THE JOURNAL OF BIOLOGK!AL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 1, Isaue of January 5, pp. 468-471,1988 Printed in L? S A .

Proteolysis of Human a2-Macroglobulin Without Hydrolysis of the Internal Thiolesters or Expression of the Receptor Recognition Site*

(Received for publication, July 27, 1987)

Fred Van LeuvenS, Peter MarynenQ, Jean-Jacques Cassiman, and Herman Van Den Berghe From the Center for Human Genetics, University of Leuven, Campus Gasthuisberg O&N, Herestraat, B-3000 Leuven, Belgium

Proteolysis of human az-macroglobulin (a2M) in the bait region is the prerequisite and necessary trigger for the trapping of the proteinase by a massive confor- mational change of azM. This labilization of the native conformation of a2M is mediated by activation of the internal thiolesters, but the underlying mechanism is unknown. We now describe observations on proteolysis of human a2M without concomitant hydrolysis of the internal thiolesters or conformational change. This proteolysis was obtained with a novel bacterial pro- teinase we recently used to isolate the receptor-binding domain from a2M (Van Leuven, F., Marynen, P., Sot- trup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). This proteinase is not inhibited by aZM, and therefore it was possible to study its effect on native azM at pH 4.5, conditions used previously to produce the receptor- binding domain (Van Leuven, F., Marynen, P., Sot- trup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). The major observations are that despite extensive proteol- ysis, azM largely retained its native conformation as shown by rate electrophoresis, the absence of binding of monoclonal antibody F2B2, and the incorporation of [‘*C]methylamine into a 145-kDa fragment of a2M. 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 a2M, and concomitantly released the receptor-binding domain. This indicated that pro- teolysis at Ly~’~’~-Glu also proceeded in native azM. At least one more major proteolysis site was deduced from the observation of a 27-kDa heat-induced frag- ment, the 145-kDa [‘4C]methylamine-labeled frag- ment, and from the presence of the SO-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 azM and the expression of the receptor-recognition site by activation of the internal thiolesters.

* This work was supported by Grant 3.0055.83 from the National Fund for Scientific Research, Belgium, by a grant “Geconcerteerde Acties” from the Belgian Government, and by a research grant from the American Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. § Research Associate of the National Fund for Scientific Research,

Belgium.

Human-az macroglobulin (azM)’ is a large (Mr 725,000) tetrameric glycoprotein which has evolved into a wide spec- trum inhibitor of virtually all endoproteinases (2). The unique steric mode by which azM binds the proteinase is made possible by a sequence of molecular events, starting with proteolysis of the bait region which activates the internal thiolesters. This, in an unknown fashion, produces a large conformational change in azM to trap the proteinase (for review, see Van Leuven (3)). In this mechanism, proteolysis of the bait region of aZM is the essential and necessary trigger for the formation of an azM-proteinase complex (4). The receptor-recognition site by which the complex is recognized, bound, and cleared by receptors on fibroblasts and macro- phages is located in the COOH-terminal domain of aZM (1).

Except for the limited proteolysis of the bait region, aZM is generally observed to be resistant to proteolysis. Our obser- vation that the receptor-recognition site is vulnerable to pro- teolysis (5, 6) was essential to map this site to the COOH- terminal end of azM (7). Our studies indirectly led to the isolation of a novel proteinase from Lysobacter enzymogenes which allowed us to isolate and characterize a 20-kDa recep- tor-binding domain of human azM (1). Proteolysis at Lys1313- Glu (numbering according to Sottrup-Jensen et al. (8)) re- leased the COOH-terminal domain from azM at pH 4.5 (1). The acid pH proved at least as important as the proteinase because, at pH 4.5, papain yielded the same fragment by cleavage at Lys1313-G1u, although papain was less active than the bacterial enzyme (9).

In this communication, we present observations on prote- olysis of native azM at pH 4.5 with this proteinase. This was made possible because this enzyme is not complexed by azM2 and because azM proved stable for at least 6 h at pH 4.5 and 37 “C. Our findings show that, despite extensive proteolysis outside the bait region of native azM, the internal thiolesters were kept intact, and inhibition of trypsin was essentially not affected. Moreover, the receptor-binding domain remained associated with azM, despite proteolysis at Lys1313-G1u, and was released upon aminolysis of the internal thiolesters by methylamine or by complex formation with trypsin. These findings are discussed in relation to the domain structure of azM and other members of the azM family.

MATERIALS AND METHODS

a2M was isolated from fresh citrated human plasma (10). Native a,M was obtained by an additional purification step involving hydro- phobic interaction chromatography on LKB TSK-phenyl5PW (11).

Rate electrophoresis under nondenaturing conditions was done on

The abbreviations used are: aZM, az-macroglobulin; azM-MA, azM inactivated with methylamine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; TLCK, 1-chloro-3-tosylamido-7-amino-2- heptanone; mAb, monoclonal antibody.

F. Van Leuven, unpublished results.

468

Proteolysis of a2M 469

5 or 8% polyacrylamide gels as indicated (10). Polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) was done in the buffer system of Laemmli (12). Gels were stained with Coo- massie Brilliant Blue. Autoradiographs of dried gels were taken on Kodak XAR-5 film.

The bacterial proteinase preparation used in the experiments re- ported here was isolated as described (1). The 20-kDA receptor- binding domain was prepared by proteolysis and isolated by gel filtration as described (1).

RESULTS

Proteolysis of native azM with the bacterial proteinase a t pH 4.5 was examined by SDS-PAGE. In unreduced samples, two small fragments of molecular mass 27 and 20 kDa were observed (Fig. L4, lune 2). The 20-kDa fragment is formed by proteolysis at Ly~'"~-Glu and was recently characterized as the receptor-binding domain of human azM (1). The forma- tion of the 27-kDa peptide was due to heat-induced fragmen- tation at the internal thiolesters, because treatment of the sample with methylamine after digestion with the bacterial protease prevented its formation during preparation for SDS- PAGE (Fig. L4, lune 3). This was confirmed by analyzing the same samples after reduction. A set of fragments was ob- served, indicating considerable proteolysis of azM (Fig. 1B). The effect of treatment with methylamine, after proteolytic digestion, is reflected by the disappearance of the 27-kDa peptide and of a 120-kDa fragment. In some experiments, a quantitatively less important 60-kDa fragment was observed occasionally. The 120-kDa and 60-kDa fragments co-migrate with the heat-induced fragments from undigested, native azM (Fig. 1B). Other fragments produced by proteolysis of native azM which were not affected by subsequent treatment with methylamine were of apparent molecular mass 145, 106, 46, 35, and 14 kDa (Fig. 1).

1 2 3

185- 120-

60-

127- 120-

1 2 3

-145 -106

-&6 -35

-1 4

FIG. 1. SDS-PAGE of human a2M digested by the bacterial proteinase. Native a2M (1 mg) was incubated with the bacterial proteinase (1.4 pg) in 150 pl of sodium acetate buffer (50 mM, pH 4.5). After 2 h a t 37"C, the mixture was made 80 mM in Tris-HCI, pH 8.0, and TLCK was added to a final concentration of 3 mM. Samples corresponding to 25 pg of a2M were analyzed by SDS-PAGE on 6-2076 linear gradient gels, unreduced ( p a n e l A ) or reduced with 2-mercaptoethanol (l%, v/v). Lane 1, native a2M treated as above with omission of the proteinase. Lane 2, digested a2M. Lane 3, digested a2M subsequently treated with methylamine (0.2 M, pH 9.0, 2 h, 37 "C). Apparent molecular mass is indicated in kilodaltons.

The observation of heat-induced fragmentation, and partic- ularly the generation of the 27-kDa fragment, indicated that the thiolesters were still intact despite extensive proteolysis. This was proven by covalent incorporation of methylamine. Native azM, digested at pH 4.5 for different time periods with the bacterial proteinase, was subsequently treated with ["C] methylamine. The products were analyzed by autoradiogra- phy after rate electrophoresis and SDS-PAGE (Fig. 2). The results showed radiolabel to be associated with aZM on rate electrophoresis (Fig. 2 A ) . Quantitation of radioactivity, by excision of the stained protein bands, showed that under the conditions of this experiment methylamine incorporation after 60 min of digestion was still 87% relative to incorpora- tion in native azM. In SDS-PAGE of reduced samples the set

1 2 3 4 5

"""

A

FIG. 2. ["C]Methylamine incorporation in digested a 2 M . Native a2M was digested with the bacterial proteinase (0.1%, w/w) in sodium acetate buffer (50 mM, pH 4.5) as described in the legend to Fig. 1. After 5 min (lane 2), 15 min (lane 3), 30 min (lane 4) , and 60 min ( l a n e 5), digestion was stopped by the addition of TLCK (5 mM final concentration) and Tris-HCI (100 mM, pH 9.0). Portions of 100 pg of undigested a2M (lane 1 ) and of the different digests were treated with ["Clmethylamine (Amersham Corp., 56 mCi/mmol) a t a final concentration of 25 mM, for 18 h at ambient temperature. In Panel A, samples of 10 pg of protein were analyzed by rate electro- phoresis on 5% gels, and in panel B portions of 20 pg were subjected to SDS-PAGE (6-2076 linear gradient gels). Autoradiographs are shown (10 days at -70°C on Kodak XAR-5 film).

470 Proteolysis of a2M

1 2 3 4

”“

* a

FIG. 3. Reaction of mAb F2B2 with a2M after digestion with the bacterial proteinase. Rate electrophoresis on 8% polyacryl- amide gel. In lane 1, native azM; lane 2, digested azM (prepared as described in legend of Fig. 1); lane 3, digested azM as in lane 2 but further treated with methylamine (as described in legend to Fig. 1); lane 4, digest of azM-MA (1). In all lanes, 5 pg of azM was treated with 5 pg of mAb F2B2 before application to the gel.

A

0.0 a -

007- I

E C o0.06 0

- CI - 0.05-

U UI

z q O.O& - m [L 0

mO.0 3 - In 4

0.0 2 -

0.01 -

L I - 10 20

3

J il 1111

10 20

FRACTION

FIG. 4. Gel-filtration of digested a2M and effect of methyl- amine. Native azM (5 mg) was digested with 5 pg of the bacterial proteinase for 2 h at 37’C in sodium acetate buffer at pH 4.5. The reaction was stopped by addition of TLCK to a final concentration of 2 mM and Tris-HC1 (100 mM, pH 9.0). Half of the material was treated with methylamine (0.2 M, 2 h, 37°C). Untreated and meth- ylamine-treated material was analyzed by HPLC-gel filtration on a Pharmacia Superose 6 column, coupled to an LKB HPLC system as described (11). The buffer used was 50 mM sodium phosphate, pH 7.5, a t 0.75 ml/min. Fractions of 0.75 ml were collected.

of fragments described above was observed, while autoradi- ography (Fig. 2B) showed a progressively decreasing incor- poration of radiolabel in the 185-kDA azM-subunit and a concomitant increase in the 145-kDa fragment (Fig. 2). Pro- longed digestion of a2M with the proteinase and subsequent reaction with [“C]methylamine proved that even after 6 h the 145-kDa fragment became labeled, while label also was

observed in fragments of 44-46 kDa (results not shown). The results thus far proved that proteolysis of native a2M

produced the 20-kDa receptor-binding domain ( l ) , while the internal thiolesters were intact. A relation between both was detected when the samples were analyzed by rate electropho- resis on 8% polyacrylamide gels. Native a2M, digested for 1 h at pH 4.5, did not bind the monoclonal antibody F2B2 (Fig. 3, lane 2). This mAb defines a neo-antigenic site on aZM complexes related to the receptor-recognition site (5). Treat- ment with methylamine induced the fast form of the digested aZM, but this form still did not react with mAb F2B2. The mobility of the latter, however, was decreased (Fig. 3, lane 3). This pattern was also observed when azM-MA was digested and analyzed for binding of mAb F2B2 under the same conditions (Fig. 3, lane 4 ) . Under these conditions the recep- tor-binding domain, which carries the F2B2 epitope, is re- leased from a2M-MA (2). The isolated 20-kDa receptor-bind- ing domain also decreased the mobility of mAb F2B2 in rate electrophoresis on 8% polyacrylamide gek2 The results there- fore indicate that the receptor-binding domain remained as- sociated with digested azM until the internal thiolesters were aminolysed and that, upon release, the domain became bound to F2B2, decreasing its mobility in rate electrophoresis (Fig. 3).

The same effect was noted when proteolyzed a2M was examined for activity as a proteinase inhibitor. When titrated with trypsin, different preparations proved to have retained almost full inhibitory capacity, ranging from 68 to 88%, relative to undigested azM, the lower values being noted with preparations digested for 6 h. Complex formation with trypsin was accompanied by transition to fast mobility on rate elec- trophoresis and by the release of the receptor-binding domain, as observed with methylamine treatment (Fig. 3).

This phenomenon was further demonstrated by gel filtra- tion. Native azM was digested at pH 4.5 with the bacterial proteinase, and equal portions of 2.5 mg were separated by HPLC gel filtration, either directly or after treatment with methylamine. In the latter separation, an extra peak eluted (Fig. 4) corresponding to the 20-kDa receptor-binding domain by criteria of SDS-PAGE and rate electrophoresis with mAb F2B2. (1). This domain constitutes the COOH-terminal por- tion of human a2M, from Glu-1314 onwards (numbering ac- cording to Sottrup-Jensen et al. (8)). This domain does not contain tryptophan residues, which explains the relatively low absorbance a t 280 nm (Fig. 4). Quantitation by radioimmuno- assay and by protein assay showed in this experiment about 170 pg of the 20-kDa domain to be present in this peak (Fig. 4). The 20-kDa receptor-binding domain eluted in the first peak when the digested apM was not treated with methyla- mine (Fig. 4A). The late peak in both chromatograms con- tained reagents like the proteinase inhibitor TLCK used to inactivate the proteinase.

DISCUSSION

The results presented here show that proteolysis of native a2M does not destroy per se the internal thiolesters. Several sites of proteolysis are apparent, but all are clearly located outside the bait region. A detailed study of the localization of these proteolysis sites is in progress, although one cleavage point is already certain: t he Ly~’~‘~-Glu bond, which was shown to yield the 20-kDa receptor-binding domain from a2M-MA (1). This domain was soluble and was readily iso- lated by gel filtration (1). I t contained the epitopes of mono- clonal antibodies F2B2 and F12A3, which were previously mapped to the COOH-terminal end of the a2M-subunit (7). These antibodies define neo-antigen sites on a2M complexes

Proteolysis of a2M 471

related to the receptor-recognition site (1, 5, 7, 13). The hypothesis of a hinge region (1) formed by the stretch of amino acid residues containing Lys-1313 gets strong support from the present results. Proteolysis at Lys1313-G1u released the receptor-binding domain from azM-MA (1). This domain is therefore only retained by the stretch of residues between cys-1298 and cys-1329 (numbering according to Sottrup- Jensen (8)). In native azM, on the other hand, proteolysis at Lys1313-G1~ did not result in dissociation of the receptor- binding domain as shown here, at least not until the thioles- ters were broken by methylamine or by trypsin. How the receptor-binding domain remains associated with azM, not- withstandingproteolysis at Ly~'~l~-Glu, is an interesting prob- lem. One is tempted to consider a special type of domain- domain interaction which might make use on the receptor- binding domain of all or some of the residues also mediating binding to the receptor. This internal complex must then dissociate concomitantly with hydrolysis of the internal thiolesters. Factors that could be directly involved or indi- rectly mediating the dissociation are metal ions, carbohydrate chains, hydrophobic effects, and proteolysis.

In one aspect, the a2M species derived here by proteolysis of native human aZM are reminiscent of azM in mouse (14, 15) and of alM in rat (16, 17). These homologues of azM are composed of a large and a small subunit. Most likely, and recently proven for rat alM (18), these are derived from a single-chain precursor by post-translational proteolytic proc- essing. This has analogy with the normal processing of human complement components C3 and C, to two- and three-chain mature proteins, respectively. The present results show that proteolytic processing of azM is possible without affecting the internal thiolesters which are crucial to a proper functioning of azM. Both the observation of the 145-kDa fragment and of the 27-kDa heat-induced fragment, as described here, are similar in the subunit structure of rat alM and mouse a2M. Clearly, however, proteolysis of human aZM by the bacterial proteinase is more complex, because several cleavage sites are anticipated from the data presented here and previously (1,

5-7). A detailed analysis, combining proteolysis and epitope mapping of monoclonal antibodies, is in progress and will eventually disentangle some of the remaining mysteries of the domain structure of azM and its homologues.

Acknowledgments-We thank L. Stas and K. Merckx for expert technical assistance and K. Rondou for the photographical artwork.

1.

2.

3. 4. 5.

6.

7.

8.

9.

10.

11.

12. 13.

14.

15. 16.

17.

18.

REFERENCES Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-

J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261,11369- 11373

Starkey, P. M., and Barrett, A. J. (1977) in Proteinases in Mum- muliun Cells and Tissues (Barrett, A. J., ed) North Holland Publishing Co., pp. 663-696, Amsterdam

Van Leuven, F. (1982) Trends Biochem. Sci. 7, 185-187 Harpel, P. C. (1973) J. Enp. Med. 138,508-521 Marynen, P., Van Leuven, F., Cassiman, J.-J., and Van Den

Berghe, H. (1981) J. Zmmunol. 127, 1782-1786 Marynen, P., Van Leuven, F., Cassiman, J.-J., and Van Den

Berghe, H. (1982) FEBS Lett. 137, 241-244 Van Leuven, F., Marynen, P., Cassiman, J.-J., and Van Den

Berghe, H. (1986) J. Bwl. Chem. 261,6933-6937 Sottrup-Jensen, L., Stepanik, T. M., Kristensen, T., Wierzbicki,

D. M., Jones, C. M., Lbnblad, P. B., Magnusson, S., and Petersen, T. E. (1984) J. Biol. Chem. 269,8318-8327

Sottrup-Jensen, L., Glieman, J., and Van Leuven, F. (1986) FEBS

Van Leuven, F., Cassiman, J.-J., and Van Den Berghe, H. (1981)

Van Leuven, F., Cassiman, J.-J., and Van Den Berghe, H. (1985)

Laemmli, U. K. (1970) Nature 227,680-685 Van Leuven, F., Marynen, P., Cassiman, J.-J., and Van Den

Hudson, N. W., and Koo, P. H. (1982) Biochim. Biophys. Acta

Saito, A., and Sinohara, H. (1985) J. Biol. Chem. 260, 775-781 Scheufele, J. T., and Koo, P. H. (1982) Biochem. Biophys. Res.

Nelles, L. P., and Schnebli, H. P. (1982) Hoppe-Seyler's 2. Phys-

Geiger, T., Tran-Thi, T. A., Decker, K., and Heinrich, P. C.

Lett. 206,20-24

J. Bwl. Chem. 256,9016-9022

Sci. Tools 32,41-43

Berghe, H. (1983) Ann. N Y Acad. Sci. 421,434-441

704, 290-303

Commun. 108,l-7

w l . Chem. 363,677-682

(1987) J. Biol. Chem. 262,4973-4977