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Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Mainz, Germany
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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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Three types of complement receptors that bind to C1q have been described to date; they differ by their ligand recognition specificity: C1qRp, a high molecular mass receptor of 126 kDa (1); cC1qR3 of 60 kDa, which binds to the collagenous portion of C1q (2); and a cell-associated protein of 33 kDa (p33) that interacts with the globular heads of C1q (3), and therefore has been dubbed gC1q receptor (gC1qR). Detailed studies have demonstrated that p33/gC1qR readily complexes with plasma proteins such as H-kininogen (4), factor XII (5), vitronectin (6), thrombin, prothrombin (7), and cC1qR (8). In vitro binding studies revealed that the binding to p33/gC1qR is specific and saturable with apparent dissociation constants (Kd) ranging from 9 nM for H-kininogen to 240 nM for C1q (4).
To serve its postulated function as a cellular C1q-binding protein, p33/gC1qR must be expressed on the cell surface. The mature form of the protein has been isolated and sequenced from total cell extracts of B cells; it comprises 209 amino acid residues and represents a highly acidic protein (28 Glu and 20 Asp residues) that is devoid of a typical hydrophobic transmembrane-spanning region (9). The corresponding cDNA sequence predicts a preprotein of p33/gC1qR that contains additional 73 residues at the amino terminus, and it has been speculated that this presequence might anchor gC1qR to the cell membrane (7, 9). Indeed, immunofluorescence studies have claimed the surface expression of p33/gC1qR for B cells, mast cells, neutrophils, platelets, and endothelial cells (9, 10, 11, 12, 13); however, recent studies with B cells, macrophages, and endothelial cells have challenged these findings (14, 15). Daha and coworkers (14) demonstrated that gC1qR is located in the cytoplasm of B cells, from which it can be released under stress conditions, whereas we have demonstrated that gC1qR is associated with the vesicular fraction of endothelial cells (15). However, the precise cellular location of gC1qR has remained elusive.
To analyze the subcellular distribution of p33/gC1qR and to follow its intracellular routing, we have overexpressed the protein in Sf9 cells. Unexpectedly, we were unable to detect p33/gC1qR on the cell surface of infected Sf9 cells. Rather, we determined that p33/gC1qR was associated mainly with the intracellular particulate fraction. Using fusion constructs with green fluorescent protein (GFP), we demonstrated that the amino-terminal portion of p33/gC1qR represents a targeting sequence that directs the protein to mitochondria. We conclude that p33/gC1qR cannot function as a binding protein for C1q in intact cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials were purchased from the following manufacturers: HUVEC
cDNA library and anti-GFP Ab from Clontech Laboratories (Palo Alto,
CA); rabbit reticulocyte lysate from Promega Corp. (Madison, WI); PCR
primers from MWG Biotech (Ebersberg, Germany); pAlpha + GFP from
Maxygen (Santa Clara, CA); pVL1392 vector and liposome kit from
Invitrogen BV (Leek, The Netherlands); standard marker proteins from
Pharmacia Biotech (Uppsala, Sweden); [
-35S]dATP
and enhanced chemiluminescence (ECL) kit from Amersham Buchler
(Braunschweig, Germany); [35S]cysteine/methionine
(Tran35S label) and [-32P]dATP from ICN
Biomedicals (Eschwege, Germany); and rhodamine 123 from Molecular
Probes Europe BV (Leiden, The Netherlands). H-kininogen was isolated
from human plasma (16).
Cloning of full-length p33/gC1qR cDNA and in vitro translation
The cloning of the nucleotide sequence encoding the mature form of p33/gC1qR (positions 74 to 282 of the protein sequence) from total RNA of HUVEC by reverse transcriptase-PCR was done as described (4). Using this PCR fragment as a probe, we screened a HUVEC cDNA library and retrieved a full-length p33/gC1qR cDNA clone. The p33/gC1qR cDNA was subcloned into the Bluescript KS+ (Bks) vector and sequenced on both strands. We transcribed p33/gC1qR sense RNA using T3 RNA polymerase and the Bks-p33/gC1qR vector that had been linearized with XbaI. For transcription of a p33/gC1qR antisense RNA, the vector was cut with EcoRI, and the T7 RNA polymerase was used. In vitro translation of 1 µg of p33/gC1qR sense and antisense RNA, respectively, was conducted using a commercial reticulocyte lysate (Promega Corp.). The polypeptides were labeled with [35S]cysteine/methionine.
Construction of GFP fusions with p33/gC1qR
To generate fusion proteins of GFP and p33/gC1qR, various parts of the p33/gC1qR DNA sequence were amplified by PCR using primers with flanking XbaI restriction sites. The linearized Bks-p33/gC1qR plasmid (3 ng/5 µl) was added to 95 µl of a polymerase mixture that contained 2 U Taq polymerase, 25 pmol each of the 5' and 3' primers, 250 µM concentrations of each dNTP, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2. The individual samples were overlaid with 70 µl of mineral oil and amplified in a thermal cycler for 45 s at 94°C, 45 s at 58°C, and 1 min at 72°C for 40 cycles. Isolated PCR fragments were digested with XbaI and ligated into the XbaI site of pAlpha + GFP vector, thus generating GFP fusions with variable amino-terminal extensions of p33/gC1qR.
The following p33/gC1qR-GFP constructs were generated: [p331–33] GFP, positions 1–33 of the p33/gC1qR protein sequence, amplified with the primers 5'-GCATCTAGACGTGTTCGCAGTCGTTTCC-3' and 5'-GGGTCTAGACTGCAGGAGCTGCCGGAAAGGCG-3'; [p331–81]GFP, positions 1–81 of p33/gC1qR amplified with the primers 5'-GCATCTAGACGTGTTCGCAGTCGTTTCC-3' and 5'-GGAAATCTAGAAGCTTTGTCTCCGTCGGTGTGC-3'; [p3332–81]GFP, positions 32–81 amplified with the primers 5'-CCTTCTAGAATGGCTCTGCAGCCGGCACCCCGGCTGTGC-3' and 5'-GGAAATCTAGAAGCTTTGTCTCCGTCGGTGTGC-3'; [p331–282]GFP, full-length p33/gC1qR precursor amplified with the primers 5'-GCATCTAGACGTGTTCGCAGTCGTTTCC-3' and 5'-GTCTTCTAGACTGGCTCTTGACAAAACTCTTGAGG-3'; and [p3374–282] GFP, maturated form of p33/gC1qR amplified with the primers 5'-CTGTTCTAGAATGGCTCTGCACACCGACGGAGACAAAGC-3' and 5'-GTCTTCTAGACTGGCTCTTGACAAAACTCTTGAGG-3'.
GFP fusions, with mitochondrial targeting sequences of human cytochrome c1 (cc1) (17) and human Rieske iron-sulfur protein (rfs) (18), were generated by reverse-transcriptase PCR using total RNA from EA.hy926 cells. The following constructs were made: [cc11–88]GFP, amino acid positions 1–88 of the cytochrome c1 sequence, amplified with primers 5'-GGCTCTAGAGAGGCCAAGATGGCGGCAGCTGCG-3' and 5'-GGGTCTAGACTCCAGGTCACTGGCACTCACAGC-3'; [rfs1–86]GFP, positions 1–86 of rfs, amplified with the primers 5'-GGATCTAGAGTCGCCATGTTGTCGGTAGCAGCC-3' and 5'-GTATCTAGAGAATTCAGGCACCTTGATGTCTGTG-3'.
Cell culture
The Spodoptera frugiperda (Sf9) cell line was grown as monolayers in ambient atmosphere at 27°C in TC100 medium (Life Technologies, Eggenstein, Germany) supplemented with 10% (v/v) FCS, penicillin (100 IU/ml), and streptomycin (100 µg/ml). EA.hy926 cells (19) were cultured in DMEM containing 4.5 g/L glucose, 10% (v/v) FCS, 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM TdR, and penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37°C. COS-7 cells were kept under the same conditions, except that RPMI medium with 10% (v/v) FCS and penicillin/streptomycin were used. The organelle fraction of EA.hy926 cells was prepared as described (15).
Construction of recombinant baculovirus and infection of Sf9 cells
The full-length p33/gC1qR cDNA was cloned into the EcoRI/XbaI restriction sites of the baculovirus transfer vector pVL1392 (Invitrogen). Recombinant baculoviruses were generated by cotransfection of Sf9 cells with the pVL1392-p33/gC1qR construct and with linearized AcMINPV DNA by the lipofection method, according to the manufacturer’s instructions (Invitrogen). Briefly, Sf9 cells grown to near confluence on a 10-cm dish were washed with PBS (PBS = 0.15 M NaCl, 10 mM sodium phosphate, pH 7.4) and starved for 2 h in TC100 medium lacking supplements and FCS. Recombinant pVL1392-p33/gC1qR plasmid (400 ng), 100 ng linearized AcMINPV DNA, 1 ml TC100 medium without supplements or FCS, and 25 µl cationic liposomes were mixed ("transfection mixture") and incubated for 20 min at room temperature. The medium was removed from the starved Sf9 cells, and the transfection mixture was added. Following incubation for 4 h at room temperature, 1 ml of complete TC100 medium was added. Cells were incubated at 27°C until morphologic changes due to infection appeared (typically after 3–5 days). Individual baculovirus clones were established by limited dilution of a high titer virus stock and infection of Sf9 cells grown in microwell plates. Baculovirus clones were identified by immunoblotting for expressed p33/gC1qR protein. For bulk production, Sf9 cells were infected with recombinant viruses at a multiplicity of infection of 2 to 5. The cells were harvested 40 to 96 h after infection and used to prepare recombinant p33/gC1qR protein.
Binding of H-kininogen to Sf9 cells and cell lysates
H-kininogen from human plasma was radioiodinated as previously described (20). Infected Sf9 cells were washed twice in 0.135 M NaCl, 2.7 mM KCl, 11.9 mM NaHCO3, 0.36 mM NaH2PO4, 14.7 mM HEPES, pH 7.35 (HEPES-Tyrode’s buffer), containing 3.5 mg/ml dextrose, and 50 µM ZnCl2, and suspended in the same buffer including 0.2% BSA. The cells (106/100 µl) were mixed gently for 1 h at 4°C with 10 nM of 125I-H-kininogen in the presence or absence of a 100-fold molar excess of unlabeled H-kininogen and recombinant p33/gC1qR, respectively. Bound 125I-H-kininogen was separated from the free ligand by centrifugation (10,000 x g, 4 min, 4°C) of the cell suspension through 500 µl of a mixture of dibutylphthalate/1-bis(2-ethylhexyl)phthalate (1.1/1, v/v) (21). Cell-bound radioactivity was measured in a gamma counter (Packard Instrument Company, Meriden, CT).
For the indirect ELISA, microtiter plates (MaxiSorb, Nunc, Wiesbaden,
Germany) were incubated overnight with 2 µg/ml of H-kininogen, C1q,
factor XII, and 2-HS glycoprotein, respectively, in 100
mM sodium acetate, 100 mM NaCl, pH 5.5. The plates were washed with PBS
and incubated with serial dilutions (2n; starting
concentration 500 µg/ml) of total cellular extracts of infected
Sf9 cells. Bound protein was detected by an Ab to p33/gC1qR (0.5
µg/ml), followed by a secondary peroxidase-conjugated Ab to rabbit Ig
(Bio-Rad Laboratories, Hercules, CA; 1:5000), and the chromogenic
substrate 2,2-azino-bis(3-ethyl-2, 3-dihydrobenzthiazoline-6-sulfonate)
(ABTS) for 30 min. The change of absorbance was read at 405 nm. All
incubations were done at 37°C, except for the coating step, which was
done at 4°C (22).
Gel electrophoresis and immunoblotting
Harvested cells were washed two times with PBS and resuspended in sample buffer (63 mM Tris-HCl, pH 6.8, containing 2.5% SDS, 5% glycerol, 5% ß-mercaptoethanol, and 0.005% bromphenol blue) for 30 min at 42°C. Proteins were resolved by SDS-PAGE (12% (w/v) total acrylamide) and subsequently electrotransferred to nitrocellulose membranes using established protocols. For immunoprinting, affinity-purified rabbit Abs to the mature form of p33/gC1qR (positions 74–282; As385) recombinantly expressed in Escherichia coli (15) were used at a concentration of 0.5 µg/ml. Alternatively, Abs to synthetic peptide RLC24 derived from the targeting sequence of the p33/gC1qR precursor (positions 37–60; As423) were used at a concentration of 1 µg/ml; the anti-GFP Ab was applied at 1:5000. Bound Ab was detected by a secondary peroxidase-labeled Ab (1:5000) and the chemiluminescent substrate assay.
Transfection of COS-7 cells and visualization of fusion proteins
COS-7 cells were transfected with the fusion constructs of pAlpha + GFP and p33/gC1qR using the DEAE-dextran method. After 60 h, the transfected cells were washed twice with ice-cold PBS and fixed with 4% (v/v) formaldehyde in PBS. For membrane permeabilization, the cells were treated with ice-cold methanol for 10 min. After washing three times with PBS, the cells were incubated with 15 µg/ml anti-p33 diluted in blocking buffer (PBS containing 0.5% BSA) for 30 min at 37°C. The cells were washed three times with PBS and incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit Ig (Sigma Chemical Co., St. Louis, MO; 1:100 in blocking buffer) for 30 min at 37°C. After washing three times with PBS, the cells were embedded in n-propyl gallate solution (5% in glycerol) and viewed with an epifluorescence microscope (Axiophot, Zeiss, Jena, Germany). To stain for mitochondria, rhodamine 123 was dissolved in water at 1 mg/ml and diluted with culture medium to a final concentration of 10 µg/ml. EA.hy926 cells grown on chamber slides (Nunc) were incubated with the dye for 30 min in a 5% CO2 incubator at 37°C. Adherent cells were rinsed three times for 5 min each with medium. The medium was removed, and the stained cells were examined by epifluorescence microscopy.
Confocal laser-scanning and light microscopy
Fixed and permeabilized EA.hy926 cells were stained for 30 min with 15 µg/ml (final concentration) of anti-p33 or with a human autoantibody directed to mitochondrial pyruvate dehydrogenase (1:1000), or both. Anti-p33 was detected by a goat anti-rabbit Ig conjugated to the fluorescent dye Cy3 (Dianova, Hamburg, Germany). The human autoantibody was visualized by FITC-conjugated goat anti-human Ig. In control stainings, no cross-reactivity of the anti-rabbit and anti-human Ab was observed (data not shown). For double labeling, the cells were first stained at room temperature with anti-p33, followed by incubation with autoantibody to pyruvate dehydrogenase. The unbound Abs were removed by washing twice with PBS for 5 min each. The cells were mounted using PBS/glycerol (1:1, v/v). Cells were sliced into horizontal optical sections at an interval of 500 nm, and examined by confocal laser-scanning microscopy (LSM10, Zeiss). Evaluation of the stored images of the horizontal optical sections was done with the LSM10 image-processing unit. For immunocytochemistry on the light-microscopy level, paraffin sections of fetal mice fixed with Bouin’s fluid were dewaxed, rinsed, and stained with 15 µg/ml anti-p33. Bound Ab was detected by the peroxidase-antiperoxidase technique and the chromogenic substrate diaminobenzidine·4HCl. Sections were lightly counterstained with Harris’ hematoxylin solution and viewed in a light microscope (Axiophot, Zeiss).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a cDNA fragment of 644 bp that had been generated by
reverse-transcriptase PCR from total RNA of human endothelial cells
(4), we screened a HUVEC cDNA library and isolated a clone of 1165 bp
encoding the full-length p33/gC1qR preprotein of 282 amino acid
residues. Sequence analysis established the full identity of the
endothelial p33/gC1qR clone with the published gC1qR cDNA sequence from
B cells (9). To estimate the size of the corresponding primary
translation product, we performed in vitro transcription/translation
experiments in the presence of 35S-radiolabeled
cysteine/methionine (Fig. 1
A). A 39-kDa protein
representing the p33/gC1qR precursor was translated from sense RNA
(lane 1), but not from antisense RNA
(lane 2). Immunoprinting with an
anti-peptide Ab, anti-RLC24, derived from the extreme
amino-terminal segment, putative presequence, of the p33/gC1qR
precursor, indicated that this 39-kDa preprotein does not accumulate in
the total cellular extracts of HUVEC or EA.hy926 cells to detectable
amounts (data not shown).
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The full-length cDNA for p33/gC1qR was inserted into the
baculovirus genome. Following infection of Sf9 cells, the expression of
p33/gC1qR precursor was monitored by immunoblotting with anti-p33
directed to the mature form of p33/gC1qR, or with anti-RCL24
targeted to positions 37 to 60 of its presequence (see above). After
40 h of infection, a 39-kDa band was detected by both Abs (Fig. 1
B, lanes 3 and 5).
Prolongation of the infection periods up to 96 h allowed the
detection of additional bands at 33 kDa (major) and 35 kDa (minor) by
anti-p33 (lane 4). The major 33-kDa band
most likely represents the mature form of p33/gC1qR precursor because
it fails to bind the anti-RCL24 (lane 6),
whereas the 35-kDa protein might represent an intermediate processing
product. Unrelated recombinant proteins failed to produce specific
bands with anti-RCL24 (lane 7) or
anti-p33 (not shown).
p33/gC1qR is not transported to the surface of Sf9 cells
Isolated p33/gC1qR binds to plasma proteins such as C1q (apparent
Kd = 240 nM), and even more tightly to
H-kininogen (Kd = 9 nM). Therefore, we
probed Sf9 cells overexpressing p33/gC1qR for the presence of newly
exposed binding sites for H-kininogen (Fig. 2A). After 40 h of
infection with recombinant baculovirus, the Sf9 cells were incubated
with 10 nM 125I-H-kininogen in the absence (hatched column)
or presence of a 100-fold molar excess of unlabeled H-kininogen
(stipled column) or p33/gC1qR (solid column). For control, Sf9 cells
expressing an unrelated protein were tested under identical conditions.
Even prolonged expression of p33/gC1qR up to 60 h did not increase
the number of specific binding sites on the surface of Sf9 cells.
Likewise, FACS studies with anti-p33 did not indicate surface
expression of p33/gC1qR on infected Sf9 cells (data not shown).
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2-HS glycoprotein
(negative control) were incubated with increasing concentrations of
total cellular extract of Sf9 cells expressing p33/gC1qR. Figure 2B demonstrates that p33/gC1qR from whole cell lysates of
Sf9 cells infected with the virus encoding p33/gC1qR readily bound
H-kininogen (Fig. 1B, diamonds) and C1q (Fig. 1B,
circles), whereas moderate or no binding was found with factor XII
(Fig. 1B, triangles) and 2-HS glycoprotein
(Fig. 1B, pentagons), respectively. No binding of
H-kininogen was observed in extracts of Sf9 cells expressing an
unrelated protein (Fig. 2B, squares). Hence, the recombinant
p33/gC1qR displays ligand-binding properties as would be expected from
a C1q receptor. The p33/gC1qR presequence targets GFP to intracellular compartments
It has been proposed, but not demonstrated, that the presequence
of 73 residues might serve as a signal sequence (amino-terminal
portion) and a membrane anchor (carboxyl-terminal portion) that tethers
p33/gC1qR to the cell surface (7, 9). Because we failed to detect
surface expression of p33/gC1qR in Sf9 insect cells, we asked whether
the presequence is involved in the intracellular targeting of p33/gC1qR
in mammalian cells. To this end, we generated fusion constructs of the
p33/gC1qR sequence or portions thereof (= amino-terminal part of
the fusion protein) and GFP (= carboxyl-terminal part) and expressed
them in COS-7 cells: [p331–282]GFP
comprising the entire p33/gC1qR sequence;
[p331–81]GFP carrying the complete
presequence; [p331–33] GFP and
[p3332–81]GFP holding the amino- and carboxyl-terminal
portion, respectively, of the presequence; and
[p3374–282] containing the complete sequence of the
mature protein, but lacking the presequence (Fig. 3).
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A) for GFP (green) and
p33/gC1qR (red). By contrast, the construct
[p331–282]GFP containing the full-length p33/gC1qR
precursor was directed to intracellular compartments (Fig. 4B). Fusion of GFP to the proper presequence, i.e.,
[p331–81]GFP, targeted GFP to vesicular structures,
where it colocalized with endogenous p33/gC1qR of COS cells (Fig. 4C). Truncation of the carboxyl-terminal portion of
the presequence, [p331–33]GFP, did not significantly
change this latter distribution pattern (Fig. 4D),
whereas the amino-terminally truncated presequence,
[p3332–81]GFP, afforded a cytoplasmic localization (Fig. 4E). For control, expression of unfused GFP was
studied and resulted in diffuse staining of the cytoplasm for GFP, and
a discrete vesicular staining for the endogenous p33/gC1qR (Fig. 4F). The combined results indicate that the
presequence represents a functional targeting sequence that directs the
reporter protein GFP to the same intracellular compartments that harbor
endogenous p33/gC1qR.
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The cellular distribution of p33/gC1qR-containing structures was
reminiscent of mitochondria that typically show perinuclear and
cytoplasmic location. Indeed, staining for mitochondria in EA.hy926
cells by rhodamine 123 fluorochrome (23) yielded staining patterns (not
shown) that strongly resembled those produced by anti-p33. To test
the hypothesis that p33/gC1qR might reside in mitochondria, we analyzed
nontransfected, endothelial EA.hy926 cells by double immunostaining and
confocal laser-scanning microscopy. Cells were stained with
anti-p33 and with a human autoantibody directed to mitochondrial
pyruvate dehydrogenase (Fig. 5).
Horizontal sections were scanned, and overlays were prepared from the
stored images of the optical sections. A similar staining pattern was
obvious for mitochondrial pyruvate dehydrogenase (Fig. 5A) and endogenous p33/gC1qR (Fig. 5B). An overlay of the staining patterns produced by
the autoantibody and anti-p33 revealed that they were essentially
congruent (Fig. 5C). We conclude that p33/gC1qR
colocalizes with mitochondrial pyruvate dehydrogenase of EA.hy926
cells.
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The colocalization of p33/gC1qR with a mitochondrial enyzme
complex prompted us to examine the amino acid composition of the
p33/gC1qR presequence. The unusual distribution of charged amino acids,
i.e., a single acidic residue (Glu), 11 basic residues (Arg), and the
presence of 7 hydroxylated residues, suggested that the leader sequence
may in fact represent a mitochondrial targeting sequence (24). To test
this hypothesis, we fused the reporter protein GFP with two established
mitochondrial targeting sequences, i.e., residues 1–88 of cytochrome
c1, [cc11–88]GFP, and
residues 1–86 of rfs, [rfs1–86]GFP. We then tested by
transiently transfecting COS-7 cells whether the mitochondrial reporter
constructs are present in the same compartments as endogenous
p33/gC1qR (Fig. 5). Double labeling revealed that the staining
patterns for [cc11–88]GFP and
[rfs1–86]GFP (Fig. 5, D and
F) are congruent with those of endogenous p33/gC1qR
in COS-7 cells (Fig. 5, E and G),
suggesting that the proteins have been routed to the same intracellular
compartments, namely mitochondria.
Mitochondrial proteins carrying cleavable targeting sequences are
matured by processing peptidases after their import into mitochondria
(25). Using anti-GFP, we followed the intracellular processing of
the p33/gC1qR precursor by Western blotting of the various fusion
constructs. For control, we transiently expressed unfused GFP in COS-7
cells and obtained a single band of 30 kDa for GFP (Fig. 6A, lane 1).
Both the [p331–81]GFP construct and the
[p331–33]GFP construct, which harbor the relevant
topogenic segments of the p33/gC1qR targeting sequence, gave rise to
mature GFP of 30 kDa (lanes 2 and
3). The additional bands of 36 and 32 kDa,
respectively, most likely represent the corresponding precursor
proteins indicative of incomplete processing. The amino-terminally
truncated form of the presequence, [p3332–81]GFP,
generated a single band of 34 kDa, but failed to produce mature GFP
protein of 30 kDa (lane 4). Both the
[p3374–282]GFP and the [p331–282]GFP
fusion constructs gave a 63-kDa protein (lanes 5 and
6). In the latter case, the appearance of an
additional 69-kDa band suggested that the [p331–282]GFP
undergoes proteolytic conversion into [p3374–282]GFP.
Hence, the combined data of Figures 4 and 6 indicate that intracellular
routing of the p33/gC1qR precursor is accompanied by the removal of its
targeting sequence.
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Using authentic mitochondrial targeting sequences, we found that
fusions of prototypical mitochondrial targeting sequences to GFP, i.e.,
[cc11–88]GFP and
[rfs1–86]GFP, produced bands of 30 and 36 kDa for mature
GFP and the corresponding precursor proteins, respectively (Fig. 6B, lanes 7 and 8). The
presence of an intermediate-size form of 32 kDa in the case of
[cc11–88]GFP is in agreement with the
bipartite targeting sequence of human cytochrome
c1, of which the first part represents a
mitochondrial import signal that is cleaved in the matrix, whereas the
second part directs the protein to the intermembrane space, where
processing to the mature form occurs (25). By contrast, no such
intermediate-size form was detected in the case of
[rfs1–86]GFP (Fig. 6B, lane
8) and [p331–81]GFP (Fig. 6A,
lane 2). Notably, the targeting sequence of human rfs
undergoes a single cleavage and directs the protein to the outer
surface of the inner mitochondrial membrane (26). Therefore, we
concluded that p33/gC1qR may be associated with the matrix and/or the
inner membrane of mitochondria. To further test this hypothesis, we
examined the presence of p33/gC1qR in bovine liver mitoplasts, i.e.,
mitochondria devoid of an intact outer membrane (Fig. 6C). Immunoprint analysis revealed that Ab to human
p33/gC1qR, anti-p33, readily detected its bovine homologue of 32
kDa in whole cell extracts of bovine aortic endothelial cells
(EA.hy926, Fig. 6C, lane 9; BAEC, lane
10). Likewise, liver mitoplasts gave a strong band of 32
kDa (lane 11), thus supporting the notion that
p33/gC1qR resides in the mitochondrial matrix and/or the inner
mitochondrial membrane.
p33/gC1qR is expressed ubiquitously in fetal mice
We asked whether p33/gC1qR is expressed predominantly in tissues
that are rich in mitochondria (Fig. 7).
Initial experiments demonstrated that anti-p33 readily cross-reacts
with its mouse homologue (data not shown). Immunostaining of paraffin
sections of fetal mice indicated that p33/gC1qR is expressed
ubiquitously in fetal mice tissues (Fig. 7A); the
corresponding preimmune serum stained negative (Fig. 7B). Most prominent staining was observed in brown
fat tissue (Fig. 7A'), liver (Fig. 7B'), kidney (Fig. 7C'), hindgut
(Fig. 7D'), pancreas, and salivary glands (not shown
at higher magnification). The cellular distribution was cytoplasmic,
particulate (Fig. 7, a'–d'). Polarized cells in
secretory structures such as hindgut (Fig. 7d') and
pancreas (not shown at higher magnification) had the strongest staining
in their apical parts. This pattern is compatible with and indicative
of p33/gC1qR location in mitochondria of metabolically highly active,
secretory tissues.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further puzzling is the fact that p33/gC1qR is similar or even identical to putative nuclear factors. A 32-kDa protein (SF2p32) that copurifies with the pre-mRNA splicing factor SF2 from HeLa cells is identical with p33/gC1qR (28). Yu et al. (29) reported the sequence of trans activator (Tat)-associated protein (TAP) of 32 kDa that facilitates the binding of HIV-1-encoded Tat to the transcription factor TFIIB. Comparison of the TAP sequence with that of p33/gC1qR reveals total sequence identity for positions 1 to 23 and 67 to 282 of the proteins. Complete sequence identity of the entire protein sequence (positions 1–282) is readily obtained by the correction of two potential sequencing errors or cloning artifacts that cause frameshifts at the points of divergency (data not shown). By the two-hybrid approach, a mouse homologue of TAP ("YL2") that associates with the HIV-1 trans activator Rev has been isolated from T cells (30). The corresponding cDNA sequence that covers only positions 74–282 of p33/gC1qR is 92% identical to the human homologue (30). Unfortunately, the subcellular localization of the putative nuclear factors SF2p32, TAP, and YL2 has not been reported to date.
Given the wide array of plasma, viral, and nuclear proteins that interact with p33/gC1qR-like proteins (4, 5, 6, 7, 8, 9, 28, 29, 30), we suggest that p33/gC1qR represents a multiligand-binding protein. Indeed, the unique amino acid composition with clusters of highly charged sequence segments makes p33/gC1qR a prime candidate for promiscuous interaction with many different proteins, including cC1qR (8). Therefore, the identification of p33/gC1qR in affinity-purified or immunoprecipitated protein isolates from whole cell extracts or in yeast two-hybrid systems warrants a careful analysis of the physiologic relevance of the observed protein-protein interactions.
Considering its mitochondrial localization of p33/gC1qR described in
this work, the proposed functions as an extracellular or cytoplasmic
anchor for plasma, nuclear, or viral proteins cannot be fulfilled by
p33/gC1qR, unless it is released from mitochondria. The secretion of
minor amounts of p33/gC1qR has been claimed for lymphocytes (14, 31);
however, our own studies do not support such a notion. Using Abs
against distinct epitopes of p33/gC1qR, we were unable to detect
significant amounts of the protein on the surface or in the nucleus of
intact endothelial cells (15). Likewise, the stimulation of EA.hy926
cells with bradykinin, epidermal growth factor, PMA, FMLP, IL-1ß,
TNF-, ionophore A23187, or shear stress failed to induce significant
secretion or translocation of p33/gC1qR (data not shown). Our
experiments do not rule out the possibility that p33/gC1qR is released
from mitochondria following cell apoptosis; however, such a mechanism
is incompatible with its postulated function as a major complement
receptor of circulating or resident cells of the host defense systems.
Rather, cellular protein(s) other than p33/gC1qR must account for the
observed binding sites for the globular heads of C1q (3). Other
mechanisms that could be invoked for an extramitochondrial location of
mitochondrial proteins include alternative splicing of the primary
transcript (32) and/or redistribution of differentially folded
translation products (33); however, the failure to immunodetect
p33/gC1qR Ag in the cytoplasm denies such possibilities.
At present, one can only speculate about the true cellular function(s) and ligand(s) of p33/gC1qR-like proteins in mammalian cells. In this context, it is most noteworthy that the yeast homologue of p33/gC1qR has been identified recently in Saccharomyces cerevisiae (34). Mitochondrial acidic matrix protein of 33 kDa (Mam33p) shares 53% sequence similarity and 24% sequence identity with human p33/gC1qR. The Mam33p precursor comprises a mitochondrial targeting sequence of 47 residues, which directs the protein to the mitochondrial matrix. Disruption of the Mam33p gene does not produce a major phenotype, e.g., cell growth is near normal (34). Hence, the role(s) of Mam33p in yeast remains obscure. Given the evolutionary conservation of p33/gC1qR-like proteins, it will be interesting to learn about the effects of a targeted deletion of the p33/gC1qR gene in multicellular species such as mouse.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Werner Müller-Esterl, Institute for Physiologic Chemistry and Pathobiochemistry, Johannes Gutenberg-University at Mainz, Duesbergweg 6, D-55099 Mainz, Germany. E-mail address:
3 Abbreviations used in this paper: cC1qR, receptor for the collagenous domains of C1q; ABTS, diammonium 2,2'-azino-bis(3-ethyl-2, 3-dihydrobenzthiazoline-6-sulfonate); cc1, cytochrome c1; gC1qR, receptor for the globular domains of C1q; GFP, green fluorescent protein; H-kininogen, high molecular mass kininogen; 125I-H-kininogen, 125I-labeled high molecular mass kininogen; rfs, Rieske iron-sulfur protein; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication October 28, 1997. Accepted for publication December 9, 1997.
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), C1q (
), factor XII (
), or 
) were incubated with serial dilutions
(2n; starting concentration 500 µg/ml) of total cell
lysates of Sf9 cells overexpressing p33/gC1qR (infection period 40
h). Bound p33/gC1qR was detected with 0.5 µg/ml of anti-p33,
followed by a peroxidase-labeled secondary Ab (1:5000) and ABTS. For
control, extracts of Sf9 cells overexpressing an unrelated protein
(endothelin receptor) were analyzed for kininogen binding (
).
