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Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan;
Division of Environmental Pharmacology, Department of Pharmaceutical Sciences, Osaka University, Osaka, Japan; and
Department of Molecular Immunology, Nara Institute for Science and Technology, Ikoma, Japan
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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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60 aa, termed the short consensus
repeat (SCR), and their genes are clustered in 1q32 (3, 4). All of these proteins physically bind C3 fragments
(3). It is currently accepted that the role of the soluble
SCR C regulatory proteins is to prevent excess C activation in the
fluid phase, whereas the role of DAF and MCP is to protect host cells
on the same membrane from attack by autologous C (3). CR1
and CR2 are involved in immune complex clearance in addition to C
regulation and enhancement of Ag-C3d-dependent activation of B cells,
respectively (5, 6, 7, 8). In rodents, these sets of SCR proteins are conserved, although their distribution profiles and predicted roles are not always consistent with those of humans. In addition, a specific SCR protein, C receptor 1-related protein y (Crry), has been identified (9, 10) that like DAF and MCP protects the rodent cells from complement attack (11, 12). However, in nonmammalian lower vertebrates the membrane-regulatory system of C has not been identified. Soluble form C regulators were presumed to exist in lower vertebrates and some invertebrates (13, 14, 15, 16), and the fluid phase C regulatory system has been hypothesized to have evolved simultaneously with C proteins. However, no membrane-associated forms of C regulators have yet been identified in lower vertebrates other than mammals.
In this study, we cloned a cDNA encoding a novel transmembrane SCR protein in the chicken, which we named chicken C regulatory membrane protein (Cremp). Chicken Cremp was first identified in a chicken cDNA library as a fragment containing the consensus sequence shared with MCP of all species reported to date (17, 18). The predicted amino acid sequence of Cremp suggests a hybrid consisting of human DAF-like and MCP-like sequences with five complete and one incomplete SCR domains. This protein was shown to protect mammalian transfectants from chicken C-mediated cytolysis. Thus, this protein is the first nonmammalian cell-associated C regulator with the ability to protect cells from attack by homologous C. We propose the evolutional importance of membrane-associated C regulatory proteins in parallel with C proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh chicken, human, and rabbit sera were obtained from each species by the standard method (19). All serum samples were immediately stored at -80°C until use. Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). RK13 cells (derived from rabbit kidney) were obtained from RIKEN Cell Bank (Wako, Saitima, Japan). DT40 cells (derived from chicken B lymphocyte) were a gift from Dr. S. Takeda (Kyoto University, Kyoto, Japan). For RNA and protein blot analysis, total RNA and proteins were obtained from each chicken tissue (excel link, 20 wk) and were stored at -80°C until use.
Screening a chicken cDNA library
Chicken thymus cDNA library was a gift from Dr. R. Goizuka
(Tokyo Scientific University, Tokyo, Japan). The cDNA fragment was
ligated into the mammalian expression vector pME18s at the
BstXI site. A partial Cremp cDNA fragment was obtained by
nested PCR using a degenerate primer and a vector (pME 18s)-specific
primer (Fig. 1
). One degenerate primer was designed from the junctional
sequence of SCR3 and SCR4 of human MCP (hMCP) as described previously
for molecular cloning of mouse MCP (18). First PCR was
performed as follows: denaturation at 94°C for 2 min, 1 cycle,
denaturation at 94°C for 30 s, annealing at 50°C for 1 min,
polymerization at 72°C for 90 s, 20 cycles. In the second PCR, 1
µl of the first PCR product was added to the PCR mixture of the
nested PCR primers, and sequential PCR was performed under the same
conditions for 35 cycles. The PCR products were cloned into the vector
pCR-2.1 (Invitrogen, San Diego, CA) and subjected to DNA sequencing
using an ABI 377 sequencer.
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The complete Cremp sequence was confirmed from chicken lung mRNA (white
leghorn) by sequencing twelve RT-PCR amplicons. Tissue RNA blotting analysis
Total RNAs (20 µg) were extracted from various chicken tissues using TRIZOL Reagent (Life Technologies, Grand Island, NY) and separated in a 1.0% (w/v) agarose gel. RNAs were transferred onto Hybond-N+ membrane (Amersham, Arlington Height, IL), which was prehybridized for 30 min at 68°C and hybridized for 1 h at 68°C in Express Hybridization buffer (Clontech, Palo Alto, CA) with 32P-labeled full-length Cremp ORF as a probe. After washing, the membrane was exposed to x-ray film at -80°C. The blot was rehybridized with a GAPDH probe as a control.
Preparation of rabbit anti-Cremp polyclonal Ab
To prepare a rabbit polyclonal Ab against Cremp (anti-Cremp Ab), RK13 cells (1 x 107) were transiently transfected with pME18s-Cremp-His tag using lipofectAMINE Reagent (Life Technologies). After 2 days, transfected RK13 cells were collected with 10 mM EDTA-PBS, washed with PBS three times, and suspended in 0.5 ml of PBS. Then, RK13 cell suspensions were mixed with 0.6 ml of Freund’s complete adjuvant (FCA) (Difco, Detroit, MI) and extensively agitated. The mixture was used to immunize rabbits. Immunization was performed four times at 7-day intervals, and boosted before drawing blood. The antisera were harvested by centrifugation. IgG was purified from the sera according to the standard method.
Tissue protein blotting analysis
Various chicken tissues were solubilized with lysis buffer containing 1% (v/v) Nonidet P-40, 0.14 M NaCl, 0.01 M EDTA, 20 mM Tris-HCl (pH 7.4), 1 mg/ml iodoacetamide (IAA), and 1 mM PMSF using a Potter type homogenizer. The tissue suspension was centrifuged at 15,000 rpm for 30 min at 4°C, the pellet was removed, and the supernatant was subjected to SDS-PAGE followed by transblotting onto polyvinylidene difluoride (PVDF) membranes by the method described previously (20). Cremp was detected with anti-Cremp Ab, peroxidase-conjugated secondary Ab (Cappel, West Chester, PA), and chemiluminescence (ECL system, Amersham).
Establishment of stable transfectants expressing Cremp
The cloned Cremp cDNA was ligated into the mammalian expression vector pCXN-2 (21), and CHO cells were transfected with this vector using lipofectAMINE. Thereafter, transfected cells were selected in medium containing 0.6 mg/ml of G418 (Life Technologies). The mean fluorescence shifts (MFS) of CHO cells expressing Cremp were assessed by flow cytometry using anti-Cremp Ab.
Flow cytometry
Transfected CHO cells (5x105) were incubated with 30 µl of 20 µg/ml anti-Cremp Ab or 30 µl of 5 µg/ml mAb against hMCP (M177), which recognizes SCR2 of hMCP, for 1 h at 4°C. After three washes, cells were treated with FITC-conjugated secondary Ab. The stained cells were analyzed using a FACSCalibur. Mean fluorescence intensity was evaluated on the attached computer software, Cell Quest.
Immunofluorescence analysis of transfected cells
CHO cells expressing Cremp (CHO/Cremp) were incubated with 100 µl of 2 µg/ml anti-Cremp Ab for 1 h at 37°C in PBS containing 1% (w/v) BSA. The cells were washed, incubated with a 1:100 dilution of FITC-conjugated anti-rabbit IgG (Cappel) for 30 min at 37°C in PBS containing 10% (w/v) Block Ace (Yukijirushi, Sapporo, Japan), washed, and mounted on glass slides in PBS containing 2.3% 1,4-diazabiccyclo-2-octane and 50% glycerol. The stained cells were visualized at x40 magnification under a FLUOVIEW (Olympus, Tokyo, Japan). Images were captured using the attached computer software, FLUOVIEW.
Deglycosylation assay of Cremp
The methods for analyses using deglycosidases were described previously (20). Briefly, each transfectant (5 x 106) was solubilized with solubilization buffer containing 1% Nonidet P-40, 50 mM Tris-maleate (pH 8.6), 10 mM EDTA, 1 mg/ml IAA, 1 mM PMSF for O-glycosidase, or 1% Nonidet P-40, 20 mM Tris-maleate (pH 6.0), 10 mM EDTA, 1 mg/ml IAA, and 1 mM PMSF for N-glycosidase. Solubilized proteins were centrifuged at 15,000 rpm for 30 min at 4°C, the pellets were removed, and the supernatants were incubated with 100 µU of neuraminidase (Sigma) for 1 h at 37°C. Then, the samples were treated with either 250 mU of N-glycosidase or 1 mU of O-glycosidase (Genzyme, Cambridge, MA) for 16 h at 37°C. The samples were subjected to SDS-PAGE followed by immunoblotting. Cremp protein was detected with anti-Cremp Ab.
51Cr release cytotoxicity assay
For cytotoxicity assay, 1 x 107 transfected CHO cells were collected in 10 mM EDTA-PBS and incubated with 100 µl (3.7 MBq) of Na251CrO4 for 60 min at 37°C in 1 ml of serum-free Ham’s F12 medium. After three washes with PBS, 2 x 104 51Cr-labeled cells in 50 µl of Ham’s F12 supplemented with 10% FCS were placed in the wells of 96-well plates and incubated with 50 µl of various concentrations of rabbit Ab against CHO cells (precipitated with 33% ammonium sulfate) in gelatin veronal buffer (GVB)2+ or 0.03 M EGTA-GVB for 30 min at 4°C. Cells were subsequently incubated with 100 µl of various concentrations of chicken, human, or rabbit serum diluted in GVB2+ or 0.03 M EGTA-GVB for 60 min at 37°C with gentle shaking. GVB2+ and 0.03 M EGTA-GVB represent the conditions for activation of the classical and the alternative C pathway, respectively, which was true in chicken C according to the criteria of C4b deposition (data not shown). The plates were centrifuged at 1500 rpm for 5 min, and radioactivity in aliquots of 150 µl of supernatants were measured with an auto gamma counter. The percentage of cytotoxicity was calculated as follows: (sample cpm - control cpm)/(MAX cpm - control cpm) x 100. Untreated CHO cells were used to measure spontaneous 51Cr release (control cpm), and cells treated with 5% Triton X-100 were used to measure maximum release (MAX cpm). The experiments were performed three times in triplicate. Because we used the different sources of C in each assay to measure percent inhibition of cytotoxicity, the data show relative C-regulatory potencies of each C-regulatory protein.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of this study was the molecular cloning of nonmammalian
MCP homolog. The chicken was chosen because a cell-level gene
disruption system is available using the chicken B cell line DT40
(22). A set of degenerate PCR primer was designed
referring to conserved sequence (Fig. 2A, long arrow) based on a
homology search for MCP of various species (17). Nested
PCR was performed with the degenerate primer as the forward primer, the
vector (pME18s)-specific primer as the reverse primer, and a chicken
thymus cDNA library as the template. After many trials, the second PCR
yielded a 280-bp cDNA fragment. This cDNA fragment was cloned,
sequenced, and found to be similar to SCR4 of hMCP. As shown in Fig. 1, consecutive rounds of PCR were
performed by the same method. Then, the nucleotide sequence of the ORF
and 3'UT of the chicken SCR protein cDNA was determined by the rapid
amplification of cDNA end (RACE) method. The presence of this message
was confirmed with chicken lung mRNA by sequencing twelve independent
RT-PCR amplicons.
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A). Cremp was a type 1 membrane
protein consisting of five SCR domains, one SCR-like domain, a
transmembrane domain, and a cytoplasmic tail. Cremp has one putative
N-glycosylation site on SCR3. According to homology search
analysis, this molecule was likely to differ from a simple MCP homolog
as well as other SCR proteins (Table I).
At the amino acid level, SCR1/SCR2 (combinational homology of two SCRs)
of Cremp was 43.6% identical with SCR2/SCR3 of human DAF, and
SCR3/SCR4 of Cremp was 45.4% identical with SCR3/SCR4 of hMCP (Fig. 2B). These two sequential SCRs of Cremp were less similar to
any two sequential SCRs of the other known SCR proteins than the above.
For example, in homology search of each SCR of the SCR proteins, mouse
Crry SCR1 and SCR5 were 31.8 and 34.0% homologous to SCR1 and SCR2 of
Cremp, respectively. The only exception was SCR3 of Cremp, which was
44.3% identical with that of mouse Crry, showing a high degree of
similarity. In total SCR pattern, Cremp was more similar to parts of
human DAF and hMCP than Crry (Table I). We hypothesized that Cremp is a
DAF/MCP hybrid molecule from which DAF and MCP, but not Crry,
evolved.
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We first analyzed the tissue distribution of Cremp mRNA by RNA
blotting analysis. RNA blotting followed by hybridization to the
full-length ORF of Cremp as a probe (1356 bp), which detects Cremp
mRNA, revealed a doublet consisting of a minor 2.2-kb band and a major
3.0-kb band. The major 3.0-kb band was expressed in all tissues,
whereas the minor 2.2-kb band showed restricted expression in the lung,
kidney, bursa, testis, and ovary (Fig. 3A). The cDNA isolated here
was 1.9 kb in length with an incomplete polyadenylation signal and
poly(A) tail. The 1.9-kb cDNA may have corresponded to the minor 2.2-kb
message, as in the case of the first identification of hMCP cDNA
(23). If this is the case, alternative usage of
polyadenylation signals, either incomplete or complete, may result in
two messages with a short or long 3'UT. Additional experiments to
clarify this point are currently in progress in our laboratory.
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B). Rabbit anti-Cremp Ab was prepared as described in
Materials and Methods. In this analysis, each lane reflected
the amount of Cremp released from 50 µg of tissue extracts with lysis
buffer. All tissues tested showed fairly high levels of Cremp. Cremp
protein had a molecular mass of 46.8 kDa, representing a putative
mature molecule with no glycosylation. Minute amounts of a 92-kDa band
were also observed in the spleen, lung, kidney, and ovary. This high
molecular mass protein appeared to be a disulfide-linked dimer of Cremp
because the band disappeared after reduction. Thus, both Cremp 3.0-kb
mRNA and 46.8-kDa protein were ubiquitously expressed in parallel. Localization of Cremp
The hydrophobicity plot suggested that Cremp protein is a type 1
membrane protein with a signal peptide (Fig. 2A). To
determine whether Cremp was localized on the cell surface, we performed
flow cytometry and immunostaining. On flow cytometry using
anti-Cremp Ab, DT40 and CHO cells expressing Cremp (CHO/Cremp) were
found to express Cremp on the cell surface (Fig. 41). Even in unstimulated DT40
cells, Cremp was highly and constitutively expressed on the cell
surface. Moreover, CHO/Cremp cells were also specifically detected by
immunostaining with anti-Cremp Ab around the cell margins (Fig. 42). Therefore, Cremp protein is largely localized on the
cell surface.
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The presence of an N-linked glycosylation site was predicted from
the amino acid sequence of Cremp (Fig. 2A). To determine
whether Cremp has N-linked and O-linked sugars,
we performed deglycosylation analysis of Cremp-expressing cells,
CHO/Cremp and DT40, using N- and O-glycosidases.
In both CHO/Cremp and DT40 cells, the equivalent mobility of Cremp
remained unchanged before and after glycosidase treatment on
SDS-PAGE/immunoblotting (Fig 5A). Therefore, Cremp on both
CHO and DT40 cells is likely to be mostly enzyme resistant. The Cremp
protein was thus suggested to be an unglycosylated or
glycosidase-resistant protein.
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To determine whether Cremp has the ability to protect host cells
from attack by C, we measured C protection activity of CHO/Cremp by
cytotoxicity assay using 51Cr-labeled CHO cells
and chicken serum (C source). We used CHO/hMCP as a positive control
and untransfected CHO cells as a negative control. The levels of
expression of Cremp and hMCP on CHO cells were 330.0 and 102.3,
respectively, expressed as mean fluorescence intensity measured by flow
cytometry. CHO cells were sensitized with an optimal concentration of
the anti-CHO Ab based on our primary tests for C protection assay.
CHO/Cremp completely inhibited chicken C attack in both of the
classical and the alternative pathways (Fig. 6A). In contrast, CHO/hMCP and
untransfected CHO induced cytolysis in a dose-dependent manner. In
contrast, human serum lysed CHO/Cremp as well as CHO control but not
CHO/hMCP in both pathways (Fig. 6B). Rabbit serum induced
cytolysis in all cells irrespective of Cremp or MCP expression in
either pathway (Fig. 6C). Therefore, Cremp selectively acted
on chicken C to protect host cells from homologous C attack. The action
of Cremp on C was thus indicated to be species specific.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Cremp cDNA isolated here was 1.9 kb in length with an incomplete
polyadenylation signal, AATTAAA, followed by a poly(A) tail. Its
message consisted of a major species of 3.0 kb and a minor species of
2.2 kb as determined by RNA blotting analysis. The 2.2-kb band was
expressed relatively thick in the lung, kidney, bursa, testis, and
ovary. The most likely interpretation of these observations is that the
3.0-kb message is ubiquitous and the 2.2 kb is tissue specific, and we
may have cloned the latter cDNA as shown in Fig. 2. Two similar
messages were obtained with human and mouse MCP; ubiquitous 4.0 kb and
testis-specific 1.5-kb species produced by alternative usage of
incomplete and complete polyadenylation signals.
The chicken C cascade has not been well delineated. Of the C components, C3 has been identified at the molecular level (26, 27). IgY has the ability to activate chicken C via the classical pathway, and in the absence of Ca2+ and IgY chicken C can be activated by the addition of foreign materials such as zymosan, suggestive of the presence of the alternative pathway (28, 29). Thus, we examined Cremp-dependent C inhibitory activity in the chicken classical and alternative pathways. The C-mediated CHO cell lysis system was used because this system worked well to test species specificity of C regulatory proteins in previous studies (19).
Cremp protects host cells from homologous C attack by inhibition of the chicken classical and alternative pathways. However, chicken Cremp hardly protects cells from heterologous C in both pathways. Cremp at least exerts host cell-protective activity against homologous C. This species specificity resembles those of DAF, MCP, and Crry. Therefore, Cremp is first identified as an SCR membrane protein of nonmammalian origin with C regulatory activity. The SCR protein-self protection theory in the C system should be adaptable to lower vertebrates including oviparous animals.
In mammals, the fetus grows in the placenta, which originates from the embryo and expresses paternal allo-Ags. One interpretation of the necessity of high levels of C regulatory proteins in organs including the placenta was that the placenta and fetal organs would be targets for allo-Ab generated by maternal lymphocytes (12). This hypothesis will not be the case in nonmammals because an oviparous animal has no direct communication between maternal and antenatal immunological factors. Yet, the chicken possesses a ubiquitously expressed membrane C regulatory protein. We favor the interpretation of our results as indicating that Cremp-like proteins must be a prerequisite for survival of lower vertebrates with sufficient C function, rather than protection of the fetus from maternal C attack via the placenta.
Cremp may represent an ancestral form of membrane C regulators. It is essentially a hybrid consisting of DAF-like and MCP-like elements participating in their functions, and like rodent Crry it contains five to six SCR domains. Most membrane SCR proteins protect the cells from C attack by two modes of action, decay-accelerating and factor I-cofactor activities. The C3 convertases, bimolecular proteases for activation of C3, are reversibly dissociated by decay-accelerating activity of SCR proteins, and are irreversibly inactivated by serine protease factor I and SCR protein-bearing cofactor activity. Although the functional profile of Cremp has not been defined, it effectively protects host CHO cells from chicken C similarly to DAF/MCP. A hybrid-like molecule of factor B/C2 was revealed in the lower vertebrate, chicken, bony fish, and lamprey (30, 31, 32). These results, together with the recent finding that chicken MHC is 10-fold more compact than that of mammals (33, 34, 35), suggest that C and its regulatory genes were more compact in the ancestral vertebrates and evolved into a complex system through gene duplication and unequal crossing over, as proposed by phylogenists. We speculate that human DAF and MCP also evolved from a single ancestor molecule by gene recombination.
The membrane SCR proteins have both or either DAF-like or MCP-like functions to regulate C3 activation on the membrane. In contrast, the SCR protein CR1 is present in B lymphocytes and phagocytes to serve as a receptor for C3 fragments deposited on opsonized cells, thereby allowing opsonized cells to be effectors for efficient Ag presentation or for phagocytosis. The SCR protein CR2 is expressed on B cells in humans and mice, and forms a complex with CD19 and TAPA-1 to signal the cells (6, 7, 8). The other SCR proteins Crry and MCP can serve as costimulators for proliferation of T cells and lead to secretion of IL-4 (36, 37). In addition, most C regulatory proteins are expressed at high levels in the testicular germ cells in mammals (18, 38), and their function may be related to fertilization (39, 40). It is still unknown whether Cremp is expressed predominantly in chicken testicular germ cells and plays some roles in fertilization as speculated in mammals. Also, it is possible that Cremp is a multifunctional protein, because almost all human SCR proteins found to date function as virus and/or bacterial receptors as well as C regulators (41, 42). Therefore, although Cremp is structurally similar to DAF and MCP, further infection-related function should be tested to verify its physiological roles.
Chicken immune-related genes appear to be simple and compact compared with those of mammals. A number of functions related to immunological phenotypes may be covered with a minimal number of molecules, and Cremp is a likely example of such a molecule. Cremp is expressed in the chicken B cell line DT40, which is a useful tool for gene disruption at the cell level (22). We will be able to analyze the functions of Cremp in future studies by gene targeting. The gene cluster of the human SCR proteins is located in a single region, 1q32 (3, 4). However, in mice, gene translocation has allowed the cluster to separate to two distinct regions (43, 44, 45). In addition, the gene regulatory profile of DAF and MCP appears to be unique compared with that of humans (46, 47). It will be of interest in future studies to identify the SCR gene cluster and its constituents in chicken chromosomes, as it may represent an ancestral SCR gene cluster. Genomic analysis will be required to settle these issues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. T. Seya, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537 Japan.
3 Abbreviations used in this paper: C, complement; CHO, Chinese hamster ovary; Cremp, C regulatory membrane protein; Crry, C receptor 1-related protein y; DAF, decay-accelerating factor; DT40, chicken B-lymphocyte cell line; hMCP, human MCP; MCP, membrane cofactor protein; RK13, rabbit kidney cell line; SCR, short consensus repeat; ORF, open reading frame; UT, untranslated region; IAA, iodoacetamide; GVB, gelatin veronal buffer.
Received for publication August 15, 2000. Accepted for publication October 10, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), CHO/hMCP (•), and control
CHO cells (
) labeled with 51Cr were sensitized with 65
µg/ml (classical pathway; CP) or 130 µg/ml (alternative pathway;
AP) of anti-CHO Ab and incubated with various concentrations of
GVB2+ (CP) or 0.03 M EGTA-GVB (AP)-chicken serum. The
percent cytotoxicity was calculated as described in Materials
and Methods. B, The same test performed with
human serum. To sensitize CHO cells, the cells were incubated with 130
µg/ml (CP) or 650 µg/ml (AP) of anti-CHO Ab. C,
The same test performed with rabbit serum. The cells were sensitized
with 65 µg/ml (both) and incubated with 10% GVB2+ or
20% EGTA-GVB-rabbit serum. Then, CHO/Cremp, CHO/hMCP, and control CHO
were represented by open, gray, and black columns, respectively. The
experiments were performed three times in triplicate, and similar
results were observed. The error bars represent SD.