Biomedical subjects

S B Easterbrook-Smith

Publications and source records attributed to S B Easterbrook-Smith.

At least 19 recordsLinked to original sources

PubMed1992

Clusterin binds by a multivalent mechanism to the Fc and Fab regions of IgG.

Clusterin was purified from human serum by IgG and monoclonal antibody affinity chromatography. SDS-PAGE and immunoblotting revealed no major differences between clusterin prepared in these two ways. An ELISA method for measuring the binding of clusterin to immunoglobulins was developed. Clusterin purified by IgG affinity chromatography bound to pooled human IgG with a similar affinity (S0.5 5.9 +/- 0.4 micrograms/ml) as clusterin purified by monoclonal antibody chromatography (S0.5 6.1 +/- 0.2 micrograms/ml). The apparent affinity of clusterin for IgG immobilised on ELISA plates increased with increasing concentrations of IgG in the coating solution. Aggregated IgG in solution was a more potent inhibitor of the binding of clusterin to immobilised IgG than was monomer IgG. Clusterin bound to all of the isotypes of human IgG, and to human IgA and IgM, with apparent affinities in the order IgG3 > IgG4 > IgM > IgG1 > IgG2, IgA. Clusterin bound to both the Fab and Fc fragments of human IgG. The clusterin binding site(s) on the Fc do not overlap with those for protein A and Clq.

Antibodies, Monoclonal↗

PubMed1992

RHP is antigenically related to factor H and binds to the globular heads of C1q.

RHP was purified from normal serum by sequential euglobin precipitation, ion exchange chromatography on DEAE-Sephacel and gel filtration using Sephacryl S-300. RHP reacted with anti-Factor H antibodies in ELISA assays and in Western blots, suggesting that it is antigenically related to Factor H. It bound to intact C1q but not to the collagen-like N-terminal half of the molecule. C1q-specific monoclonal antibody BUS-1, which blocks the binding of C1q to immune complexes, did not block the binding of RHP to C1q. This implies that the binding sites on C1q for IgG and RHP do not overlap.

Binding Sites↗

PubMed1991

Clusterin enhances the formation of insoluble immune complexes.

Clusterin was purified from human serum by sequential affinity chromatography over IgG-, protein A- and Con A-Sepharose. The protein was approximately 70 kDa by SDS/PAGE under nonreducing conditions and was resolved into approximately 35 kDa bands under reducing conditions. The protein reacted with clusterin-specific Mabs in ELISA and in Western blots. Its N-terminal sequences agreed with those published for clusterin. An antiserum specific for clusterin made by the above method detected it in complement membrane attack complexes on rabbit erythrocyte membranes. The interaction of clusterin with IgG was physiologically relevant because it was found to increase the rate of formation of insoluble immune complexes.

Amino Acid Sequence↗

PubMed1991

The Fab/c fragment of IgG produced by cleavage at cyanocysteine residues.

The intra- and inter-heavy chain disulfides of rabbit IgG were cleaved by mild reduction with either dithiothreitol or sulfite and cyanocysteines generated by treatment with either 2-nitro-5-thiocyanobenzoic acid or KCN, respectively. When cleavage occurs at a cyanocysteine residue in the hinge region of one heavy chain alone the Fab/c fragment is produced. Fab/c was also produced by papain digestion of IgG. Fab/c made by papain digestion was able to active complement in haemolytic assays; this activity was lost after cleavage of its accessible disulfide bonds. Fab/c made by cyanylysis of sulfite-reduced IgG was also active in these assays, but Fab/c made by cyanylysis of dithiothreitol-reduced IgG was not. Treatment of the latter fragment with cysteine and cystine resulted in partial reformation of cleaved disulfide bonds. Fab/c was also made from human IgG and from murine IgG2a and IgG2b.

Animals↗

PubMed1990

Single-step purification of immunoglobulin M on C1q-Sepharose.

A rapid and simple affinity chromatography method for purifying IgM from myeloma serum and ascites fluid is described. Complement protein C1q is coupled to Sepharose with an efficiency of 35%, giving 1.7 mg of C1q bound/ml of gel. This C1q-Sepharose selectively binds IgM from crude samples at 5 degrees C, with a capacity of 0.4 mg of IgM/ml of gel. The bound IgM may be eluted simply and isocratically by bringing the gel to room temperature for 2 h, or by washing with buffer containing 0.5 M KI. The eluted IgM is highly pure by SDS-PAGE and double immunodiffusion analysis, although IgG may be a potential contaminant. The C1q-Sepharose is stable for at least 18 months.

Chromatography, Affinity↗

PubMed1990

Carbodiimide crosslinking of human C1q and rabbit IgG.

The water soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to covalently link carboxyl groups on rabbit IgG to lysyl groups on complement protein C1q. The interaction between C1q and IgG was disrupted by varying the pH, modifying essential residues in the IgG binding site of C1q and by reducing the interchain disulfides of IgG. Under each of these conditions the correlation found between binding and crosslinking indicated a strong requirement for the proteins to bind normally in order for crosslinking to occur. SDS-PAGE analysis of the crosslinked material showed a 210 kDa band consistent with one IgG crosslinked to two disulfide linked C1q chains. Blotting and autoradiography showed the crosslinking involved the A and/or B and C chains of C1q. The lysines flanking the intrachain half cystines are proposed as the likely candidates for crosslinking to IgG, thus delineating the immunoglobulin binding site of C1q.

Animals↗

PubMed1989

Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q.

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.

Amino Acid Sequence↗

PubMed1988

Enhancement of the binding of C1q to immune complexes by polyethylene glycol.

The binding of 125I-labelled human C1q to insoluble rabbit IgG:ovalbumin immune complexes was enhanced by polyethylene glycol (PEG, Mr 8 x 10(3)) in the concn range 0-2.5% (w/v). C1q with native immunoglobulin bindings sites rendered inactive by diethylpyrocarbonate treatment did not bind to immune complexes in the presence of PEG. The ionic strength dependence of the binding was independent of the presence of PEG. There was a linear relationship between the logarithm of the apparent affinity constant of the C1q:immune complex interaction and PEG concn.

Animals↗

PubMed1988

The role of Fc:Fc interactions in insoluble immune complex formation and complement activation.

The initial rate of formation of insoluble immune complexes from rabbit IgG and ovalbumin was approximately 12 times that for formation of F(ab')2-ovalbumin complexes. At low IgG concns, in the range 0.7-2.7 nM, the formation of insoluble immune complexes was characterised by a lag phase, especially for complexes formed in low antigen excess, compared to antibody excess. Guanidine HCl (at concns up to 0.5 M) and urea (at concns up to 1 M) decreased the initial rates of formation of IgG immune complexes more than F(ab')2 immune complexes. Pre-formed IgG immune complexes were solubilised at lower guanidine HCl concns than were F(ab')2 immune complexes. C1q enhanced the initial rate of formation of IgG immune complexes at C1q:IgG ratios up to 1:1. Higher C1q concns decreased the initial rate of formation of the complexes. Urea (1 M) blocked the C1q mediated enhancement of immune complex formation.

Animals↗

PubMed1987

High-performance liquid chromatography-based assay for the quantitation of protein maleylation at the picomole level.

A new HPLC-based assay for the direct determination of the extent of maleylation of proteins is described. The rationale is founded on the HPLC detection of maleic acid that results from cleavage of maleyl lysyls by low pH treatment. The method, employing a C-18 reverse-phase column eluted isocratically with 0.01 M acetic acid, pH 5.4, and absorbance monitored at 210 nm, is sensitive (lower limit 1 pmol) and linear in the range 1 pmol-0.5 mumol and allows recovery of the native protein.

Chromatography, High Pressure Liquid↗

PubMed1987

The presence of histidine residues at or near the C1q binding site of rabbit immunoglobulin G.

Treatment of covalently crosslinked rabbit IgG oligomers with diethylpyrocarbonate resulted in the loss of their C1q binding activity. The inactivation was a first-order process with respect to time in the range 0-8 min, and modifier concentration from 0 to 2.39 mM. Hydroxylamine treatment of diethylpyrocarbonate-treated IgG oligomers led to 80% recovery of their C1q binding activity. Diethylpyrocarbonate treatment of IgG oligomers had little effect on their absorbance at 278 nm, but led to an increase in their absorbance at 242 nm. The apparent pKa of the modified residues was 6.91 +/- 0.12. These data are consistent with diethylpyrocarbonate modification of histidine residues leading to loss of C1q binding activity in rabbit IgG oligomers. Modification of four histidine residues per IgG molecule was associated with the loss of C1q binding activity. Thus, there may be two histidine residues at or near the C1q binding sites in the CH2 domains of rabbit IgG.

Animals↗

PubMed1987

Activation of the classical complement pathway by BioRex-70.

The cation exchange resin BioRex-70 was able to activate the classical complement pathway in human serum at 37 degrees C over the resin concentration range 0-5% (v/v). Using zymosan-treated human serum, it was found that the activation proceeded as far as complement protein C3.

Cation Exchange Resins↗

PubMed1986

Conformational changes in C1q upon binding to IgG oligomers.

The interaction between C1q and immune complexes is inhibited by 1-anilino-8-naphthalenesulfonate (ANS) in the concentration range of 2-4 mM. ANS binds to Clq with a 20-fold higher affinity than to IgG [(1986) Mol. Immunol. 23, 39-44] and therefore it is possible to label only C1q with ANS in the presence of IgG. Under such conditions no inhibition is observed. Addition of monomer IgG to a solution of C1q-bound ANS did not significantly alter the fluorescence of the ANS. However when oligomeric IgG was added there was a 2-fold increase in fluorescence over the same IgG concentration range. When C1q was pretreated with diethylpyrocarbonate there was little change in the fluorescence when IgG oligomers were added to C1q:ANS solutions. These results suggest that C1q undergoes conformational changes upon binding to IgG oligomers.

Anilino Naphthalenesulfonates↗

PubMed1986

Binding of complement component C1q by spectrin.

125I-labelled human C1q was found to bind to human spectrin. Scatchard plots for the binding process were non-linear, indicating the possible presence of multiple classes of binding sites for C1q on spectrin. The binding was ionic-strength-dependent; the extent of binding decreased with increasing ionic strength. Chemical modification of arginine and histidine residues on C1q as well as pretreatment of C1q at pH 4.45 or at 56 degrees C reduced its spectrin binding activity. The amount of 125I-labelled C1q bound to immune complexes was reduced by the presence of spectrin. Spectrin was also able to deplete the complement haemolytic activity of human serum in a dose-dependent manner.

Complement Activating Enzymes↗

PubMed1986

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine.

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.

Animals↗

PubMed1985

Evidence for arginine residues in the immunoglobulin-binding sites of human Clq.

The immune complex binding activity of human Clq was lost following treatment of the protein with the arginine-selective reagents cyclohexane 1,2-dione and phenylglyoxal. Both inactivations followed pseudo-first-order kinetics. The affinity of Clq for immune complexes was reduced 7-fold following cyclohexane-1,2-dione treatment, and could be substantially restored by treatment of the modified protein with hydroxylamine. Heat-aggregated IgG protected Clq against inactivation by both reagents. Incorporation of 25 molecules of [7-14C]phenylglyoxal per Clq molecule completely inactivated the protein. These data are consistent with the presence of arginyl residues in the immunoglobulin recognition sites of human Clq.

Antigen-Antibody Complex↗

PubMed1985

C1q binding to mitochondria: a possible artefact?

Mitochondrial preparations, obtained from human tonsils and from rat spleen, liver, heart, and kidney tissues, bound [125I]C1q with affinities of 10(7)-10(8)M-1. The binding of C1q was not affected by treatment of the mitochondrial preparations with pronase, trypsin, or phospholipase D, but it was lowered 5-6-fold following treatment of the mitochondria with DNase and RNase. Binding of C1q to mitochondrial preparations was also greatly diminished by limited chemical modification of C1q with cyclohexane-1,2-dione. It is suggested that the reported binding of C1q to mitochondria may have arisen from the protein binding to DNA and/or RNA contaminating the mitochondrial preparations.

Animals↗

PubMed1985

Binding of complement component C1q by rat adipocyte membranes.

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.

Adipose Tissue↗