A model for the formation and interconversion of protein A-immunoglobulin G soluble complexes

We present a model for the formation and interconversion of the soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies. The basic elements of the model are developed from reported hydrodynamic and electron microscopic studies of these complexes (see accompanying companion paper), together with established structural and binding properties of IgG and SpA. The model includes specific symmetry and binding requirements for IgG-SpA combination, and a steric constraint between neighboring IgG molecules. We discuss how such a constraint could influence the assembly and distribution of equilibrium complexes. After formulating a convenient symbolism for representing IgG-SpA complexes, the suggested model is used to construct plausible structures for the four predominant complexes observed in moderate SpA excess. Distributions of these stable complexes at different IgG:SpA ratios, together with LeChatelier's principle and a straightforward thermodynamic derivation, are used to predict likely arrangements of equilibrium structures. Also, a scale model of the unique IgG4-SpA2 complex formed in IgG excess is constructed from reported x-ray diffraction and amino acid sequence data. An intuitive thermodynamic argument is used to show that the suggested steric constraint could cause the rather unprecedented reversible transformation of the four 7 to 15S complexes into the unique 17S complex. A computer simulation is used to predict equilibrium concentrations of the various proposed complexes at different IgG:SpA ratios. In support of the suggested structures, the calculated thermodynamic distributions agree surprisingly well with those measured with the ultracentrifuge. We point out how the proposed arrangements of the complexes, and in particular the 17S complex, can account for many of their novel properties, such as antigen-induced conformational changes. Reported differences in complement activation and precipitate formation by SpA complexes formed with antibodies from various species are also discussed with regard to possible differences in structural arrangements of the complexes.     

Separation of complexes containing protein A and IgG or Fcγ fragments by high-performance liquid chromatography

Protein A of Staphylococcus aureus is a bivalent Fc receptor that can form complexes with immunoglobulin G (IgG) or Fcγ fragments that activate humoral (e.g., complement) and cellular (e.g., lymphocyte) components of the immune system both in vitro and in vivo. To obtain complexes formed between protein A of Staphylococcus aureus (SpA) and rabbit IgG or Fcγ fragments for purposes of characterizing their compositions and studying their biological activities, we have used high-performance liquid chromatography to separate complexes in 20 min. Complexes were prepared with trace amounts of ¹²⁵I-SpA and ¹³¹I-IgG or ¹³¹I-Fcγ to simplify the analyses. With excess molar amounts of IgG or Fcγ the complexes have the molecular formulas [(IgG)₂SpA]₂ or [(Fcγ)₂SpA]₂. With excess SpA, complexes corresponding to (IgG)(SpA) or (Fcγ)(SpA) are formed, perhaps with other complexes that have different ratios of components. Since SpA is a rod-shaped molecule it elutes at a molecular weight corresponding to 240,000 rather than the true value of 42,000. This behavior is reflected in the elution of certain complexes at shorter retention times than expected on the basis of actual molecular weights, and facilitates separation of complexes from free IgG or Fcγ. The true molecular weights and molecular formulas of complexes isolated by HPLC were verified by ultracentrifugation. This HPLC method was used to study the interconversion and stability of complexes.     

Variations in the size of growth hormone-antibody complexes

Guinea pigs were immunized with extracted human growth hormone. Human sera were obtained after treatment with biosynthetic methionyl hGH. The size of hGH-anti hGH antibody complexes was determined from the sedimentation velocity at 100,000 g. At an excess of hGH over antibodies 8 S complexes were uniformly observed in human and guinea pig sera. S values between 11.8 and 15.6 were observed at antibody excess in individual guinea pig sera. Antibodies from humans treated with methionyl hGH formed smaller complexes (7.5 S). One patient with GH-deficiency developed resistance to treatment. Complexes of 12.3 S were formed by his antibodies. HGH sustains the formation of antibody complexes containing more than three IgG molecules (15.6 S). It is discussed that human antibodies of higher diversity may form complexes larger than trimers which initiate the complement cascade.     

Sedimentation Analysis of the Interacting Mixture of Dextran Sulfate and Low Density Lipoprotein

The reversible interaction between dextran sulfate (D) and the low density lipoprotein of human serum (P) was investigated by sedimentation velocity. Analysis of the velocity patterns of dextran sulfate—lipoprotein mixtures revealed that the maximum number of binding sites on dextran sulfate molecule is approximately 6. It was also shown that the species of the complex formed is affected by the mixing ratio of the two constituents: at the molar ratio (P/D) 0.69, the complex exists in average as DP_1.6 and at 0.98 as DP_2.2. The linear increment of sedimentation coefficient of the complex due to the binding of one lipoprotein molecule was 7.8S. Finally, the mechanism of precipitation of the complexes was discussed.     

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.     

Preparation and characterization of chemically defined oligomers of rabbit immunoglobulin G molecules for the complement binding studies.

Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at -70 degrees C.     

I-protein forms cage-like aggregates of myosin in vitro

I-protein was mixed with myosin before or after myosin filaments were reconstituted. In both cases, I-protein seemed to accelerate the myosin assembly. The binding of I-protein to myosin filaments was tested by sedimentation experiments and SDS-polyacrylamide gel electrophoresis. In a low ionic strength solution at pH 6.5, the binding ratio of I-protein to myosin was 1:40 by molar ratio when the I-protein molecules highly specifically bound to myosin filaments. I-protein could maximally bind to myosin filaments at the molar ratio of 1:2.7. In this case, excess I-protein molecules remained in the supernatant after sedimentation, although the unbound I-protein could still bind to myosin filaments. Electron microscopic observations revealed that I-protein bundled myosin filaments in the low ionic strength solution (pH 6.5). Cage-like structures which were very similar to the Mg-paracrystals of non-muscle myosins were formed at pH 7.2. In gel filtration, the apparent molecular mass of I-protein was 100 kDa, while it was 50 kDa in SDS gel electrophoresis. Therefore, I-protein is regarded to be a homodimer of a 50 kDa subunit and can divalently bind to myosin molecules.     

Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments

We have examined the structure of actin-binding molecules in solution and interacting with actin filaments. At physiological ionic strength, actin-binding protein has a M_r value of 540 × 10³ as determined by direct and indirect hydrodynamic measurements. It is an asymmetrical dimer composed of 270 × 10³ dalton subunits. Viewed in the electron microscope after negative staining or low angle shadowing, actin-binding protein molecules assume a broad range of conformations varying from closed circular structures to fully extended strands 162 nm in contour length. All configurations are apparently derived from the same structure which consists of two monomer chains connected end-to-end. The radius of gyration determined from the electron microscopic images was 21.3 nm in agreement with the value of 17.6 nm calculated from hydrodynamic assays. The average axial ratio from hydrodynamic measurements was 17:1, whereas fully extended dimer molecules in the electron microscope would have an axial ratio of 54:1. All of these observations indicate that actin-binding protein dimers are extremely flexible. The flexibility parameter λ (Landau &; Lifshits, 1958) for actinbinding protein is 0.18 nm^?1.As determined by sedimentation, actin-binding protein binds to actin filaments with a K_a value of 2 × 10⁶m^?1 and a capacity of one dimer to 14 actin monomers in filaments. After incubation of high concentrations (molar ratio to actin ≥ 1:10) of actin-binding protein with actin filaments, long filament bundles are visible in the electron microscope. Under these conditions, actin-binding protein molecules decorate the actin filaments in the bundles at regular 40 nm intervals or once every 15 monomers, approximately equivalent to the binding capacity measured by sedimentation. Low concentrations of actin-binding protein (molar ratio to actin ≥ 1:50) which promote the gelation of actin filaments in solution, did not detectably alter the isotropy of the actin filaments. Direct visualization of actinbinding protein molecules between actin filaments in the electron microscope showed that dimers are sufficient for crossbridging of actin filaments and that actinbinding protein dimers are bipolar, composed of monomers connected head-to-head and having actin-binding sites located on the free tails.We conclude that actin-binding protein is a dimer at physiological ionic strength. Each dimer has two actin filament binding sites and is therefore sufficient to gel actin filaments in solution. The length and flexibility of the actin-binding protein subunits render this molecule structurally suited for the crosslinking of large helical filaments into isotropic networks.     

Interaction of α-lactalbumin with dimyristoylphosphatidylcholine vesicles. III. Influence of the temperature and of the lipid-to-protein molar ratio on the complex formation

We investigated the interaction between α-lactalbumin and sonicated dimyristoylphosphatidylcholine at pH 4 and different temperatures. (1) At 23°C and lipid-to-protein molar ratios below 170, the interaction results in a disruption of the original vesicles to form smaller complex particles. By the sedimentation velocity method we determined for this particle a molar mass of (1.05 ± 0.16) · 10⁶ g·mol^?1. The lipid-to-protein molar ratio within the complex particle is 70/1, as earlier estimated. It follows that there are approximately 1200 lipid and 17 α-lactalbumin molecules per particle. At molar ratios above 170, α-lactalbumin strongly associates with the vesicles. In this case the vesicle entity remains. The ability of α-lactalbumin to break up the vesicles at this temperature is determined by the number of protein molecules which are required in the complex particle. (2) By means of fluorescence polarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene and energy transfer of the tryptophan groups of the protein to 1,3-(1,1′-dipyrenyl)propane located in the hydrocarbon region of the vesicles, it is shown that with increasing temperature above 25°C, complexes of decreasing internal lipid-to-protein molar ratio are formed. However, by electron microscopy we show that the overall size of these complexes remains approximately the same, i.e., bars with dimensions $\text{70 × 220}\text{A}\text{?}$. A temperature-reversible transformation occurs between these complexes, which cannot be isolated by gel chromatography. In contrast, the complex of molar ratio 70/1 remains stable at lower temperatures.     

Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

An octamer of histones H3 and H4 forms a compact complex with DNA of nucleosome size.

Equimolar mixtures of histones H3 and H4 have been reconstituted onto DNA of nucleosome core size. Two distinct complexes are formed in a relative abundance that depends on the starting ratio of H3 + H4 to DNA. One of these complexes contains two H3-H4 dimers for each DNA molecule, and has a sedimentation coefficient of 7.5S. The other complex contains an octamer consisting of four H3-H4 dimers, and has a sedimentation coefficient of 10.4S. On the basis of these measurements, we conclude that the octamer complex (but not the tetramer complex) is a fully folded, compact structure resembling the nucleosome.     

Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na(2)S(2)O(4) at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)-(Fe+Mo-containing protein).     

Monoclonal alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex: production of antibody, optimization of activity, and use in immunostaining

A mouse monoclonal antibody, FMC55 (an IgG1), to alkaline phosphatase was prepared and evaluated in immunostaining. Clones producing antibody to alkaline phosphatase were selected using a micro-ELISA which identified antibodies forming active soluble complexes (APAAP) with the enzyme. Conditions that influenced the formation of the complex were investigated by using a quantitative assay in which the complex was captured by a bridging anti-mouse antibody. The ratio of FMC55 to enzyme had a major influence on the activity of the complex. Although all complexes had some activity, those that contained excess antibody had reduced ability to bind to anti-mouse antibody because of competition with excess unlabeled antibody. The optimal complex was formed with 3 micrograms of FMC55 per unit of enzyme. This complex contained neither free enzyme nor free antibody. The molecular weight by gel permeation chromatography was 600,000, giving a composition of two enzyme and two antibody molecules or one enzyme and three antibody molecules. The size of the complex was not altered by adding excess antibody or excess enzyme. Immunoblotting showed that FMC55 bound only to the Mr 140,000 homodimeric form of alkaline phosphatase. The APAAP complex was used in combination with biotin-streptavidin-peroxidase reagent to detect two antigens labeled with two different mouse monoclonal antibodies in the same tissue preparation.     

Size and structure of antigen-antibody complexes: thermodynamic parameters

The role of antigen-antibody (Ag-Ab) complexes in the immune response depends, in part, on the size of the complexes. Previously, we combined electron microscopy with classical and quasi-elastic light scattering to characterize the molecular weight distribution and the conformation of Ag-Ab complexes made from bovine serum albumin (BSA) and pairs of anti-BSA monoclonal antibodies at a single concentration and Ag:Ab molar ratio. In this report, the molecular weight distribution of Ag-Ab complexes was determined by classical light scattering at a single Ag:Ab ratio and over a range of concentrations, and binding of BSA to pairs of MAb was determined by radioimmunoassay at several Ag:Ab molar ratios. A thermodynamic model was developed for the equilibrium size distribution of Ag-Ab complexes formed between a pair of MAb, each with unique affinity and specificity, and an Ag containing a single epitope for each of the pair of MAb. The combined experimental data were used in conjunction with the model to determine the values of cyclization and polymerization constants. Successful determination of the parameters required data from both classical light scattering and electron microscopy. Cyclization constants were lower than those reported in other studies of Ag-Ab complexes; this may be attributable to our use of a protein Ag, as compared to a divalent hapten. In two out of three cases, cyclization constants increased with increasing number of Ab in the complex, in contrast to previous assumptions. The validity of the thermodynamic model was further shown by its ability, in combination with conformational and hydrodynamic model, to predict the hydrodynamic radius of the complexes over a wide range of experimental conditions.     

Recombinant models of lipoproteins. Apolipoprotein A-I/phosphatidylcholine/cholesterol complexes formed in a 2-chloroethanol-water mixture

Apolipoprotein A-I can spontaneously associate with phosphatidylcholine and cholesterol in 2-chloroethanol-water mixture. It was demonstrated, using a spin label technique, that dissolved molecules participate in complex formation. The apolipoprotein A-I/phosphatidylcholine/cholesterol complexes were isolated by gel chromatography. Complexes of three types were prepared and characterized: type A, large heterogeneous aggregates with molecular weight 600 000, sedimentation coefficient 10 S and the following molar composition - protein/phosphatidylcholine/cholesterol, 1:(70-100):(10-12); types B and C, with weight average molecular weights 140 000 and 110 000, average sedimentation coefficients 3.6 S and 1.7 S, respectively. Both types have the same molar composition - protein/phosphatidylcholine/cholesterol, 1:25:8. The dissimilar sedimentation coefficients between complexes B and C may be explained by the difference in the monomer/tetramer ratio (monomer molecular weight 50 000). The spin label sn-1-O-stearoyl-2-O-9'-spiro(4',4'-dimethyloxazolidine-3'-oxyl) heptadecanoylglycero-3-phosphocholine introduced into the complexes A and B showed different thermal properties of these complexes, which may be due to differences in the lipid-protein interactions.     

Immunoglobulin M synthesized by human lymphoblastoid cells: interaction with Staphylococcus aureus and protein A.

Immunoglobulin M synthesized by a human lymphoblastoid cell line, LA173, was found to bind specifically to the protein A-bearing Cowan I strain of Staphylococcus aureus. The (3H)-leucine-labeled, secreted IgM from these LA173 cells also formed precipitin complexes with purified protein A. Soluble complexes formed at high protein A/IgM ratios retained the ability to bind to the bacterial surface. Precipitin complexes also were observed in double diffusion Ouchterlony gels with a line of identity formed between the IgM, protein A, and anti-IgM in adjacent wells. Intracellular IgM species from detergent-lysed LA173 cells were bound to S. aureus. Labeled 19S pentamers, 8S monomers, and HL subunits were eluted from the bacteria and identified by velocity sedimentation and SDS agarose-acrylamide gel electrophoresis. In addition, several intermediates migrating between 8S and 19S were detected and shown to contain authentic H and L chains. Binding of the labeled IgM 19S pentamers to staphylococci was not inhibited by prior treatment of the bacteria with an excess of unlabeled human IgG. However, S. aureus saturated with unlabeled IgG did not bind either labeled IgM monomers or labeled IgG. The interaction of this human IgM with S. aureus exhibited a high degree of specificity with quantitative recovery of secreted 19S IgM. Intracellular IgM species were bound selectively by the bacteria with little if any contamination by other cytoplasmic proteins.     

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.     

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.