Image analysis and three-dimensional model of chymotrypsin-transformed human alpha 2-macroglobulin complexed with a monoclonal antibody specific for this conformation

Alpha 2-macroglobulin (alpha 2M) is a plasma inhibitor of proteinases, the steric mechanism of which is based on a considerable conformational change. The typical and distinct H-like shape of alpha 2M-chymotrypsin (alpha 2M-chy) complexes seen by electron microscopy led us to an ultrastructural study of the binding of a monoclonal antibody (Mab) specific for this conformation of alpha 2M. The epitope of this Mab is located near the extremities of the 4 arms of the H-like alpha 2M-chy, at a site that is not accessible on the native molecule. The identical binding of the Mab on the 4 arms of the tetrameric molecule demonstrates that these arms are equivalent portions of the 4 monomers. Various types of immune complexes between alpha 2M and IgG are described, and images of individual immune complexes were processed by correspondence analysis. This extracts new information concerning the organization of chymotrypsin-transformed alpha 2M. The molecule appears asymmetrical, presents 2 conformational states (which we describe as relaxed and twisted), and has flexible arms. These intramolecular motions are supposed to be related to IgG binding. The results are discussed in comparison with previously published models of proteinase-transformed alpha 2M.     

Proteolysis of human alpha 2-macroglobulin without hydrolysis of the internal thiolesters or expression of the receptor recognition site

Proteolysis of human alpha 2-macroglobulin (alpha 2M) in the bait region is the prerequisite and necessary trigger for the trapping of the proteinase by a massive conformational change of alpha 2M. This labilization of the native conformation of alpha 2M is mediated by activation of the internal thiolesters, but the underlying mechanism is unknown. We now describe observations on proteolysis of human alpha 2M without concomitant hydrolysis of the internal thiolesters or conformational change. This proteolysis was obtained with a novel bacterial proteinase we recently used to isolate the receptor-binding domain from alpha 2M (Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). This proteinase is not inhibited by alpha 2M, and therefore it was possible to study its effect on native alpha 2M at pH 4.5, conditions used previously to produce the receptor-binding domain (Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). The major observations are that despite extensive proteolysis, alpha 2M largely retained its native conformation as shown by rate electrophoresis, the absence of binding of monoclonal antibody F2B2, and the incorporation of [14C]methylamine into a 145-kDa fragment of alpha 2M. Moreover, the derivative still bound trypsin to 88% of control values. Treatment of the derivative with trypsin or methylamine produced the conformational change as with intact alpha 2M, and concomitantly released the receptor-binding domain. This indicated that proteolysis at Lys1313-Glu also proceeded in native alpha 2M. At least one more major proteolysis site was deduced from the observation of a 27-kDa heat-induced fragment, the 145-kDa [14C]methylamine-labeled fragment, and from the presence of the 20-kDa receptor-binding domain. These results demonstrate indirectly the particular relation of the bait region to the internal thiolesters and illustrate further the domain-structure of alpha 2M and the expression of the receptor-recognition site by activation of the internal thiolesters.     

The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.     

Image processing of proteinase- and methylamine-transformed human alpha 2-macroglobulin. Localization of the proteinases

Comparative x-ray scattering experiments and electron microscopic observations have been performed on native S-form, and on different F-forms of human plasma alpha 2-macroglobulin (alpha 2M), obtained by proteinase (chymotrypsin, plasmin, and thrombin) or methylamine treatment. Image processing of electron micrographs of the alpha 2M molecules transformed by chymotrypsin, plasmin, and methylamine displayed average images which could be compared. The proteinase-complex alpha 2M molecules exhibited the usual H-like structure, but the methylamine-inactivated ones showed a different organization, with almost no stain-excluding material in the central region of the molecule, which therefore presented a central cavity filled with stain. By subtracting average images of alpha 2M-methylamine from alpha 2M-chymotrypsin or alpha 2M-plasmin, a putative localization of the proteinases inside the alpha 2M molecule, very close to its center was revealed. The values of the radii of gyration for the S- and F-forms obtained by x-ray scattering were very different (78 and 67.7 A, respectively). All four scattering curves of the F-forms were comparable in shape and showed maxima and minima different from that of the S-form alpha 2M. Image processing of electron micrographs and x-ray scattering have provided independent results which indicate that a large cavity exists in the alpha 2M-methylamine molecule and that the proteinases might be located in a very central position inside the alpha 2M-proteinase molecules.     

Identification of alpha 2-macroglobulin conformational intermediates by electron microscopy and image analysis. Comparison of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin reacted with cis-dichlorodiammineplatinum(II) and trypsin.

Human alpha 2-macroglobulin (alpha 2M) exists in two well defined, highly distinct conformations and in less well described intermediate conformations. In this study, previously characterized reactions were used to partially or completely transform the conformation of alpha 2M. Electron micrographs of each preparation were subjected to image analysis. Ternary alpha 2M-trypsin (2 mol of trypsin/mol of alpha 2M) was analyzed as a control for the fully transformed state. Correspondence analysis (CORAN) and hierarchical ascendant classification (HAC) generated five image clusters from 330 aligned alpha 2M-trypsin complexes. Average images of each cluster resembled the letter "H" with four nearly equivalent lateral arms. Abnormally shaped lateral arms were not demonstrated by HAC, using a variety of factor sets. In a native polyacrylamide gel electrophoresis system, alpha 2M-thrombin migrated in a diffuse band partially behind alpha 2M-trypsin, suggesting conformational heterogeneity. CORAN and HAC of 733 alpha 2M-thrombin complexes identified two neighboring clusters, the average images of which showed an H-like structure in which one arm was replaced by a globular stain-excluding body. The two alpha 2M-thrombin clusters included 125 images (17.1% of image population). The complete absence of atypical lateral arm structure in the alpha 2M-trypsin clusters suggests that this variation is not the result of orientation or staining artifact. Native alpha 2M was reacted with cis-dichlorodiammineplatinum(II) and then with trypsin to form alpha 2M-Pt-trypsin, a preparation that includes partially transformed alpha 2M structures. CORAN and HAC of 580 alpha 2M-Pt-trypsin complexes generated five clusters, the average images of which showed atypical lateral arm structure equivalent to that demonstrated with alpha 2M-thrombin. The five alpha 2M-Pt-trypsin clusters accounted for 15.2% of the image population. These studies suggest that alpha 2M conformational change intermediates demonstrate common structural characteristics, permitting an elucidation of the steps involved in this complex transformation.     

Structural details of proteinase entrapment by human alpha2-macroglobulin emerge from three-dimensional reconstructions of Fab labeled native, half-transformed, and transformed molecules

Three-dimensional electron microscopy reconstructions of native, half-transformed, and transformed alpha2-macroglobulins (alpha2Ms) labeled with a monoclonal Fab Fab offer new insight into the mechanism of its proteinase entrapment. Each alpha2M binds four Fabs, two at either end of its dimeric protomers approximately 145 A apart. In the native structure, the epitopes are near the base of its two chisel-like features, laterally separated by 120 A, whereas in the methylamine-transformed alpha2M, the epitopes are at the base of its four arms, laterally separated by 160 A. Upon thiol ester cleavage, the chisels on the native alpha2M appear to split with a separation and rotation to give the four arm-like extensions on transformed alpha2M. Thus, the receptor binding domains previously enclosed within the chisels are exposed. The labeled structures further indicate that the two protomeric strands that constitute the native and transformed molecules are related and reside one on each side of the major axes of these structures. The half-transformed structure shows that the two Fabs at one end of the molecule have an arrangement similar to those on the native alpha2M, whereas on its transformed end, they have rotated. The rotation is associated with a partial untwisting of the strands and an enlargement of the openings to the cavity. We propose that the enlarged openings permit the entrance of the proteinase. Then cleavage of the remaining bait domains by a second proteinase occurs with its entrance into the cavity. This is followed by a retwisting of the strands to encapsulate the proteinases and expose the receptor binding domains associated with the transformed alpha2M.     

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.     

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen.

The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.     

Ig-binding bacterial proteins also bind proteinase inhibitors

Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.     

A conserved region in alpha-macroglobulins participates in binding to the mammalian alpha-macroglobulin receptor

Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of syntenin binding protein in the black tiger shrimp Penaeus monodon

Shrimp exhibit a diverse response to viral infection that is manifested in drastic up- and down-regulations of a variety of genes. In our previous work, we identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly upregulated in the acute phase of the infection. In order to further explore the link between Pm-syntenin and viral infection, we performed a yeast two-hybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. One of the molecules that specifically interacted with Pm-syntenin was the receptor-binding domain of alpha-2-macroglobulin (alpha2M). A GST pull-down assay showed that GST-alpha2M, but not GST alone, was capable of co-precipitating syntenin. Another GST pull-down assay showed that GST-syntenin, but not GST alone, was capable of co-precipitating alpha2M. In addition, mutant analyses showed that the N-terminal 131 amino acids of syntenin were both necessary and sufficient to bind the C-terminus receptor-binding domain of alpha2M. Furthermore, WSSV-infected Pm showed a significant upregulation of the alpha2M message, suggesting that both syntenin and its protein partner alpha2M are upregulated in the acute phase of a WSSV infection. Taken together with a previous report showing the co-localization of alpha2M and syntenin in the exosome of a dendritic cell line, it is likely that syntenin, through its interaction with alpha2M, plays an important role in the immune defense mechanisms of viral infections of shrimps.     

The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase

Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.     

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.     

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of beta2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasabeta2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M-methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M-trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significantly increased fractions of transformed to total alpha2-macroglobulin concentrations in plasma from patients with multiple sclerosis

We examined the proteinase inhibitor alpha2-macroglobulin (alpha2M) in plasma from patients with multiple sclerosis (MS); a neurological disease of the central nervous system. The plasma concentrations of native and transformed alpha2M were measured in 90 patients with clinically definite MS, 73 with relapsing-remitting and 17 with secondary progressive MS, and 132 healthy individuals. Significantly lower concentrations of native alpha2M and significantly higher concentrations of transformed alpha2M were found in MS patients. A significant correlation between the concentrations of native and transformed alpha2M was found. The fraction of transformed to total alpha2M in the MS patients was 36% higher than in the healthy individuals. The results suggest an important involvement of alpha2M in regulation of increased proteolytic activity occurring in MS disease.     

The three-dimensional structure of the human alpha 2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin alpha 2-macroglobulin complexes

Three-dimensional electron microscopy reconstructions of the human alpha(2)-macroglobulin (alpha(2)M) dimer and chymotrypsin-transformed alpha(2)M reveal the structural arrangement of the two dimers that comprise native and proteinase-transformed molecules. They consist of two side-by-side extended strands that have a clockwise and counterclockwise twist about their major axes in the native and transformed structures, respectively. This and other studies show that there are major contacts between the two strands at both ends of the molecule that evidently sequester the receptor binding domains. Upon proteinase cleavage of the bait domains and subsequent thiol ester cleavages, which occur near the central region of the molecule, the two strands separate by 40 A at both ends of the structure to expose the receptor binding domains and form the arm-like extensions of the transformed alpha(2)M. During the transformation of the structure, the strands untwist to expose the alpha(2)M central cavity to the proteinase. This extraordinary change in the architecture of alpha(2)M functions to completely engulf two molecules of chymotrypsin within its central cavity and to irreversibly encapsulate them.     

Molecular heterogeneity of Fc alpha receptors detected by receptor-specific monoclonal antibodies.

Fc alpha receptors (Fc alpha R) were isolated from a human monocytic cell line and used to raise four mAb with receptor specificity. The antibodies were used to identify the types of white blood cells that express Fc alpha R and the molecular heterogeneity of the receptor molecules. Nonpolymorphic epitopes, outside of the Fc alpha-binding site, were recognized only on blood cells of granulocyte and monocyte/macrophage lineages. The molecules identified, both by the antibodies and by the IgA ligand, were glycoproteins ranging in relative molecular mass from 55 to 75 kDa. However, one antibody detected a subpopulation of Fc alpha R molecules characterized by relatively restricted size heterogeneity. A complex glycosylation pattern was revealed by the resolution of discrete 32- and 36-kDa molecular species after removal of N-linked oligosaccharides and by evidence for O-linked carbohydrate moieties on at least a portion of the Fc alpha R molecules. In biosynthetic studies, all four anti-Fc alpha R antibodies and the IgA ligand bound a single 32-kDa core protein present in tunicamycin-treated cells, and the exceptional antibody again recognized molecules with relatively restricted glycosylation in the nontreated cells. These antibodies and native IgA ligands thus provide complementary reagents for definition of the complex structure and function of Fc alpha R in systemic IgA antibody responses.     

alpha 2-macroglobulin traps a proteinase in the midregion of its arms. An immunoelectron microscopic study

alpha 2-Macroglobulin, one of the major plasma proteinase inhibitors with Mr = 720,000, is known to inhibit proteinases of all four classes through the "trap mechanism" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724), but the proteinase binding site of alpha 2-macroglobulin has not been identified precisely. We localized bound proteinase molecules on the electron microscopic images of alpha 2-macroglobulin, using anti-proteinase IgG. Serratial Mr = 56,000 proteinase produced by Serratia marcescens was chosen as the antigenic probe in this study because its affinity to specific antibodies was retained in its bound state to alpha 2-macroglobulin. Dimers of alpha 2-macroglobulin/Mr = 56,000 proteinase complexes cross-linked with anti-Mr = 56,000 proteinase IgG were prepared and subjected to electron microscopic observations. The electron microscopic image of alpha 2-macroglobulin complexed with Mr = 56,000 proteinase had four straight arms with an overall shape looking like the character "H." From the way anti-Mr = 56,000 proteinase IgG linked two alpha 2-macroglobulins, it was concluded that the proteinase existed in the midregion of one of the arms. This result helps us to form a more concrete view of the trap mechanism in that one of the arms of alpha 2-macroglobulin wraps the trapped proteinase and holds it isolated from high molecular weight substrates in the surrounding medium.