Structural Details of Proteinase Entrapment by Human alpha 2-Macroglobulin Emerge from Three-dimensional Reconstructions of Fab labeled Native, Half-transformed, and Transformed Molecules*

Usman QaziDagger , Peter G. W. Gettins§, Dudley K. Strickland, and James K. StoopsDagger parallel

From the Dagger  Dept of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, Texas 77030, § Department of Biochemistry, University of Illinois at Chicago, Chicago, Illinois 60612, and  Department of Vascular Biology, J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Three-dimensional electron microscopy reconstructions of native, half-transformed, and transformed alpha 2-macroglobulins (alpha 2Ms) labeled with a monoclonal Fab Fab offer new insight into the mechanism of its proteinase entrapment. Each alpha 2M binds four Fabs, two at either end of its dimeric protomers approximately 145 Å apart. In the native structure, the epitopes are near the base of its two chisel-like features, laterally separated by 120 Å, whereas in the methylamine-transformed alpha 2M, the epitopes are at the base of its four arms, laterally separated by 160 Å. Upon thiol ester cleavage, the chisels on the native alpha 2M appear to split with a separation and rotation to give the four arm-like extensions on transformed alpha 2M. 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 alpha 2M, 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 alpha 2M.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Human alpha 2-macroglobulin (alpha 2M1, Mr = 720,000) is an essential protein present at a high concentration in the serum (~2 mg/ml) that has the unusual physiological role of a nonspecific proteinase scavenger (1-3). In a presently poorly understood mechanism, native alpha 2M irreversibly traps almost all known endoproteinases by undergoing a structural change that involves a large alteration in its shape. Evolutionarily related proteins performing a similar physiological function, termed alpha -macroglobulins, are present in all vertebrates and several invertebrates (1). Recently, an impairment in the alpha 2M gene has been implicated in the etiology of Alzheimer's disease (4).

alpha 2M is a glycoprotein assembled from four identical 180-kDa subunits that are disulfide-linked in pairs to form two protomers, which, in turn, are noncovalently associated (1). Each subunit contains an approximately 40-residue-long sequence termed the "bait" region, which displays target sequences for a variety of proteinases (5). Bait region cleavage by a proteinase in turn causes the activation of a functionally important internal thiol ester bond between Cys949 and Glx952 of the subunit, which rapidly undergoes a nucleophilic attack (1). Cleavage of the thiol ester moiety triggers a major shape change, aptly termed the "mousetrap mechanism," that causes alpha 2M to internally sequester the proteinase, which typically retains its catalytic activity but is inaccessible to its target proteins (6).

An attacking proteinase cleaves two of the four bait regions of alpha 2M in rapid succession (1). alpha 2M can therefore entrap up to two proteinases the size of chymotrypsin (Mr = 25,000). Significantly, a direct nucleophilic attack by methylamine on the thiol esters of native alpha 2M results in a structural change similar to cleavage by a proteinase (1). Thus, thiol ester cleavage has a pivotal role in the shape change that accompanies the entrapment of the proteinase. Transformed alpha 2M obtained by either mechanism exposes receptor binding domains (RBDs) that allow its rapid endocytosis by cell-membrane receptors principally displayed by hepatocytes but also by a variety of other cells (1, 7).

Native and transformed alpha 2Ms display significantly different physico-chemical properties, including migration speeds on nondenaturing gels (8) and Stokes radii (9). Three-dimensional electron microscopy reconstructions have shown that the two forms also exhibit markedly different shapes (10-12). The ambiguity in relating structural features between the native and transformed molecules has therefore led to conflicting models for the structural rearrangement involved in the transformation of alpha 2M (10, 12). The recently published structure of half-transformed alpha 2M (alpha 2M-HT), which has only two cleaved bait regions and two hydrolyzed thiol esters in its bottom half, has provided an important link in understanding the process leading from native to transformed alpha 2M (13). In the present study, we have employed cryoelectron microscopy to obtain structures of native alpha 2M (alpha 2M-N), alpha 2M-HT, and alpha 2M-methylamine (alpha 2M-MA) labeled with four monoclonal Fab fragments that bind to a common epitope on all three structural forms of the molecule and permit an assignment of related features.

    EXPERIMENTAL PROCEDURES

Protein Preparations-- Purification protocols for native, half-transformed, and methylamine-transformed alpha 2Ms have been previously described in Kolodziej et al. (12), Qazi et al. (13), and Schroeter et al. (11), respectively. Monoclonal antibody 6E8 binds to a 55-kDa fragment obtained from a papain digest of transformed alpha 2M that lies principally within the N-terminal half of the 180-kDa alpha 2M monomer (12, 14). Fab fragments from 6E8 were previously shown to recognize both native and fully transformed alpha 2M with a stoichiometry of approximately 4 mol of Fab bound/mol of alpha 2M (12). This electron microscopy study has further confirmed the binding of four Fabs to the native, half-transformed, and fully transformed alpha 2Ms.

Electron Microscopy-- A 6-8 M excess of 6E8 monoclonal Fab was added to the alpha 2M-N, alpha 2M-HT, or alpha 2M-MA (transformed alpha 2M) preparations so that the resulting alpha 2M concentration was 0.1 mg/ml. A 3-µl sample of each Fab-labeled alpha 2M was added to a glow-discharged carbon-coated holey grid for cryoelectron microscopy. After removing the excess sample by blotting with filter paper, the grid was rapidly cooled by immersion in liquid ethane. A Gatan cold-holder was used to maintain the specimens below -170 °C. Images were then acquired with a JEOL JEM 1200 electron microscope operating at 100 kV with an underfocus of ~1.7 µm and an exposure of ~9 e/Å2 on Kodak SO 163 film (15).

Digitization and Particle Extraction-- Micrographs were digitized using an Eikonix 1412 scanner with a 12-bit dynamic range and a pixel size of 5.7 Å on the specimen scale. Power spectra from the untilted micrographs were analyzed for astigmatism and drift. Micrographs showing frost, significant astigmatism, or drift were rejected. Representative particles were selected in 64×64-pixel boxes using the SUPRIM software package (16). The alpha 2M-N, alpha 2M-HT, and transformed alpha 2M data sets contained 2567, 3498, and 2900 particle images, respectively.

Three-dimensional Alignment and Classification-- Previously obtained unlabeled reconstructions (15) from single-particle images using the methylamine tungstate stain and carbon support film (17) were used as initial models for the alignment and refinement, i.e. three-dimensional projection alignment and iterative reconstruction, using the SPIDER software (18, 19).

In the case of alpha 2M-N and alpha 2M-HT, the particles were initially aligned to isotropic projections from the unlabeled stain models spaced 20° apart (19). Correspondence analysis, followed by hierarchical ascendent classification (20) using the SUPRIM software, was then used to identify and remove misaligned particles in each projection direction. The edited particle data sets (1564 particles, alpha 2M-N; 3026 particles, alpha 2M-HT) were then aligned to projections of these reconstructions at 2° intervals and used to obtain reconstructions with resolutions of 41 Å (alpha 2M-N) and 38 Å (alpha 2M-HT) using a Fourier shell correlation criterion of 0.67 (21).

For alpha 2M-HT, a further pass of refinement was carried out with 2-fold symmetry imposed on its major axis. This was done to give equal prominence to all Fabs. The final reconstruction, which was not further symmetrized, had a resolution of 38 Å.

The alpha 2M-MA data set was initially examined using a reference-free, K-means clustering algorithm in SPIDER with 50 clusters and 100 iterations (22). Of these, 41 average cluster images were retained and aligned to a recently obtained, refined alpha 2M-MA stain model.2 A reconstruction from these averages, which prominently displayed the Fabs, was used to obtain a final refined reconstruction (39-Å resolution) from the entire 2900 particle data set.

Display-- The reconstructions were corrected for the contrast transfer function of the electron microscope as described previously (23-25). Bandpass Fermi filtering (26) was applied to retain information between Fourier space radii of 12 pixels (1/30.4 Å-1; close to the contrast-transfer function cutoff and providing the best match with the unlabeled structures) and 1 pixel (1/364.8 Å-1), with a temperature parameter of 0.01. alpha 2M-N and alpha 2M-MA reconstructions revealed 2-fold symmetry along their major axes, and consequently they were 222-symmetrized for display. The distal ends of the Fabs appeared wedge-shaped in the average images (see Fig. 2) and mushroom-shaped in the surface-rendered structures (data not shown), presumably because their site of attachment is not rigid. In the solid-shaded structures, their external ends were trimmed to give them a rod-like appearance so that their contact with the surface can be more readily discerned.

    RESULTS

Electron Microscopy-- In the galleries of frozen-hydrated images (Fig. 1), four Fab fragments from the monoclonal antibody 6E8 clearly bind to each of the three structural variants of alpha 2M. On most particles, the Fabs can be discerned as rod-shaped protrusions with knob-like extremities (Fig. 1). Typical shapes such as "lip" views of alpha 2M-N (particles a, c, and g) (12), "pseudo-lip" views of alpha 2M-HT (particles j and m) (13), as well as "H" views of alpha 2M-MA (particles t and u) (11) can be identified and are similar to their unlabeled counterparts (11-13). Thus, the Fabs do not appear to perturb the alpha 2M molecules (11-13).


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Fig. 1.   Galleries of frozen-hydrated specimens of Fab-labeled variants of human alpha 2M. The four Fabs appear as rod-like extensions with bead-like ends that bind to the external surface of the molecules. The particle data sets, represented in reverse contrast, contain a number of off-axis views. The scale bar in this and subsequent figures corresponds to 100 Å, and the gray scale bar denotes relative protein density from white (high) to dark (low).

Cryoelectron microscopy was utilized as the imaging technique in the present study because it permits the use of higher protein concentrations in the sample preparation than stain electron microscopy. This assured that the four Fab binding sites are occupied (Fig. 1) (dissociation constants of the alpha 2M-Fab complexes are in the nM range (12)). As often observed in the imaging of nonviral proteins in vitreous ice, the alpha 2Ms tended to assume a preferred orientation in relation to the air-water interface (27-29). However, we obtained a sufficient number of off-axis, "rocking" views in all data sets, which permitted the computation of three-dimensional structures with significant spatial information in all directions (27).

Three-Dimensional Alignment and Reconstruction-- Our previous reconstructions of the unlabeled alpha 2Ms (11-13) (with resolutions near 30 Å) were obtained from single particles imaged in an amorphous layer of methylamine tungstate stain containing 10 µg/ml bacitracin over carbon support film (17). Specimen application by the spray method (17) and the inclusion of bacitracin minimized the interaction of the particles with the support film and provides multiple orientations of the molecules (28). Our technique provides high contrast images of well preserved molecules, and the multiple orientations of the particles result in reconstructions with uniform spatial resolution (11-13). The stain structures (11-13) were used to align (19) data sets of untilted, frozen-hydrated, single particles of Fab-labeled alpha 2M-N and alpha 2M-HT or the averages representing the data set for alpha 2M-MA, obtained using the reference-free technique of K-means clustering (see "Experimental Procedures" (22)). The model-based three-dimensional projection alignment method is a powerful procedure that bypasses technical difficulties encountered in three-dimensional reconstructions from tilted frozen-hydrated specimens. The method of using methylamine tungstate stain reconstructions to align and reconstruct from untilted frozen-hydrated data sets has been previously validated (13, 27, 28), and the present study further demonstrates its utility in determining structures of the corresponding Fab-labeled molecules.

A comparison of the projections of the Fab-labeled three-dimensional structures and the corresponding two-dimensional average images (Fig. 2) clearly indicate that the processes of alignment and reconstruction have correctly preserved the location of the Fab labels. The Fabs are somewhat less prominent on the alpha 2M-N structure, probably because of the predominance of lip views in the data set.


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Fig. 2.   Solid-shaded views of Fab-labeled native, half-transformed, and transformed alpha 2Ms. Surface renderings, projections, and matching average images for the three structural variants prominently display the Fabs as rod-like extensions. The arrangement of the four Fabs confirms the anti-parallel orientation of the individual 180-kDa subunits in the tetramer. The three-dimensional structures were filtered and thresholded to best match their unlabeled stain analogs shown at the top of the figure at a volume that corresponds to the molecular weight. The close agreement between the projections of the structures and their corresponding average images indicates that the reconstructions are reliable. The height, width, depth of the structures are: alpha 2M-N, 200 × 150 × 135 Å; alpha 2M-HT, 195 × 135 × 130 Å; and alpha 2M-MA, 200 × 155 × 140 Å.

Three-dimensional Structures and Location of Fab Labels-- All three Fab-labeled reconstructions of alpha 2M closely resemble their unlabeled stain analogs that were used as the initial models for three-dimensional alignment, further supporting the reliability of the reconstructions (Fig. 2). The 222-symmetric alpha 2M-N structure presents an overall twisted appearance. In its characteristic lip view, the structure appears as two Z-shaped protein strands that merge at the top and bottom to form regions of high protein density. The strands exhibit a 90° clockwise rotation about their major axis and form the walls of the cavity (Fig. 4a). The two dense ends of the molecule clearly display a chisel-like appearance as observed in the unlabeled stain structure (Figs. 2 and 3). Furthermore, the elbow-shaped bends of the two Z-shaped strands are superficially connected by low density, bridge-like features. Four small (~20-Å diameter) openings to the interior of the structure are located above and below each of the surface bridges on the front and back of the molecule.


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Fig. 3.   Views of solid surface renderings of the three-dimensional structures of native, half-transformed and methylamine-transformed alpha 2Ms. The structures are displayed at a reduced threshold to enhance the presentation of the Fabs. The four Fabs are shown in black.

The end view of alpha 2M-N shows that the Fabs bind on the elbow-like bends of the Z-shaped protein strands 120 Å apart and protrude diagonally outward from either side of the base of the chisel (Fig. 3). A closer examination of the "figure-8" end views shows that the Fabs are arranged in a staggered configuration, i.e. located slightly away from the symmetry planes bisecting the structure along its major axis. On each Z-shaped protomer, the two Fabs are located near the ends of antiparallel-linked subunits and consequently form an angle greater than the 90° rotation exhibited by the strands (Figs. 3 and 4).


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Fig. 4.   Protein density distribution in slices of the alpha 2M structures (a) and the proposed arrangement of the protomers (b). a, slices 5.7 Å thick were cut perpendicular to the major axis of the alpha 2M-N and alpha 2M-HT stain structures and the labeled alpha 2M-MA ice structure (with the Fabs removed) as shown at the top of the figure. An extensive comparison of the slices has been previously presented (13). Two major strands that appear to split in alpha 2M-HT are the putative dimeric pro tomers that constitute all three structures. The proteinase-entrapping mechanism appears to involve rotation and separation of the two strands at the top and bottom of alpha 2M. The two Z-shaped protomers of alpha 2M-N rotate 90° clockwise between slices 2 and 6 and merge at each end of the structure to form chisel-like features. In alpha 2M-MA, the two protomers rotate by 45° in the anticlockwise sense between slices 1 and 7, forming significant connections with each other near the ends of the structure, giving it a cage-like appearance. In alpha 2M-HT, the chisel-shaped top splits into two relatively untwisted strands that split further (slice 4), remerge, and undergo a 45° rotation accompanied by a broadening near the bottom of the structure without the separation seen in alpha 2M-MA (b). The arrangement of the protomers in alpha 2M and alpha 2M-MA is apparent upon displaying the structures at a high threshold level. Each protomer has been shaded at a different gray level for ease of viewing. The arrows indicate the approximate locations of the Fab epitopes.

alpha 2M-HT is a functional intermediate between the native and transformed molecules in which two of the four bait domains and thiol ester moieties have been cleaved by chymotrypsin bound to Sepharose (30). Its top has a chisel-like shape analogous to alpha 2M-N and is similarly flanked by two Fab labels 120 Å apart on either side, analogous to the native structure. However, the center of the molecule shows an approximately 2-fold widening of the openings to its internal cavity, whereas the superficial, bridge-like features of alpha 2M-N are absent (Fig. 3). The bottom of alpha 2M-HT presents a bulbous, rounded appearance. The two strands that comprise the native molecule appear to have split into four that exhibit little twist (Fig. 4a). The two Fabs that bind at the bottom appear to be rotated ~50° with respect to the Fabs at the top of the structure and are located laterally 120 Å from each other as in alpha 2M-N (Fig. 3).

alpha 2M-MA forms a more compact, cage-like structure, with four arm-like features that extend, two from each end of the molecule (Fig. 3). As seen in the H and X views, the molecule is formed by two relatively straight strands of protein density that form major connections near the two ends of the structure. The H view exhibits a groove, which appears as a pronounced gap at an increased threshold and separates the body of the structure into two strands of protein density that twist 45° counterclockwise (Fig. 4, a and b). The side X view shows that each strand is broad at the center and tapers at the two ends to form the arm-like extensions (Fig. 3). This structure is similar to the alpha 2M-MA reconstruction reported previously without the Fab labels (11). Two Fabs are located at each end near the tapered base of its arm-like extremities 160 Å apart, in a noticeably staggered fashion. The Fabs show the same ~45° rotation exhibited by the two strands (Figs. 3 and 4a). In the three structures, the epitopes are located near the upper and lower ends of their internal cavities with a vertical separation of ~145 Å.

    DISCUSSION

Structural Organization of the alpha 2M Variants-- A variety of hypotheses have been proposed for the structural transformation that links the quite dissimilar structures of the native and transformed alpha 2Ms (10, 12). The bases for these proposals have ranged from a correlation of surface features (10) to a comparison of the protein density distributions in the two structures (12, 13). As discussed below, previous immunoelectron microscopy studies of individual stained particles have provided significant structural information for the location of domains in transformed alpha 2M (31, 32). However, a structural correspondence was not established with antibody-labeled alpha 2M-N, because the images of stained specimens exhibited variable shapes, making their interpretation questionable. The acidic uranyl salts (pH ~ 4) used in these studies seemingly perturbed the more labile native molecule. We have shown that a reconstruction obtained from a specimen stained with the neutral pH methylamine tungstate (27) gives excellent correspondence with that obtained from frozen-hydrated specimens (Fig. 2).

Our Fab-labeled three-dimensional structures have provided the first definitive structural comparisons of antibody-labeled alpha 2Ms, making it possible to relate the morphological changes upon transformation by methylamine or chymotrypsin. An initial consideration of the locations of the four Fab binding epitopes, near the ends of all three structural forms of alpha 2M, clearly shows that individual 180-kDa subunits are present in an antiparallel or head-to-tail orientation within the two disulfide-linked protomers that noncovalently associate to form the tetrameric alpha 2M. This is a structural validation of the proposal from previous sequencing studies that the two 180-kDa subunits in each protomer are linked antiparallel by two disulfide bonds near their N termini (1, 33) and a previously reported comparison of two-dimensional average images of Fab-labeled alpha 2Ms (12).

A comparison of the three structures of alpha 2M (Fig. 3) indicates that in each case, the top and bottom pairs of Fab epitopes are separated along the major (long) axis by a constant distance of approximately 145 Å. However, the lateral distance between the epitopes in each of these pairs is variable, ranging from 120 Å in the native and half-transformed structures to 160 Å in the transformed molecule (Fig. 5). This indicates that the transformation of alpha 2M, which allows the physical entrapment of a proteinase after the bait domains and thiol ester moieties of alpha 2M have been cleaved, involves a rearrangement of protein density about its major axis to this regard, the "accordion folding" model in which a lateral compression and vertical stretching of the molecular was proposed for this transformation of alpha 2M is inconsistent with the disposition of the Fab labels on the native and transformed structures (10). Our observation agrees with the proposed localization of the two disulfide-linked dimers in the three variants of alpha 2M reported here (12, 13), one on either side of the major axis of the structure, and is supported by studies that showed dimeric variants are incapable of trapping proteinases (34). Slices of alpha 2M and alpha 2M-MA obtained perpendicular to their major axes (Fig. 4a) reveal two twisted strands of high protein density that form major connections near the ends of the molecules thereby leading to the structural division proposed (Fig. 4b).


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Fig. 5.   Location of receptor binding domains in alpha 2M-N and alpha 2M-MA. The chisel-shaped features at the two ends of the native molecule sequester the RBDs (hatched oval). After thiol ester cleavage, the chisels split and rotate, exposing the RBDs near the tops of the arm-like features of alpha 2M-MA. For clarity, the rotation and translation are depicted with arrows only at the top of the molecule. The Fabs at the bottom rotate in the opposite direction.

Relatedness of Structural Features in the alpha 2M Variants and Locations of the RBDs-- The Fabs used in the present study bind transformed alpha 2M near the base of the arm-like extensions on the top and bottom of the molecule. Previous immunoelectron microscopy studies with other monoclonal antibodies showed that the RBDs are located near the tips of these arms and allow for the rapid endocytosis of alpha 2M-proteinase complexes (32, 35, 36). It was further shown that an antibody to the RBD prevented the binding of alpha 2M to its receptor (32, 35). However, the antibody did not bind alpha 2M-N, indicating that the RBDs are internally sequestered (35). As the antibody 6E8 used in the present study binds native, half-transformed, and transformed alpha 2Ms, its binding site appears to be distinct from the RBD.

The disposition of the epitopes of 6E8 on alpha 2M-N and alpha 2M-MA (Fig. 5) reveals a structural relationship of major significance in the biology of alpha 2M. A comparison of the end views of the native and transformed structures (Fig. 5) shows that the two protomers that merge to form the chisels in alpha 2M-N undergo a separation of 40 Å, along with an opposite 90°-rotation at each end. This results in the emergence of arms and exposure of the RBDs at the two ends of the arm-like features of the transformed structure. It appears that the chisels of alpha 2M-N enclose the RBDs (Fig. 5), and this finding nicely correlates with the inability of the RBD binding antibody (7H11D6) to bind to the native molecule (35).

Structural Basis of Proteinase Entrapment-- alpha 2M-HT is a functional intermediate that was obtained by reacting an excess of alpha 2M-N with immobilized chymotrypsin so that bait domain cleavage and thiol ester hydrolysis occurred in only two of its four subunits (30). In the top half of alpha 2M-HT, the chisel-shaped feature and the arrangement of the two Fabs closely resemble alpha 2M-N, whereas the bottom half is broad and bulbous, and the two Fabs have rotated ~45° (Fig. 6). Therefore, we conclude that the bait domain and thiol ester on one subunit in each dimer that may be in close proximity have reacted with chymotrypsin in the bottom portion of the structure. Therefore, the minor axis of alpha 2M-N represents its functional division. The intact subunit in each protomer maintains the shape of the chisel-like feature of the native molecule. This proposal agrees with the antiparallel arrangement of the subunits described above.


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Fig. 6.   Structural relationship between alpha 2M-N and alpha 2M-HT. The Fabs indicate that the strands rotate 45° in the bottom portion of the molecule after cleavage of two bait and thiol ester domains by chymotrypsin. Consequently, the openings to the cavity are enlarged, permitting the entrance of the proteinase (Figs. 3 and 4).

The untwisting of the strands in alpha 2M-HT (Fig. 6) appears to lead to an approximately 2-fold widening of the openings to the cavity (Fig. 4, cf. alpha 2M-N and HT), making the cavity accessible to proteinase entry. However, the Fabs show that there is no separation of the strands at the ends of the molecule, and the arm-like features of alpha 2M-MA have not formed. Consequently, the RBDs are not exposed (Fig. 3). In an apparent contradiction to this proposal, a binary chymotrypsin alpha 2M complex that may share the cleavage arrangement of alpha 2M-HT (two bait domains and thiol esters cleaved) appears to interact with an anti-RBD antibody (35, 37). However, it was noted (30, 37) that thiol ester cleavage is more extensive in the binary complex preparation, and this may have resulted in the IgG binding to some of the alpha 2M molecules seen in immunoelectron microscopy (35). We propose that transformed alpha 2M, which is targeted for endocytosis, requires cleavage of intact thiol esters in both ends of the molecule. Such an arrangement would ensure the ability of alpha 2M-HT to encapsulate a second molecule of proteinase before the RBDs are exposed and the complex is removed from the circulation. Thus, the presence of two intact bait domains and thiol ester moieties in the native half of the intermediate structure imposes constraints on the complete transformation of alpha 2M (13). In this regard, a recent study (38) suggested that the four bait regions are in contact with one another near the center of the structure. It was reported that disulfide cross-links between two dimeric protomers blocked the structural change induced by thiol ester cleavage that, however, occurred upon cleavage of bait domains. The need to cleave bait regions in both halves of the alpha 2M tetramer to enable complete transformation of the molecule shown in this study may, therefore, arise from the need to allow for complete reorganization of the dimer-dimer interface.

The entrapment of two proteinases by alpha 2M is known to occur in two steps with a fast reaction occurring between alpha 2M-N and the first targeted proteinase (1). The proteinase may enter the cavity through the enlarged openings where the epsilon -amino group of its lysine moiety is typically cross-linked to the Glx952 of alpha 2M (39). The thiol esters are therefore analogous to harpoons that tether the proteinase to the interior of the cavity of the alpha 2M and, thus, may have a role in maintaining its irreversible attachment. The intermediate or half-transformed alpha 2M can subsequently undergo further cleavage by a second proteinase at a reduced rate (30) in the native portion of the structure, resulting in a similar entrapment, followed by a 45° counterclockwise rotation of the strands to encapsulate the proteinases and expose the receptor binding domains (Fig. 5).

    ACKNOWLEDGEMENT

The authors thank Norman Nolasco and Jay R. Henderson III for contributions to the image processing of Fab-labeled alpha 2M-MA. They would further like to thank Steven Kolodziej, Dr. Z. Hong Zhou, and Dr. John Schroeter for useful discussions and help with the manuscript.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants HL 42886 (to J. K. S.), HL 50744 (to D. K. S.), and GM 54414 (to P. G. W. G.). A preliminary presentation of the results was given at the Microscopy Society of America annual meeting, Atlanta, July 12-16, 1998 (40). The contents of this publication are part of the dissertation to be presented to the University of Texas Graduate School of Biomedical Sciences at Houston in partial fulfillment of the Ph.D. degree (by U. Q.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Texas Medical School, 6431 Fannin St., Houston, TX 77030. Tel.: 713-500-5345; Fax: 713-500-0730; E-mail: stoops{at}casper.med.uth.tmc.edu.

2 U. Qazi, P. G. W. Gettins, D. K. Strickland, and J. K. Stoops, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: alpha 2M, human alpha 2-macroglobulin; alpha 2M-N, native alpha 2M; alpha 2M-MA, methylamine-transformed alpha 2M; alpha 2M-HT, half-transformed alpha 2M; Fab fragment, Fab 50-kDa fragment; RBD, receptor binding domain.

    REFERENCES
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ABSTRACT
INTRODUCTION
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