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A Recombinant Homotrimer, Composed of the Helical Neck Region of Human Surfactant Protein D and C1q B Chain Globular Domain, Is an Inhibitor of the Classical Complement Pathway¹

Uday Kishore^*, Peter Strong, Michael V. Perdikoulis and Kenneth B. M. Reid^2,

^* Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington, United Kingdom; and Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB₃, formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB₃ indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB₃ is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
C1q plays a key role in the recognition of immune complexes, thereby initiating the classical complement pathway (CCP).³ The human C1q molecule (460 kDa) is composed of 18 polypeptide chains (6A, 6B, and 6C). The A chain (223 residues), B chain (226 residues), and C chain (217 residues) each have a short (3–9 residues) N-terminal region (containing a half cysteine residue involved in interchain disulfide bond formation), followed by a collagen-like sequence of 81 residues and a C-terminal globular region (gC1q domain) of 135 residues (1). Comparison of the mature chains shows that there are four conserved cysteine residues in each chain (at positions 4, 135, 154, and 171, as per the B chain numbering). The cysteine residues at position 4 in each of the chains are involved in the interchain disulfide bridging yielding the A-B and C-C subunits; the other three are considered to yield one intrachain disulfide bond and one free thiol group per C-terminal globular region. The interchain disulfide bonding yields six A-B dimer subunits and three C-C dimer subunits. The collagen-like sequences in the A and B chains of an A-B subunit form a triple-helical collagen-like structure with the equivalent sequence in one of the C chains present in a C-C subunit, to form a structural unit, of the composition ABC-CBA, which is therefore held together by both covalent and noncovalent bonds. Three of these structural units are then considered to associate, via strong noncovalent bonds in the fibril-like central portion, to yield the hexameric C1q molecule (2). The first component of complement C1 is a complex of three glycoproteins, C1q, C1r, and C1s. C1s and C1r interact to form a tetrameric proenzyme complex, C1r₂-C1s₂, which makes contacts with the C1q collagen region. Many activating ligands for C1, including immune complexes, bind to the gC1q domains; however, a number of nonimmune substances, such as DNA, C-reactive protein (CRP), serum amyloid protein (SAP), decorin, and some putative C1q receptors are thought to bind C1q via the collagen region. Binding of C1q to immune complexes (IgG or IgM) via the gC1q domain is considered to induce a conformational change in the collagen region of C1q, which leads to the autoactivation of C1r, which in turn activates C1s. The activated C1 complex then cleaves components C2 and C4 in the CCP. After C1 activation and removal of activated C1r₂-C1s₂ by C1 inhibitor, the collagen region appears to interact with cell surface receptors.

Human C1q shows only weak binding to the Fc regions of nonaggregated IgG (4 x 10³ to 5 x 10⁴ M^-1). Upon presentation of multiple, closely spaced Fc regions, as are found in immune complexes, the strength of binding of the hexameric C1q to IgG increases dramatically (10⁷ to 10⁸ M^-1) (3, 4). The precise binding region of the IgG molecule for C1q is considered to be located in the C-terminal half of the C2 domain of IgG and, specifically, to three amino acids, Glu³¹⁸, Lys³²⁰, and Lys³²², which are highly conserved in different IgG isotypes (5). The charged residues Asp⁴¹⁷, Glu⁴¹⁸, and His⁴²⁰ in the Cµ3 region of IgM have been proposed to form the binding site for the gC1q domain (6). Recent reports of recombinant production and characterization of the globular region of all three chains of C1q suggest that the gC1q domain is likely to be composed of three structurally and functionally independent modules, which retain multivalency in the form of a heterotrimer (7, 8, 9). The gC1q-like modules are also found in a variety of noncomplement proteins, which include the C-terminal globular regions of the human type VIII and type X collagen, precerebellin, the chipmunk hibernation proteins, multimerin, Acrp-30, and the sunfish inner-ear specific structural protein, called saccular collagen (9, 10). In several of these proteins, the chains containing gC1q modules appear to form a homotrimeric structure. The crystal structure of the homotrimeric Acrp-30 (11) suggests that gC1q modules may assemble as C-terminal appendages to the collagen regions in the same way as the carbohydrate recognition domains (CRDs) present in the family of proteins called collectins. The members of the collectin family include mannose-binding lectin (MBL), surfactant protein A (SP-A), surfactant protein D (SP-D), bovine conglutinin (BC), and collectin 43 (CL-43). Collectins have an N-terminal, C1q-like collagen region that is linked to the C-terminal CRDs via an helical, coiled-coil neck region, which acts as a nucleation center for the trimerization of the CRDs (12). However, the gC1q domain, which has a very different fold than CRD, leads directly into the collagen region with no intervening neck region. The specific hydrophobic bonds within the sequence of the globular regions are considered to facilitate the interchain recognition and alignment of the three chains to yield a heterotrimeric (ghA, ghB, ghC) globular head structure, which, in turn, could act as a nucleation center for the trimerization of the triple-helical collagen region.

To further dissect the modular organization of the gC1q domain of human C1q, we addressed the question of whether a homotrimeric structure containing one type of globular region, as is seen in other members of the C1q family, can retain some biological functions. We made an upstream fusion of the trimerizing, helical coiled-coil neck region of human lung SP-D with the C-terminal globular head region of the human C1q B chain (ghB) and expressed in Escherichia coli linked to maltose-binding protein (MBP). The expressed recombinant polypeptide, composed of MBP, the factor Xa protease site, and the neck and ghB regions, was affinity purified using an amylose resin column and then cleaved with factor Xa to release a hybrid molecule (neck/ghB), designated ghB₃. The ghB₃ formed a soluble homotrimer in solution, preferentially bound aggregated IgG in ELISA, and inhibited C1q-dependent hemolysis of sensitized SRBCs. The finding that the recombinant ghB₃ is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation of the CCP at a very early stage, and is consistent with the view that the globular region of C1q B chain is an independently folding module. The generation of a monomeric module as a homotrimeric structure also highlights the potential of the neck region of human SP-D as a trimerizing/multimerizing agent.

	Materials and Methods

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Purification of human C1q

Hemolytically active C1q was purified from human serum, using procedures previously described by Reid (13), with few modifications. Briefly, serum was dialyzed against a 50-fold excess of distilled water overnight at 4°C. The resulting precipitate was harvested, solubilized in TE buffer (20 mM Tris-HCl and 5 mM EDTA, pH 7.4) containing 500 mM NaCl, and passed through a Q-Sepharose column (Pharmacia, Piscataway, NJ), which retained C1r/C1s and IgM. The C1q-enriched flowthrough was then applied to an SP-Sepharose column (Pharmacia), extensively washed with TE containing 150 mM NaCl to remove IgG. C1q was eluted using a 150- to 500-mM NaCl gradient, and the peak fractions were concentrated by ultrafiltration and further purified by Superose 6 gel filtration chromatography. The purity of C1q was assessed by SDS-PAGE (15% w/v) under reducing conditions where it appeared as three bands, corresponding to the A, B, and C chains of 34, 32, and 27 kDa, respectively. The final yield of purified C1q from 100 ml serum was 2 mg.

Construction of plasmid encoding the neck region of human SP-D and globular head region of C1q B chain

The expression vector pMal-c2 (New England Biolabs, Beverly, MA), which contains the E. coli malE gene under the isopropyl -D-thiogalactoside (IPTG)-inducible P_tac promoter (coding for MBP), was used for expression. A Bluescript plasmid containing neck and CRD regions of human SP-D (12) was used to PCR-amplify the neck region as an XbaI-MscI fragment (170 bp). Using B chain cDNA as a template (1), the globular head region of the C1q B chain, corresponding to the residues 90–226, was PCR amplified as an SmaI-HindIII fragment (FP, 5'-GGGGACTACAAGGCCACCCAGAAA-3', universal reverse primer, 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, 30 cycles). In a three-piece ligation reaction, pMal-c2 (XbaI-HindIII backbone), neck region (XbaI-MscI), and the globular region of C1q B chain (SmaI-HindIII) yielded a new construct, designated pKBM-b₃, which comprised of the neck region/ghB linked to MBP and a factor Xa cleavage site.

Expression and purification of the recombinant ghB₃

E. coli BL21 containing pKBM-b₃ was grown in Luria-Bertani medium with ampicillin (100 µg/ml) to A₆₀₀ of 0.8 at 37°C and induced with 0.4 mM IPTG for 3 h. Cells from 1 L of culture (3 g of cell pellet) were pelleted by centrifugation, suspended in 50 ml lysis buffer (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1% v/v Triton X-100, 1 mM EGTA, 1 mM EDTA, and 5% v/v glycerol) containing lysozyme (100 µg/ml) and PMSF (0.1 mM), and incubated over ice for 30 min. The cell lysate was then sonicated at 60 Hz for 30 s with an interval of 1 min (15 cycles) to disrupt the cells and shear the bacterial chromosomal DNA. After centrifugation at 16,000 x g for 30 min, the supernatant was collected and diluted 5-fold using column buffer I (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1% v/v Triton X-100, 1 mM EDTA, and 5% v/v glycerol) and loaded on to an amylose resin column (50-ml bed volume; New England Biolabs) equilibrated with the column buffer. The column was washed successively with 3 bed volumes of column buffer I and 5 bed volumes of column buffer II (column buffer I without Triton X-100). The fusion protein was eluted with 100 ml of column buffer II containing 10 mM maltose. The peak fractions were pooled and dialyzed against factor Xa buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂, 5% v/v glycerol), and the concentration was adjusted to 1 mg/ml. Factor Xa (1 U/ml; New England Biolabs) was added (1 U factor Xa per 50 µg of fusion protein) and incubated overnight at 4°C. The factor Xa digest was loaded over a Q-Sepharose column and washed extensively with column buffer II (to remove unbound MBP, which elutes at 150 mM NaCl), and then ghB₃ was eluted using a 0.15–1 M NaCl gradient. The peak fractions containing ghB₃ eluted between 0.3 and 0.45 M. This pool was concentrated to 1 ml and loaded onto a Superose 12 gel filtration column (Pharmacia) equilibrated with 20 mM Tris-HCl, 100 mM NaCl, and 1 mM EDTA, pH 7.4. Fractions that eluted with an apparent molecular mass 60 kDa were pooled.

Western blot analysis of the recombinant ghB₃

The ghB₃ (2 µg) was electrophoresed on a 15% (w/v) SDS-PAGE under reducing conditions and electrotransferred to polyvinyl difluoride transfer membrane. After blocking with 2% (w/v) BSA, the membrane was probed with rabbit anti-human C1q or anti-ghB polyclonal Abs (7) (1:5000 dilution), followed by incubation with a goat anti-rabbit IgG-alkaline phosphatase conjugate (1:10,000 dilution). The blot was developed using the substrates, p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. The globular head region of native C1q (prepared after collagenase treatment) was used as positive control. BSA and human properdin were used as negative control proteins.

N-terminal sequencing

To confirm the N-terminal sequence of the recombinant protein, the purified ghB₃ was applied to an Applied Biosystems (Foster City, CA) 470A protein sequencer with an on-line Applied Biosystems 120A analyzer for the amino acid derivatives.

Chemical cross-linking and SDS-PAGE analysis

The recombinant ghB₃ (300 µg/ml concentration) was dialyzed overnight against 10 mM HEPES buffer, pH 7.5, containing 100 mM NaCl and 1 mM EDTA. An aliquot of dialysate (45 µl) was incubated with 5 µl of various concentrations of bis-(sulfosuccinimidyl) suberate (BS³; Perbio Science U.K., Chester, U.K.) for 30 min at room temperature. The cross-linking reactions were electrophoresed on a 15% (w/v) SDS-polyacrylamide gel under reducing conditions and stained with Coomassie blue R-250.

Binding specificity of ghB₃ for heat-aggregated IgG and IgM

C1q and ghB₃ (0–1 µg/ml) in sodium carbonate buffer, pH 9.6, were coated to the wells of polysorb ELISA plates overnight at 4°C. After blocking with 2% (w/v) BSA for 2 h and subsequent washing, the plates were incubated with heat-aggregated IgG (10 µg/ml) or IgM (20 µg/ml) in TBS-NTC (50 mM Tris-HCl, 150 mM NaCl, 0.05% w/v NaN₃, 0.05% v/v Tween 20, and 5 mM CaCl₂) at 37°C. Following a 2-h incubation, the plates were washed, and anti-human IgG and IgM, which had been conjugated to alkaline phosphate, were added (at a 1:10,000 dilution) to the appropriate wells. Following incubation for 2 h, the microtiter wells were developed using the substrate p-nitrophenyl phosphate, and A₄₀₅ was measured. BSA was used as a control protein.

Inhibition of C1q-dependent hemolysis by ghB₃

C1q hemolytic assays were essentially performed as previously described (7, 14). SRBC sensitized with either IgG (EA_IgG) or IgM (EA_IgM) were prepared in DGVB²⁺ (isotonic Veronal-buffered saline containing 0.1 mM CaCl₂, 0.5 mM MgCl₂, 0.1% w/v gelatin, and 1% w/v glucose). The addition of human C1q (1 µg/ml) back to C1q-deficient serum (1:40 dilution in DGVB²⁺) was sufficient to lyse >95% SRBC (EA_IgG or EA_IgM). Using a 1-µg/ml concentration of C1q, the experiments were performed to examine whether the binding of ghB₃ to EA_IgG or EA_IgM resulted in inhibition of C1q-dependent hemolysis.

Aliquots of EA_IgG or EA_IgM (10⁷/100 µl) were coincubated with ghB₃, MBP-ghB, MBP, and rSP-D (0–10 µg) for 1 h at 37°C. The pretreated cells were then gently pelleted by centrifugation at 3000 x g for 2 min, washed, and resuspended in 100 µl of DGVB²⁺. Each aliquot of SRBC was added to a mixture composed of 1 µg of C1q in 10 µl of DGVB²⁺, 2.5 µl of C1q-deficient serum, and 87.5 µl of DGVB²⁺. After a 1-h incubation at 37°C, the reaction was stopped by transferring the tubes to an ice bath and adding 0.6 ml ice-cold DGVB²⁺. The unlysed cells were pelleted by centrifugation, and A₄₁₂ values of 100-µl aliquots of the supernatant were measured. Total hemolysis was assessed as the amount of hemoglobin released upon cell lysis with water (100%). The C1q-dependent hemolytic activity was expressed as a percentage of total hemolysis. Purified MBP-ghB (7) was used as positive control, whereas MBP and a recombinant fragment of human SP-D, composed of trimeric neck and CRD regions (rSP-D; Ref. 12), were used as negative control proteins.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression and purification of ghB₃

The globular head region of human C1q B chain, together with the neck region of human lung SP-D, was expressed as a soluble fusion to E. coli MBP. As shown in Fig. 1A, upon induction with 0.4 mM IPTG, the fusion protein accumulated as an overexpressed protein of 60 kDa (lane 3). Following one-step affinity purification over an amylose resin column (lane 4), the neck/ghB fragment was cleaved away from MBP by using factor Xa (lane 5). MBP migrated at 40 kDa and ghB₃ at 20 kDa, which is its monomeric size. After completion of factor Xa cleavage (as judged by SDS-PAGE analysis), the free ghB₃ was further purified using Q-Sepharose column chromatography (Fig. 1C, lane 1). The final recovery of ghB₃ from a start culture of 1 L bacterial cells was 2–3 mg.

View larger version (45K): [in this window] [in a new window]   FIGURE 1. A, SDS-PAGE (15% w/v) analysis of the expression and purification of the ghB3 that was expressed as a fusion to MBP. Lane 1, molecular mass markers; lane 2, lysate from cells before IPTG induction; lane 3, upon induction with 0.4 mM IPTG, the fusion protein, MBP-ghB3, accumulated as an overexpressed protein of 60 kDa; lane 4, the MBP-ghB3 was affinity purified using amylose resin; lane 5, the MBP-ghB3 was then cleaved using site-specific factor Xa protease that yielded MBP (40 kDa) and ghB3 (20 kDa). B, Western blots of ghB3. Purified ghB3 was run on a SDS polyacrylamide gel (15% w/v) under reducing conditions, electrotransferred to polyvinyl difluoride membranes, and probed with specific Abs. Blot on left, lane 1, human gC1q (prepared after collagenase digestion of intact human C1q) probed with a rabbit anti-human C1q polyclonal Ab (lane 1). Blot in the middle is ghB3 probed with rabbit anti-human C1q polyclonal Ab. Lane 2, BSA; lane 3, human properdin; lane 4, ghB3. Blot on right is ghB3 probed with rabbit anti-recombinant ghB Ab (7 ). Lane 5, BSA; lane 6, human properdin; lane 7, ghB3. C, SDS-PAGE (15% w/v) analysis of purified ghB3 under reducing and nonreducing conditions. The ghB3 was separated away from MBP using Q-Sepharose ion-exchange chromatography, which migrated at 20 kDa under reducing (lane 1) as well as nonreducing conditions (lane 2).

The ghB₃ was recognized by rabbit anti-human C1q as well as anti-ghB polyclonal Abs (7), as judged by the Western blot (Fig. 1B). Automated N-terminal amino acid sequencing of the recombinant protein confirmed the presence of the neck region of human SP-D, preceded by eight Gly-Xaa-Yaa triplets, derived from the collagen-like region of human SP-D. When applied to a Superose 12 gel-filtration column, ghB₃ eluted as an apparent trimer of 60 kDa, immediately after BSA (data not shown). When examined by SDS-PAGE, the ghB₃ ran as a monomer even under nonreducing conditions (Fig. 1C, lane 2), suggesting that the trimerization was not because of aberrant disulfide bridges between the ghB regions. The fact that the neck region was responsible for the homotrimerization was further confirmed by the chemical cross-linking experiment, where a spectrum of monomer (20 kDa), dimer (40 kDa), and trimer (60 kDa) bands could be seen (Fig. 2). Upon reaction with a cross-linking agent (BS³), the highest oligomeric species seen was a trimer when the reaction progressed to completion, whereas protein bands corresponding to monomeric, dimeric, and trimeric species were seen in partially cross-linked reactions. Higher oligomers were never observed. A range of BS³ concentration was used to monitor the progression of cross-linking reaction.

View larger version (61K): [in this window] [in a new window]   FIGURE 2. SDS-PAGE (15% w/v) analysis of ghB3 under reducing conditions, following cross-linking in the presence of BS3. Lane 1, molecular mass markers; lane 2, ghB3 with no BS3 showing only monomer at 20 kDa; lane 3, ghB3 with 0.01 mM BS3 showing some dimer at 40 kDa; lane 4, ghB3 with 0.1 mM BS3 showing some dimer at 40 kDa and trimer at 60 kDa; lane 5, ghB3 with 1 mM BS3 showing mostly trimer at 60 kDa.

In the direct binding ELISA, different concentrations of C1q and ghB₃, coated on microtiter wells, were allowed to interact with fixed concentration of either heat-aggregated IgG or IgM. As shown in Fig. 3A, aggregated IgG bound, in a saturable manner, to C1q as well as ghB₃, whereas only background levels of IgG bound to BSA-coated wells. Although IgM bound C1q-coated wells in a dose-dependent manner, it did not bind solid-phase ghB₃ significantly (Fig. 3B). These results appeared to suggest that ghB₃ bound IgG preferentially and in a dose-dependent manner compared with IgM, which is consistent with our previously published data on the globular head region of human C1q B chain (7).

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Binding of heat-aggregated IgG and IgM to solid-phase-bound C1q and ghB3. Various concentrations of C1q and ghB3 were coated to ELISA plates and then incubated with a fixed concentration of heat-aggregated IgG (10 µg/well, A) or of IgM (20 µg/well, B) for 2 h at 37°C. The plates were washed, and the amount of each Ig binding to C1q and ghB3 was measured. All experiments were performed in duplicate, and BSA was used as a control protein. Both aggregated IgG and IgM were bound equally well by C1q, whereas ghB3 bound with more preferential affinity to the aggregated IgG than to IgM.

To examine whether the ghB₃ may have an inhibitory effect on the C1q-mediated complement activation, SRBC were sensitized with hemolysin (anti-SRBC IgG or IgM) to yield EA_IgG- or EA_IgM-sensitized cells. Reconstitution of C1q-deficient serum with 1 µg/ml of C1q was found to completely lyse (>95%) the sensitized SRBC. EA_IgG and EA_IgM were pretreated with various concentrations of ghB₃, MBP-ghB, MBP, and rSP-D before adding C1q- and C1q-deficient serum. The dimeric MBP-ghB (7) was chosen as a positive control for the assay because it has been previously shown to inhibit C1q-dependent hemolysis. As shown in Fig. 4A, the addition of 3.5 µg (58.3 pmol) ghB₃ brought hemolysis of EA_IgG down to <50%, whereas it required 5 µg (41.7 pmol) of MBP-ghB to bring about similar effect. In case of EA_IgM, it required 7.5 µg (120 pmol) ghB₃ to bring C1q-mediated hemolysis down to <50%, whereas it required 10 µg (83.4 pmol) of MBP-ghB to compete with C1q to a similar extent. Normal serum (1:20 dilution) and C1q-deficient serum (1:40 dilution) were used as control for complete and background lysis, respectively. MBP did not interfere with C1q-dependent hemolysis. We also included rSP-D, containing trimeric neck and CRD regions, to rule out the possibility that the neck region of SP-D, on its own, could interfere with the hemolytic assay.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Inhibition of the C1q-dependent hemolysis of sensitized sheep erythrocytes by ghB3. SRBC (1 x 107), EAIgG (A), or EAIgM (B) were pretreated with various concentrations of ghB3 for 1 h at 37°C, and then spun, washed, and resuspended in the assay buffer. C1q (1 µg), together with a C1q-deficient serum (diluted 1:40), was then added to the pretreated SRBC for an additional 1 h. The nonlysed cells were pelleted, and 200 µl DGVB2+ was added to the supernatant and A412 values were measured. , ghB3; , MBP-ghB; •, MBP; , rSP-D. The percentage of lysis was determined relative to complete (100%) hemolysis. The mean of three experiments is presented for each set of experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The three chains of human C1q show only 30% sequence identity on comparison with each other. When conservative replacement amino acids are also included, the conserved regions vary in length from a single to five or six residues. These conserved regions of hydrophobic amino acid residues are considered to be responsible for the general maintenance of the overall structure of the gC1q domain, rather than involved in binding to immune complexes (9). When the C-terminal sequences of human C1q A, B, and C chains (residue 90 onwards, based on the B chain numbering) are compared, 27% of the residues are found to be completely conserved. These include three cysteine and several hydrophobic and neutral residues that form the scaffold of the gC1q domain and impart upon it a largely sheet structure, as has been predicted from Fourier transform infrared spectroscopy and averaged structure predictions (31). The recently described crystal structure of recombinant, homotrimeric Acrp-30 has revealed an asymmetric trimer of sandwich protomers, each of which has a ten-strand jelly-roll folding topology, which is also seen in the TNF family of proteins (11). This fold appears to be common to all the members of the C1q family of proteins. The trimer is bell shaped, with a wide base. The trimer contacts take place through a cluster of hydrophobic interactions near the base, whereas the trimer interface near the apex is largely hydrophilic. These features are in common with TNF family trimers (32). The Acrp-30 structure shows that the globular region forms stable trimers, stabilized by a central hydrophobic interface, suggesting a structural basis for their role in triple-helical assembly of collagen regions. Four residues are conserved throughout the members of C1q and TNF families: Tyr¹⁶¹, Gly¹⁵⁹, Phe²³⁷, and Leu²⁴² (based on Acrp-30 numbering). Each residue seems important for the correct packing of the hydrophobic core of the protomer. Chemical modification studies have implicated two regions of the C1q globular domain in IgG binding (33); these are in the C1q B chain (site 1, localized to residues 114–129) and in the A and C chains (site 2, both around residue 160). Each of these maps to the exterior of the Acrp-30 trimer. These two C1q sites can also be mapped to two separate loops in the Acrp-30 crystal structure, although site 1 appears more attractive as a candidate binding surface. These observations (11, 33), together with previous studies (7, 8), strongly favored ghB as a candidate for homotrimerization (9). A few general conclusions can be drawn from the results described in this study: 1) a single globular head module of C1q does not appear to homotrimerize on its own, unlike other members of the C1q family; 2) engineering of a trimerizing, helical coiled-coil, neck region of human SP-D, upstream to single-chain globular head, can yield a stable homotrimer; 3) the homotrimeric globular head of C1q B chain can block C1q-dependent hemolysis of erythrocytes; and 4) the physical and functional behavior of ghB₃ implies that the B chain globular head is an independently folded module.

The expression and functional characterization of the ghB₃ has also indicated that the C-terminal regions of C1q A, B, and C chains, which form the globular head region, are likely to have some independence of structure and function (7, 8) and there is a major contribution of the globular region of the C1q B chain in binding to Ig. The production of ghB₃ also highlights the significance of the neck peptide in the trimerization of a heterologous module. This opens up the possibility of using the neck region (of human SP-D) to trimerize other low-affinity domains or modules, such as selectins, single-chain Abs, receptor molecules, etc., to generate high-affinity multimeric molecules. Although complement is an important line of defense against pathogens, its uncontrolled activation may lead to host tissue damage. Complement has been implicated in the pathogenesis of several diseases including autoimmune diseases, adult respiratory distress syndrome (ARDS), stroke, heart attack, burn injuries, and complications of cardiopulmonary bypass and xenotransplantation (34). The generation of ghB₃ opens up the possibility of blocking the CCP at a very early step (35).

	Acknowledgments

 
We thank Tony Willis for N-terminal sequence analysis and Dr. Sanjeev Gupta, Visiting Scientist to the Medical Research Council Immunochemistry Unit, Oxford, for help in the preparation of the revised manuscript.

	Footnotes

 
¹ This work was supported by the European Commission Biotechnology Program and the Medical Research Council, U.K.

² Address correspondence and reprint requests to Dr. Kenneth B. M. Reid, Department of Biochemistry, Medical Research Council Immunochemistry Unit, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.

³ Abbreviations used in this paper: CCP, classical complement pathway; ghA, ghB, and ghC, carboxyl-terminal, globular head region of the C1q A, B, and C chains, respectively; MBP, maltose-binding protein of E. coli; SP-D, surfactant protein D; EA_IgG, SRBC sensitized with IgG; EA_IgM, SRBC sensitized with IgM; DGVB²⁺, isotonic Veronal-buffered saline containing 0.1 mM CaCl₂, 0.5 mM MgCl₂, 0.1% w/v gelatin, and 1% w/v glucose; gC1q, globular domain present in the proteins belonging to the C1q/TNF- superfamily; CRD, carbohydrate recognition domain; IPTG, isopropyl -D-thiogalactoside; BS³, bis-(sulfosuccinimidyl) suberate.

Received for publication October 19, 1999. Accepted for publication October 6, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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