JOURNAL

OF SI’RUC!l’URAL

BIOLOGY

106,

237-242 (1991)

Immunoelectron Microscopy Studies with a Monoclonal Antibody Directed against a Receptor Recognition Site Epitope in Human a,-Macroglobulin NANCY L. FIGLER,*

DUDLEY K. STRICKLAND,? MARGAREWA ALLIETTA,*

AND STEVEN L. GONIAS*‘~

*Departments of Pathology and Biochemistry, University of Virginia Health Sciences Center, Churlottesville, Virginia 22908; and tThe Biochemistry Laboratory, American Red Cross Biomedical Research and Development, Rockville, Maryland 20855 Received April 2, 1990, and in revised form December 11, 1990

Starkey, 1973). The reaction of azM with proteinase causes a major conformational change in the inhibitor (SottrupJensen, 1987; Barrett et al., 1979; Gonias et al., 1982). As a result of this conformational change, the proteinase is irreversibly trapped by the azM (Gonias and Pizzo, 1983; Crews et al., 1987). Each azM subunit has one p-cysteinyl-y-glutamyl thiol ester bond (four per molecule) (Sottrup Jensen, 1987). The thiol ester bonds react with compounds such as methylamine in the absence of proteinase causing a conformational change that is nearly equivalent to the change caused by trypsin (Gonias et al., 1982, 1988; Boisset et al., 1989). a,M conformational change has been demonstrated by electron microscopy (Tapon-Bretaudiere et al., 1985; Nishigai et al., 1985; Gonias and Figler, 1989). Native azM (before reaction with methylamine or proteinase) is somewhat heterogeneous in appearance; however, a “doughnut-shaped” center is observed in most molecules. Two to four small spherules may be observed surrounding the central doughnut; these structures are stabilized by reacting a,M with the crosslinking agent cis-dichlorodiammineplatinum (II) @is-DDP) Gonias and Figler, 1989). A representative field showing cis-DDP treated-native a,M is presented in Fig. 1A. After reaction with proteinase or methylamine, a,M is converted into a structure resembling the letter “H.” As an example of the “II” structure, azM-trypsin is shown in Fig. 1B. Each mole of azM can bind up to 2 mole of proteinase (SottrupJensen, 1987). When only one of the two proteinase binding sites is occupied, as in binary azM-thrombin (Boche and Pizzo, 1988; Steiner et al.,

a,-Macroglobulin (a,M) 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 a,M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7HllD6 is exposed. The association of a,M-proteinase or a,Mmethylamine with a,M cellular receptors is prevented by 7HllD6. In this investigation, the binding of 7IIllD6 to a,M was studied by electron microscopy. 7HllD6 bound to a,M-methylamine and %M-trypsin but not to native a,M. The structure of a,M after conformational change resembled the letter “H.” 7HllD6 epitopes were identified near the apices of the four arms in the a,M “H” structure. 7HllD6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to a,M-trypsin but not to native a,M). azM conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete a,M conformational change is not necessary for 7HllD6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary a,M-trypsin (1 mole trypsin per mole azM 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. These incompletely transformed structures were similar to the a,M conformational intermediates described previously (S. L. Gonias and N. L. Figler (1989) J. BioZ. Chem. 264, 9565-9570). We propose that lateral arm extension is a critical step in a,M conformational change. Failure of lateral arm extension is probably a common property of different a,M conformational intermediates. 0 1991 Academic Press, Inc. INTRODUCTION

’ To whom correspondence should be addressed at University of Virginia, Health Sciences Center, Depta. of Pathology and Biochemistry, Box 214, Charlottesville, VA, 22908. Fax: 804924-8060.

az-Macroglobulin (azM) is a tetrameric plasma glycoprotein (M, 718 000) that inhibits numerous proteinases from all four major classes (Barrett and 237

1047~3477191 $3.00 Copyright 8 1991 by Academic F’rese, Inc. All rights of reproduction in any form reeerved.

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FIG. 1. Electron micrographs of native a,M and a,M-trypsin. (A) Native aeM stabilized by crosslinking with 1.0 m&f cis-DDP for 6 hr at 37°C. (B) aeM-trypsin complex prepared by reacting a,M with a twofold molar excess of trypsin. The bars represent 25 nm.

1985; Strickland et al., 1988) or binary azM-trypsin (Strickland et al., 1988), the azM conformational change may be incomplete. The term “binary” indicates 1:l (proteinase:cr,M) binding stoichiometry as distinguished from ternary (2:l) complexes. Binary o,M-trypsin and azM-thrombin have not been studied by electron microscopy. azM conformational change intermediates have been prepared using chemical modification procedures (Gonias and Figler, 1989; Cunningham et al., 1990). When ozM is treated with the protein crosslinker, cis-DDP, and then with trypsin, the proteinase causes only a partial azM conformational change (Gonias and Pizza, 1981; Roche et al., 1988a). The resulting intermediate structures have been characterized by electron microscopy (Gonias and Figler, 1989). o,M-proteinase complexes and azM-methylamine are recognized by specific receptors on hepatocytes, fibroblasts, and macrophages (Pizza and Gonias, 1984). Marynen et al. (1981) described a monoclonal antibody, F2B2, that binds to a,M only after conformational change and blocks receptor binding. The sequence recognized by F2B2 is located in a 20-kDa

fragment derived from the C-terminus of each arzM subunit (Van Leuven et al., 1988). By electron microscopy, F2B2 epitopes have been identified near the tips of the four arms in the crzM “H” structure (Delain et al., 1988; Boisset et al., 1988). Strickland and co-workers recently prepared monoclonal antibody 7HllD6 which also recognizes an epitope exposed in human a,M only after conformational change (Strickland et al., 1988). 7HllD6 has been used to generate an anti-idiotypic IgG that binds directly to the o,M receptor (Isaacs et al., 1988); this anti-idiotypic antibody provides direct evidence that 7HllD6 recognizes residues in the receptor recognition site. The association of 7HllD6 with ol,M-trypsin and o,M-methylamine is sensitive to the integrity of the inhibitor (Roche et al., 198813); peptide fragmentation of azM-methylamine causes a significant reduction in the affinity of the antibody for azM. Therefore, the primary structure recognized by 7HllD6 has not been defined. Roche et al. (1988b) suggested that 7HllD6 may recognize a structure that is not included in the cx,M C-terminal 20-kDa fragment, thereby raising the possibility that F2B2 and 7HllD6 bind to separate regions of the cwzM structure. The first major goal of the present investigation was to study the binding of 7HllD6 to ozM by electron microscopy and to compare these results with those previously presented for F2B2 (Delain et al., 1988; Boisset et al., 1988). 7HllD6 was then adducted to colloidal gold and used to study epitope exposure in binary ozM-trypsin and in ozM conformational intermediates prepared with cis-DDP. EXPERIMENTAL

PROCEDURES

Proteins. a,M was purified from human plasma as previously described Umber and Pixzo, 1981). aaM-methylamine was prepared by reacting a,M with 200 mM methylamine (Sigma) at pH 8.2 for 6 hr followed by extensive dialysis. Trypsin was purchased from Sigma. The concentration of active trypsin was determined by the method of Chase and Shaw (1967). Ternary a,M-trypsin was formed by reacting 2.0 pM a,M with 4.0 p,M trypsin for 30 min at 22°C. A complete characterization of monoclonal antibody 7HllD6 is presented elsewhere (Strickland et al., 1988). cis-DDP reactions. aeM was reacted with 0.6, 1.0, or 1.6 mM cis-DDP (Aldrich) for 6 hr at 37°C and then subjected to extensive dialysis as previously described Klonias and Figler, 1989). The preparation treated with 1.6 m&f cis-DDP was designated a,MW Each mole of a,M, incorporated 17-20 mole Pt, as determined by atomic absorption spectrophotometry. aaMp, was reacted with a twofold molar excess of trypsin for 30 min at 22°C. The trypsin was inactivated with 0.05 mM p-nitrophenyl-p’-guanidinobensoate-HCl (PNPGB) (Sigma). The preparation was designated a,MR-T. 7HllD6-colloidal gold adducts. Colloidal gold was prepared from HAuCl, (Sigma) using the procedure of Muller and Baigent (1980). Adducts of 7HllD6 and colloidal gold were formed as previously described (Gonias et al., 1988) and designated 7HAu. Three separate preparations of 7HAu were studied. 7HAu was incubated with different preparations of a,M

a,M IMMUNOELECTRON

MICROSCOPY

239

(asM-methylamine, a,M-trypsin, a,Mm, and aeM,-T) for 30 min at 22°C. The concentration of a,M was 50 pg/ml. Free a,M and a,M bound to 7HAu were resolved by chromatography on Sepharose CL-4B equilibrated in 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS) or by ultracentrifugation. In the latter procedure, the 7HAu and bound a,M were pelleted for 1 min at 60 kPa in a Beckman Airfuge. The pellet was then resuspended in 200 pl PBS and subjected to ultracentrifugation again. Washed preparations were adsorbed to carbon films immediately after recovery. Electron microscopy. Thin carbon films were floated for 1 min on 400~)11 aliquots of different asM or 7HAu preparations. The films were then transferred to a solution of buffered 2.0% glutaraldehyde for 10 min, washed with water, stained with 2.0% uranyl formate (Polysciences, Inc.) for 60 set, and transferred to 300-mesh nickel grids. The glutaraldehyde enhanced the quality of the stained images but did not affect the structure of the a,M (Conias et al., 1988; Gonias and Figler, 1989). Air-dried grids were studied by conventional transmission electron microscopy using a Zeiss 902 electron microscope at 80 kV. Photomicrographs were obtained at a direct magnification of X 140 000 or x 250 000 enlargement. RESULTS

AND DISCUSSION

7HllD6 epitope identification. 7HllD6 bound readily to a,M-trypsin and asM-methylamine. The antibody binding sites were equivalent in both forms of the proteinase inhibitor. ol,M-trypsinantibody complexes are shown in Fig. 2. The 7HllD6 epitopes were located near the apices of the four arms in the cxsM “II” structure. When the concentration of asM was equal to the concentration of antibody, the two binding sites of a single antibody usually engaged epitopes on separate a,M molecules. Crosslinking of two arms in a single “H” structure by one antibody was not observed. 7HllD6 did not bind to native aaM as determined by electron microscopy. Delain et al. (1988) described two classes of monoclonal antibodies that bind to a,M only after conformational change. The first class (including F2B2) binds to the 20-kDa C-terminal fragment of the aaM subunit and blocks asM receptor binding (Van Leuven et al., 1986, 1988). In electron micrographs, these antibodies bind to the adjacent arms of a single (rsM, “crosslinking” or “closing” one side of the inhibitor structure shut. The second class of antibodies does not bind to the 2OkDa C-terminal CQM fragment and does not inhibit CQM receptor binding Wan Leuven et al., 1988). By electron microscopy, the epitopes for the second class of antibodies are located more laterally in the CQM structure and crosslinking of adjacent arms is not observed (Delain et al., 1988). Based on the appearance of the a,M-7HllD6 complexes by electron microscopy in Fig. 2 (no intramolecular crosslinking), 7HllD6 is most similar to the second class of previously described antibodies; however, since 7HllD6 prevents olzM receptor binding,

FIG. 2. a,M-trypsin binding to 7HllD6. a,M-trypsin complex (2 mole trypsin per mole inhibitor) was incubated with 7HllD6 for 30 min at 22°C. The molar ratio of aaM-trypsin to antibody was 1:l. The bars represent 25 nm.

this antibody cannot be placed into either of the two previously described groups. azM Binding to 7HAu. 7HAu formed complexes with ol,M-trypsin and a,M-methylamine but not with native 02M. While the gold partially obscured the antibody structure in some projections, the epitope was clearly identified near the apices of the CQM arms, as shown in Fig. 3. These studies demonstrate that 7HAu can be used to recognize the 7HllD6 epitope with specificity and accuracy. The 7HAu preparation also demonstrated the redundancy of the 7HllD6 epitope in cxsM-trypsin. When the concentration of 7HAu was sufficiently high, a single OL~M frequently engaged more than one 7HAu particle (Fig. 3, top panel). This mode of

240

FIGLER ET AL.

mediate, 7HAu was used to identify the “transformed-conformation specific” 7HllD6 epitope. As shown in Fig. 4, significant binding of a,MR-T to 7HAu was demonstrated. The 7HAu-IX,M~-T complexes were heterogeneous in appearance reflecting variability in orientation and probably azM structural heterogeneity; however, the typical transformed-ozM “H” structure was rarely present as expected. In control experiments, complex formation was not observed between ozMp, (no trypsin treatment) and 7HAu. Since 7HllD6 does not recognize a,M in the native “unchanged” conformation, these studies suggest that opMpt-T is a true azM conformational change intermediate. Approximately 40 representative fields were analyzed in order to quantitate the binding of cr,Mp, and azM,-T to 7HAu. The number of gold particles and crzM molecules were counted in each field. Before reaction with trypsin, 0.15 azM molecules were counted per gold particle (40% of the CY,M was apparently associated with the 7HAu). After reaction with trypsin, 1.47 azM molecules were counted per

FIG. 3. Binding of 7HAu to transformed a,M. u,M-trypsin and a,M-methylamine were incubated with 7HAu for 30 min. The 7HAu-a,M complexes were partially purified by chromatography. When the concentration of a,M was similar to the concentration of 7HAu, aggregates tended to form (top). Individual 7HAu-transformed a,M complexes are shown in the bottom two rows. There were no apparent differences in the binding of 7HAu to a,M-trypsin and a,M-methylamine. The bars represent 25 nm.

binding resulted in the formation of large transformed azM-7HAu aggregates. In these aggregates, two 7HAu bound either to the same side or opposite sides of the same a,M “I-I” structure suggesting that a single complete epitope is present on each a,M arm (four equivalent epitopes per intact ol,M molecule) . 7HAu Binding to cy2MPt. a,M that is treated with high concentrations of cis-DDP (02MrJ still reacts with trypsin; however, the platinum crosslinks prevent complete crzM conformational change (Gonias and Pizzo, 1981; Roche et al., 1988a). In electron micrographs of trypsin-treated ozMpt (dzMpt-T), well defined “H” structures are absent (Gonias and Figler, 1989). In order to determine whether ozMR-T is a true ozM conformational change inter-

FIG. 4. Binding of 7HAu to aaM,-T. a,MB-T was incubated with 7HAu for 30 min. Ultracentrifugation was used to separate free and bound a,MR-T. The final preparation is shown in the figure. The bar represents 25 nm.

a,M IMMUNOELECTRON

gold particle (60% of the asM was associated with the 7HAu). It has been suggested that only a partial conformational change is required in asM in order to expose the receptor recognition site (Roche et al., 1989). Our experiments with 7HllD6 and aaMR-T support this hypothesis. Binary a&-trypsin. asM (2.0 p.iW) was reacted with trypsin (0.2 m for 10 set at 22°C. The trypsin was then inactivated with PNPGB. As shown in Fig. 5A, the majority of the azM (at least 90%) remained in the native conformation due to the low concentration of proteinase. Since binding of one proteinase to a,M does not promote the binding of a second proteinase (Gonias and Pizza, 1983; SottrupJensen and Hansen, 1983; Larsson et al., 19891, at least 97% of the a,M-trypsin was binary (1 mole proteinase per mole inhibitor) and less than 3% was ternary (2 mole proteinase per mole inhibitor). 7HllD6 binds both binary and ternary asM-proteinase complexes with similar affinity (Strickland et al., 1988). Therefore, 7HAu was used to separate the small amount of primarily binary a,M-trypsin from the unreacted inhibitor. After ultracentrifugation to remove unbound asM, preparations such as that shown in Fig. 5B were obtained. Many of the asM-trypsin complexes that bound or copelleted with the 7HAu showed the “H” structure similar to ternary a,M-trypsin. The 7HAu binding sites were located near the apices of fully extended inhibitor arms. A second population of atypical molecules was identified in the binary a,M-trypsin-7HAu preparations (arrow in Fig. 5B and Fig. 6, row A). These molecules were similar to the usual “H” structure except for the incomplete extension of at least one of the four lateral arms. Similar structures have been generated previously by crosslinking a,M with a

241

MICROSCOPY

low concentration of cis-DDP (0.6 mM for 4 hr) (Gonias and Figler, 1989). These reaction conditions cause less auzM structural rigidity compared with the a,M, complexes (1.6 miU cis-DDP) described above. Trypsin treatment causes the 0.6 m&f cisDDP-treated aaM molecules to adopt conformations that closely resemble the “H” structure except for incompletely extended lateral arms. Examples of “H-like” cis-DDP-a,M conformational intermediates are shown in row B of Fig. 6. Since the azM conformational intermediates identified in the binary asM-trypsin and cis-DDP-azM preparations both demonstrate incompletely elongated lateral arms, we propose that unfolding or extension of the lateral arms is an important step in a,M conformational change. CONCLUSIONS

The studies presented here demonstrate that a 7HllD6 epitope is located near the apex of each of the four lateral arms in conformationally transformed aaM. The inability of 7HllD6 to crosslink adjacent arms in a single a,M-trypsin suggests a more lateral position for the binding site compared with F2B2, the antibody studied by Delain et al. (1988). Studies with 7HAu demonstrate that the epitope recognized by 7HllD6 is exposed in incompletely transformed a,M species referred to as conformational intermediates. Since 7HllD6 binds to the a,M receptor-recognition site, these studies also suggest that a full conformational change may not be necessary to reveal the signal for receptor uptake of azM. Finally, studies with binary asM-trypsin and with cis-DDP-modified asM-trypsin indicate that elonga-

FIG. 5. Binding of binary a,M-trypsin to 7J3Au. a2M was incubated with trypsin for 10 set at 22°C. The molar ratio of proteinase to inhibitor was 1O:l. The initial preparation is shown in A. This preparation was incubated with 7HAu for 30 min. 7HAu- a,M complexes were resolved from unbound a,M by ultracentrifugation. The final preparation is shown in B. The bar represents 25 nm.

242

FIG. 6. Comparison of a,M conformational intermediates in binary a,M-trypsin and cis-DDP-treated a,M preparations. (A) Atypical molecules identified in 7HAu- binary a,M-trypsin preparations. (B) a,M Conformational intermediates prepared by reaction with a low concentration of cis-DDP (0.6 nn%f), followed by trypsin. Note the incomplete extension of at least one lateral arm in each structure from either preparation. The bar represents 25 nm.

tion of the lateral arms may be an important step in (r2M conformational change. Failure of one or more arms to elongate may provide a common structure seen in different preparations of au,M conformational intermediates. This work was supported by the Pew Scholars Program in the Biomedical Sciences and Grant HL-30200 from the National Heart, Lung and Blood Institute. S.L.G. is the recipient of a Besearch Career Development Award (HL-02272). REFERENCES BARRETP, A. J., AND STARKEY, P. M. (1973) B&hem. J. 133,705~ 712. BARREIT, A. J., BROWN, M. A., AND SAYERS, C. A. (1979) Biothem. J. 181, 401-418. BOISSET, N., TAVEAU, J.-C., BARRAY, M., VAN LELJVEN, F., DELAIN, E., AND LAMY, J.N. (1988) Bid. Cell 64, 45-55. BOISSET, N., TAVEAU, J.-C., POCHON, F., TARLXEU, A., BARRAY, M., LAMY, J. N., AND DELAIN, E. (1989) J. Biol. Chem. 264, 12,04612,052. CHASE, T., AND SHAW, E. (1967) B&hem. Biophys. Res. Commun. 29, 508-514. CREWS, B. C., JAMES, M. W., BETH, A. H., GE’ITINS, P., AND CUNNINGHAM, L. W. (1987) Biochemistry 26,5963-5967. CUNNINGHAM, L. W., CREWS, B. C., AND GETTINS, P. (1990) Biochemistry 29, 1638-1643. DELAIN, E., BARRAY, M., TAPON-BRETAUDIERE, J., POCHON, F., MARYNEN, P., CASSIMAN, J. J., VAN DEN BERGHE, H., AND VAN LEWEN, F. J. Biol. Chem. (1988) 263.2981-2989. GONIAS, S. L., AND PIZZO, S. V. (1981) J. Biol. Chem. 256, 12,478 12,484. GONIAS, S. L., AND PIZZO, S. V. (1983) J. Biol. Chem. 258, 14,68% 14,685. GONIAS, S. L., AND FIGLER, N. L. (1989) J. Biol. Chem. 264,95659570. GONIAS, S. L., REYNOLDS, J. A., AND PIZZO, S. V. (1982) B&him. Biophys. Acta 705, 306314.

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