusion protein was purified by protein A affinity
chromatography (Prosep A, Bioprocessing, Consett, U.K.) and was used to
immunize mice. Hybridomas were generated using established protocols.
Supernatants were screened by ELISA on plates coated with the pig
DAF-Ig fusion protein or an irrelevant Ig-fusion protein as a negative
control. Positive clones were recloned to obtain monoclonal
populations.
Expression of pig DAF in CHO cells
Insert containing full-length cDNA encoding transmembrane pig
DAF was released from the pBluescript vector by digestion with enzymes
XbaI and NheI and was purified by agarose gel
electrophoresis. Full-length cDNA encoding GPI-anchored DAF was
obtained by RT-PCR from granulocyte mRNA using VENT polymerase (New
England Biolabs, Hitchin, U.K.) and primers PDAF-X (GGT TCT AGA GCG GTG
AGG CGC CTA ATG G) and PDAF-GPI (GGT GGA TCC CTT TGG CTA AGT CAG CTA
GCC). The primers contained, respectively, XbaI and
BamHI restriction sites. The PCR product was digested with
these enzymes. Each of the cDNAs obtained was ligated into
pDR2EF1 previously digested with XbaI and
BamHI. After electroporation into DH5, colonies were
picked, and the plasmids were purified. The fidelity and orientation of
each of the pig DAF cDNA in the vector were confirmed by sequencing.
CHO cells were Lipofectamine transfected with DAF-containing vector,
with empty expression vector (negative control), or with expression
vector containing human DAF cDNA as described previously
(24). Surface expression and the nature of the membrane
anchor were demonstrated by flow cytometry as described below.
Expression was confirmed by SDS-PAGE and Western blotting of cell
lysates using anti-pig DAF mAb MBC-PD1. Lysates from pig
erythrocytes, leukocytes, granulocytes, and platelets were blotted in a
similar manner.
Flow cytometry
Cells were harvested, washed three times with PBS, and resuspended at 106/ml in FACS buffer (1% BSA and 0.2% NaN3 in PBS). All staining steps were conducted on ice. Cells were incubated with primary Ab (2–5 µg/ml) for 30 min, washed three times with FACS buffer, and incubated for 30 min with a PE- or FITC-conjugated secondary Ab. Cells were washed three times in FACS buffer and fixed with 1% formaldehyde in FACS buffer. Fluorescence was measured on a Becton Dickinson FACScalibur (San Jose, CA).
Susceptibility to cleavage by PIPLC was assessed by incubation of cells (2 x 106/ml in PBS) at 37°C for 30 min with PIPLC (0.14 U/ml final concentration; Roche, Lewes, U.K.). Cells were washed, stained as described above with mAb MBC-PD1 (anti-pig DAF), BRIC216 (anti-human DAF), JM4C8 (anti-pig MCP), or MEL-2 (anti-pig CD59), and analyzed by flow cytometry.
C3 deposition assay
CHO cells transfected with transmembrane pig DAF, human DAF, or vector control were harvested by incubation in PBS, 20 mM EDTA, and 1% (w/v) BSA. Cells (106/ml) were incubated for 30 min at 4°C with 10% (v/v) rabbit anti-CHO antiserum (for activation of human C) or 0.7% (v/v) guinea pig anti-CHO antiserum (for activation of pig C). Both anti-CHO antisera were heat-treated (56°C, 30 min) to inactivate C before use. Cells were washed twice in C fixation diluent (CFD; Oxoid, Basingstoke, U.K.) containing 1% BSA, resuspended at 106/ml, and incubated with an equal volume of a dilution in CFD of either C8-depleted human serum or normal pig serum for 25 min at 37°C. Cell viability was monitored by trypan blue exclusion. Cells were washed twice in FACS buffer and stained for C3 deposition using mAb anti-human C3 (C3-30; 2 µg/ml) for detection of human C3 fragments or rabbit polyclonal anti-pig C3 (1/250 dilution) for detection of pig C3, followed by either rabbit anti-mouse Ig-FITC (Dako) or goat anti-rabbit Ig-FITC (Harlan Seralab, Crawley Down, U.K.). C-activating antisera were selected for negligible cross-reactivity with these secondary Abs. C3 deposition was measured by flow cytometry.
Calcein release assay
CHO cells were seeded into 24-well plates 48 h before the assay. Hygromycin B was omitted from the culture medium, and cell density was adjusted to yield approximately 70% confluence at the time of assay. Cells were incubated at 37°C for 30 min with 200 µl of calcein-AM (Molecular Probes, Portland, OR) at 2 µg/ml in culture medium containing 5% FCS. Calcein-loaded cells were rinsed once in ice-cold CFD/1% BSA and incubated at 4°C for 30 min in 175 µl of the same buffer containing 10% heat-inactivated rabbit anti-CHO antiserum. Cells were rinsed once with CFD/1% BSA and incubated at 37°C for 30 min with 200 µl of a dilution of either normal pig or human serum. Plates were chilled on ice, and supernatant was transferred to a flat-bottom 96-well plate for analysis. Calcein remaining in cells was released by incubation for 15 min with 200 µl of 0.1% Triton X-100 in CFD/1% BSA. Calcein released by serum or detergent was measured in a Wellfluor fluorometer (Life Sciences International, Basingstoke, U.K.) with excitation at 485 nm and emission at 530 nm. The percent calcein released by serum was calculated as follows: % release = 100 x calcein released by serum/(calcein released by serum + calcein released by detergent). Background release was assessed by incubation with buffer only. For function blocking studies, 20 µg/ml MBC-PD3 function-blocking anti-DAF mAb or MBC-PD1 nonblocking control mAb (both IgG1 isotype) was included with the sensitizing Ab.
Function blocking studies on pig erythrocytes
Classical pathway. Pig E were sensitized by incubation of E (2% (v/v) in PBS) for 30 min at 37°C with a 1/20 dilution of a cross-reactive rabbit polyclonal antiserum raised against pig aortic endothelial cells (prepared in-house). Ab-sensitized E were washed in CFD/1% BSA and resuspended to 1% (v/v). AB-sensitized E (50 µl) was incubated for 20 min at 37°C with 50 µl of CFD/BSA containing MEL3 (function-blocking anti-pig CD59) (24), 6C5 (function-blocking anti-MCP) (23), and PD2 (anti-pig DAF, nonfunction blocking), PD3 (anti-pig DAF, function blocking), or an irrelevant isotype control Ab (20 µg/ml of each Ab). Pig or human serum (50 µl of an appropriate dilution) was added to the cells, mixed, and incubated for 45 min at 37°C. Cells were pelleted by centrifugation, and hemoglobin release was assessed by absorbance of the supernatant at 415 nm. Background release (0%) and 100% release were measured by incubating cells with buffer only or with 0.05% Triton X-100, respectively; absorbance of each serum dilution was also measured: % lysis = 100 x (A415 sample - A415 serum - A415 0% control)/(A415 100% - A415 0%).
Alternative pathway. Pig E (10% (v/v) in 50 mM sodium acetate (pH 5.5), 150 mM NaCl, 0.1% CaCl2·2H2O) were incubated with neuraminidase (0.1 U/ml; Roche) for 1 h at 37°C. Cells were washed and resuspended at 1% (v/v) in alternative pathway buffer (5 mM sodium barbitone, pH 7.4, containing 150 mM NaCl, 7 mM MgCl2, 10 mM EGTA, and 1% BSA). The assay set-up and calculations were identical with those described above, except that MEL-2 was used to block the function of CD59.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Screening of a porcine muscle cDNA library identified a single
positive clone from a total of 6 x 105
screened. The clone comprised 1546 bp and contained a single open
reading frame encoding a 37-aa signal peptide, a 187-aa region
containing three SCR domains, a 61-aa ST-rich region, a 19-aa sequence
compatible with a transmembrane anchor, and a 37-aa putative
cytoplasmic tail (Fig. 1). Comparison with the sequence of human DAF
revealed amino acid identity of 64% through the three SCRs (67% for
SCR1, 67% for SCR2, 57% for SCR3), with all Cys residues conserved
between species (Fig. 2). These data
strongly suggested that the cDNA isolated was a pig analogue of DAF. No
sequence resembling SCR4 in human DAF was found. The first 20 aa of
the ST-rich region were 55% identical with the first 20 aa of ST-a in
human DAF. The next 34 aa of the ST-rich sequence were 39% identical
with sequence in ST-b of human DAF. The final 7 aa of the ST-rich
sequence were 57% identical with ST-c in human DAF. The putative
transmembrane segment was 42% identical with the transmembrane region
in guinea pig DAF. The cytoplasmic domain also had some homology with
that in guinea pig DAF (38%). This isoform is henceforth referred to
as pDAFtm.
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). This new sequence was 56%
identical with the carboxyl terminus of human DAF and provided a
putative GPI anchor addition signal; this isoform is henceforth termed
pDAFgpi. The predicted site of GPI anchor addition, based upon the
consensus GPI signal and comparison with human DAF, is at S241. Another
cDNA isolated from granulocytes was identical with that described
above, except that the 21-bp sequence encoding ST-c was deleted. The
GPI anchor addition consensus signal was retained in this isoform with
the predicted anchor addition site at S234. All DAF isoforms contained
three potential N-glycosylation sites, the first at the
junction of SCR1 and SCR2 (N60), the second in SCR2 (N113), and the
third in SCR3 (N140). Only the first of these is present in human
DAF. Pig DAF mRNA is heterogeneous and broadly expressed in tissues
To estimate the size of pig DAF mRNA, total RNA freshly extracted
from pig spleen, liver, and testis was analyzed by Northern blotting.
The probe used contained sequence corresponding to SCR1–3 of pig DAF
and should detect all forms of pig DAF. As shown in Fig. 3A, multiple bands were
detected in each of the tissues. The major bands were present at
approximately 3.3, 2.8, and 1.6 kb. These same bands were present in
mRNA isolated from pig cell lines (Fig. 3B). The GAPDH probe
gave similar intensity of staining in each tissue sample (not
shown).
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A) or a primer from a portion
of the 3'-untranslated region common to both transmembrane and GPI
isoforms (Fig. 4B). The predicted length of the product for
pDAFtm in the first reaction was 458 bp. The predicted lengths of the
products for pDAFtm and pDAFgpi in the second reaction were 571 and 395
bp, respectively. The pDAFtm-specific reaction (Fig. 4A)
demonstrated that mRNA encoding pDAFtm was widely distributed and was
present in all tissues tested, albeit very weakly in granulocytes. In
contrast, pDAFgpi expression was abundant in granulocytes and also in
lung, but was essentially absent from testis and kidney (Fig. 4B). In each reaction the major products were of the
predicted size, but several larger products were obtained. Southern
blotting of the gels using a probe specific for the common ST region
demonstrated that all the bands obtained were derived from pig DAF
mRNAs (data not shown). Additional large bands (
1 and 1.2 kbp) were
particularly abundant in granulocytes, and these products were
sequenced. None contained sequence suggestive of a fourth SCR. Instead,
each contained 606 bp of additional ST-a sequence encoding 202 aa,
including 10 copies of a 17-residue repeating unit (Fig. 5). A 34-aa homology unit was also
apparent in this novel ST sequence, each pair of 17 residue repeats
being highly homologous with the next (Fig. 5). The larger band (1.2
kbp) encoded a transmembrane-anchored isoform, and the smaller (1 kbp)
encoded a GPI-anchored isoform.
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Pig DAF protein is expressed on all circulating cells, but anchoring differs between cell types
None of the available mAb against human, rat, or mouse DAF stained
pig cells. mAb specific for pig DAF were therefore generated using a
pig DAF-Ig fusion protein as immunogen. Several mAb were obtained,
three of which (MBC-PD1, MBC-PD2, and MBC-PD3; all IgG1 isotype),
were shown by flow cytometry to stain pig cells and CHO cells
transfected with cDNA encoding pDAFtm. The expression of pig DAF
was strong on erythrocytes, and treatment with PIPLC failed to release
the protein, indicating that erythrocyte pig DAF was transmembrane
anchored (Fig. 6). As a control, the same
dose of PIPLC was shown to efficiently release GPI-anchored pig CD59
from pig erythrocytes (Fig. 6). The transmembrane nature of pig
erythrocyte DAF was further confirmed by demonstrating that the
purified protein failed to incorporate into sheep erythrocytes (data
not included). Unfractionated PBMC gave a broad staining distribution
for pig DAF, and PIPLC treatment caused a small reduction in
expression. In contrast, isolated granulocytes strongly expressed DAF,
and treatment with PIPLC reduced expression by almost 90% (Fig. 6).
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). From erythrocyte lysates, pig DAF ran
as a broad band of approximately 45–52 kDa; from lysates of
mononuclear cells and platelets, pig DAF gave a sharp band with an
apparent Mr of 52 kDa; granulocyte
lysates gave a broad band of 45–50 kDa. High
Mr bands were apparent, particularly
in erythrocytes and platelets, which presumably represent DAF
aggregates. None of the mAb recognized pig DAF following reduction
(data not shown).
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Stable populations of CHO cells expressing pDAFtm, pDAFgpi, the
ST-c-deleted isoform of pDAFgpi, or human DAF were generated. The
pDR2EF1 vector was chosen because it is reported to give
reproducible high levels of expression of different cDNAs in a given
cell type (29, 30). It was therefore anticipated that
similar levels of expression of human and pig DAF would be achieved.
Expression was assessed by flow cytometry and Western blotting as
described above, using BRIC216 anti-human DAF and MBC-PD1
anti-pig DAF. Uniformly high levels of expression were obtained for
all proteins (Table I). Neither of the
mAb recognized vector control cells. BRIC216 was negative on pig
DAF-transfected cells, and MBC-PD1 was negative on human
DAF-transfected cells. Although precise comparison of expression level
based upon staining with different reagents is not possible, the data
suggested that human DAF and the different pig DAF isoforms were
expressed at similar levels (Table I). The level of expression of pig
DAFtm in the transfected CHO cells was 14-fold that of endogenous DAF
on pig erythrocytes, as assessed by flow cytometry (data not shown).
Treatment of human DAF transfectants with PIPLC decreased the mean cell
fluorescence by 75%; PIPLC treatment caused no reduction in expression
of pig DAFtm, but caused a 65–75% decrease in expression for each of
the pDAFgpi isoforms, confirming the predicted modes of anchoring
(Table I).
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) identified a major band at approximately 55 kDa and
a minor band, presumably unglycosylated protein, at 36 kDa. Blotting of
CHO expressing each of the pDAFgpi isoforms gave a band of
approximately 50 kDa (data not shown). Functional analysis of pDAFtm
Protection against C was assessed in transfected CHO cells by
measurement of C3b deposition and inhibition of C lysis. Deposition of
either pig or human C3b on CHO cells transfected with pDAFtm was
measured by flow cytometry and compared with C3b deposition on human
DAF-transfected CHO cells and cells transfected with vector alone as a
control. Following incubation with pig serum, high levels of C3b were
detected on vector control cells whereas cells expressing human DAF
bound almost no C3b, even at the highest serum doses (Fig. 8A). Cells expressing pDAFtm
did not differ significantly from vector control cells, suggesting that
pDAFtm had little or no effect on accumulation of pig C3b. Following
incubation with human serum, human DAF again efficiently prevented
accumulation of C3b compared with vector controls (Fig. 8B).
Expression of pDAFtm also significantly reduced accumulation of human
C3b, albeit less efficiently than human DAF (Fig. 8B).
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). Expressed human DAF markedly
inhibited lysis by human and pig C, whereas expressed pig DAF inhibited
human C, albeit less efficiently, but had negligible activity against
pig C (Fig. 9). To confirm that the observed lytic protection against
human C was DAF mediated, pDAFtm-expressing cells were preincubated
with the function-blocking anti-pig DAF mAb MBC-PD3 or the
nonblocking mAb MBC-PD1. Preincubation with the function-blocking mAb
eliminated the observed protective effect against human C, whereas
incubation with the isotype-matched nonblocker did not (Fig. 9C).
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, A and
B). These data support the results obtained in CHO
transfectants. Under AP conditions, blocking pig DAF had no effect on
lysis of pig E by either pig or human serum (Fig. 10, C and
D), indicating that pig DAF does not function as an AP
regulator.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We here describe the identification and characterization of the pig analogue of human DAF. Several isoforms of pig DAF were identified, both tm and GPI anchored and with variability in the length of the ST region. All isoforms of pDAF contained only three SCR domains, homologous with the first three SCRs in human DAF. The ligand binding site described above, comprising three tandem Lys residues in the groove between SCR2 and SCR3 and adjacent hydrophobic residues (8), is well conserved in pig DAF. Several approaches, including direct sequencing of cell and tissue-derived mRNA, and RT-PCR or 3'-RACE followed by sequencing, were used in an attempt to find a DAF isoform containing a fourth SCR in a large panel of cells and tissues and from different strains of pig (large white and laboratory mini-pigs). No such sequence was identified. From these data we conclude that DAF in pigs is unique in that it lacks SCR4. The process by which SCR4 has been lost is not yet known. In man SCR4 is encoded in a single exon, exon 6 of the Daf gene. Two possibilities exist to explain the loss of SCR4 in pigs, either the coding exon has been lost from the gene or it is present but not transcribed. Screening of a pig genomic library with a human SCR4-derived probe has yielded negative results. Current efforts are focussed on sequencing between exons encoding the carboxyl-terminal portion of SCR3 and ST-a in genomic clones obtained from the library, a distance in excess of 11 kbp.
The longest ST region, obtained from granulocyte RNA, contained an
insert of 202 aa of additional ST-a sequence, composed almost entirely
of 10 repeats of a 17-aa unit homologous with sequence in ST-a of human
DAF (Fig. 5). A second homology unit comprised sequential pairs of the
17-mer repeats, yielding five highly homologous 34-aa repeats. A
similar 17-aa ST repeating unit was described in guinea pig DAF and was
shown to be responsible for variation in ST region length
(14). The tm region in pDAFtm was homologous with the tm
regions in guinea pig and mouse tm DAF. Of note, the predicted
membrane-spanning region in pig DAF includes the last four amino acids
of ST-c. The cytoplasmic region was homologous with that in guinea pig
DAF and contained a consensus ATP/GTP binding site (P-loop) motif
(GFWHYGKS), the significance of which is unknown (Fig. 2) (15, 36).
Northern and RT-PCR analysis of RNA extracted from pig tissues and cell
lines indicated that pig DAF was widely expressed, although the
expression levels varied between tissues (Figs. 3 and 4). These
analyses also identified the presence of multiple isoforms of DAF,
differentially expressed in the various tissues. To identify the
expression of the major isoforms in tissues and cells, RT-PCR was
performed using primers specific for pDAFtm or capable of amplifying
all pDAF isoforms. These studies demonstrated that pDAFtm was broadly
expressed, whereas pDAFgpi was significantly expressed only in
granulocytes and lung (Fig. 4). Granulocytes expressed predominantly
pDAFgpi.
Flow cytometric analyses using mAb generated against a pDAF-Ig fusion
protein confirmed the abundant expression of pDAF on circulating cells
(Fig. 6). Pig erythrocyte DAF was not released by PIPLC treatment,
although pig CD59 on the same cells was efficiently released. The
transmembrane anchoring of DAF on pig erythrocytes was confirmed by
immunoaffinity purification from erythrocytes, demonstrating that the
protein did not incorporate into guinea pig erythrocytes, a property of
the GPI anchor in human DAF. In sharp contrast, treatment of
granulocytes with PIPLC efficiently released pDAF, as predicted from
the mRNA analyses. Mononuclear cells expressed predominantly pDAFtm.
This result parallels the situation described for Fc
RIII (CD16) in
humans. GPI-anchored CD16 is expressed by granulocytes, whereas a tm
form is expressed by macrophages and NK cells (37). Early
studies on mouse DAF suggested that the protein on erythrocytes was tm
anchored (15). However, recent studies contradict this;
mice in which the gene encoding GPI-anchored DAF has been deleted
apparently lack DAF on erythrocytes, which are consequently susceptible
to C lysis (38).
The most abundant form of pig DAF thus differs from human DAF in two
important respects: it is tm anchored, and it lacks SCR4.
Transmembrane-anchored forms of human DAF have been engineered and
expressed, and C regulatory activity compared with that of GPI DAF
(39). Both forms were equally effective at inhibiting C,
making it unlikely that tm anchoring adversely affects function in pig
DAF. The degree to which deletion of SCR4 in human DAF compromises C
regulatory activity is unclear. One study reported that human DAF
lacking SCR4 had no C inhibitory activity when expressed on CHO cells
(6). A more recent study in which DAF lacking SCR4 was
examined as a soluble protein or after incorporation into erythrocytes
described inhibition of the classical, but not the alternative,
activation pathway (7). To assess the capacity of pDAFtm
to regulate C we generated stable transfectants in CHO cells abundantly
expressing the molecule. CHO cells expressing either pig or human DAF
showed a single homogeneous population with high expression of the
regulator. Transmembrane anchoring of the expressed pDAFtm was
confirmed by the failure of PIPLC to cause release. Cells were compared
in assays of classical pathway C regulation. In C3b deposition assays,
pDAFtm inhibited human C, albeit less efficiently than human DAF
expressed in the same system, but had no significant activity against
pig C (Fig. 8). In lytic assays, pDAFtm inhibited human C, but again
had a negligible influence on pig C lysis, whereas human DAF was
strongly inhibitory for both human and pig C (Fig. 9). A
blocking anti-pig DAF mAb reversed protection conferred by
expressed pig DAF.
To confirm these findings in a different setting, the effect upon lytic susceptibility of blocking DAF on pig erythrocytes was assessed. The blocking mAb enhanced CP lysis by human serum, but not by pig serum; a control nonblocker had no effect. These data confirm that pDAFtm, the sole isoform expressed on pig erythrocytes and the major isoform expressed on most other cells, can inhibit human C, but has no significant C regulatory activity against pig C when tested in vitro in a classical pathway assay. These findings have been replicated in a classical pathway hemolysis assay using soluble recombinant pig DAF; regulation of human, but not pig, C was obtained (data not included). Treatment of pig E with neuraminidase rendered the cells activating for the AP. Blocking of pig DAF with mAb had no effect on lysis under AP conditions regardless of the serum source, indicating that pig DAF does not regulate the AP of pig or human C. These data are not surprising, in that others have reported that SCR4 is essential for AP regulatory activity of human DAF (7).
The demonstration that pig DAF does not significantly regulate pig C is most unexpected. Pig erythrocytes, unlike human erythrocytes, abundantly express MCP, and MCP is an important C regulator on these cells (23). It is therefore possible that pig erythrocytes and other plasma-exposed cells are adequately protected from C activation by MCP. An alternative possibility is that pigs might express another membrane regulator, perhaps a molecule analogous to Crry in rodents. Further, it remains possible that other isoforms of pig DAF may retain regulatory capacity for pig C. We are currently examining C regulatory function in pDAFgpi and in isoforms with different lengths of STP. Guinea pig DAF isoforms with long STP regions were better regulators of C (40), and it has previously been reported that deletion of the ST region in human DAF ablates C regulatory activity (6). The role of the ST region is to act as a spacer to place the active sites contained within the SCRs at an appropriate distance from the membrane. It is possible that the long ST isoforms of pig DAF will show increased pig C regulatory activity. We are also engineering pDAFtm to express a fourth SCR to examine whether this confers pig C regulatory capacity on the molecule.
What, then, is the role of DAF on pig erythrocytes? One may speculate that pig DAF serves roles other than C regulation on pig erythrocytes and other circulating cells, possibly serving as a C receptor with roles in handling immune complexes. In man this role is served by CR1, and no analogue of CR1 has yet been discovered in the pig. Pig DAF may have evolved to fill this role and, in the process, lost the capacity for intrinsic C regulation. The long ST isoforms, projecting the SCRs far from the membrane, might be particularly relevant to immune complex handling. Experiments to test this hypothesis are currently in progress.
It remains puzzling that pigs alone of all the species studied to date have lost SCR4 in DAF. In humans, SCR4 is implicated in the binding of E. coli adhesins and enteroviruses (33, 34). It is possible that loss of SCR4 in pigs has been selected in evolution, because it renders cells resistant to infection with these pathogens. Our preliminary data support this hypothesis in that echoviruses that use human DAF as receptor cannot bind pig DAF. Precedent exists in that MCP, which contains a measles virus binding site in SCR1, is expressed in New World monkeys as a three SCR molecule lacking SCR1 and unable to bind measles virus (41). A concern emerging from these studies is that pig organs engineered to hyperexpress human DAF for use in xenotransplantation (42) might be rendered susceptible to infection by these common human pathogens with potentially disastrous consequences.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. B. Paul Morgan, Complement Biology Group, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom CF14 4XX.
3 Abbreviations used in this paper: CRP, complement regulatory protein; CFD, C fixation diluent; CHO, Chinese hamster ovary cells; DAF, decay-accelerating factor; MCP, membrane cofactor protein; PIPLC, phosphatidylinositol-specific phospholipase C; SCR, short consensus repeat; ST, serine/threonine; tm, transmembrane; UTR, untranslated region; AP, alternative pathway; RACE, rapid amplification of cDNA ends.
Received for publication February 8, 2000. Accepted for publication June 16, 2000.
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.
Arrows indicate the predicted sites of GPI anchor addition in isoforms
of pDAFgpi with and without ST-c sequence. GenBank accession numbers
are 
) or pig DAF (
) or vector control
cells (
) were incubated with dilutions of normal pig serum
(A) or C8-depleted human serum (B). C3b
deposition was assessed by flow cytometry using anti-pig C3 or
anti-human C3 Abs, respectively. Results are the means of three
determinations ± SD. No lysis of cells was observed using
C8-depleted human serum; up to 60% lysis of vector control and pig
DAF-expressing cells was observed following incubation with a 1/4
dilution of normal pig serum, whereas cells expressing human DAF were
not lysed.
) or in the presence of a nonblocking (
) or blocking
(
) mAb anti-pig DAF. All cells were then incubated with a 1/8
dilution of human serum. Calcein released by serum was calculated.
Results are the means of three determinations ± SD. 
), or an irrelevant isotype control Ab (