405Biochem. J. (1982) 203, 405-411Printed in Great Britain

Relation of internal thioesters to conformational change and receptor-recognition site in a2-macroglobulin complexes

Fred VAN LEUVEN, Peter MARYNEN, Jean-Jacques CASSIMAN andHerman VAN DEN BERGHE

Division ofHuman Genetics, Department ofHuman Biology, University ofLeuven, Minderbroedersstraat 12,B-3000 Leuven, Belgium

(Received I October 1981/Accepted 6 January 1982)

The unique steric type of inhibition of endopeptidases by human a2-macroglobulin(a2M) and the inactivation of the latter by methylamine were examined in relation to theinternal thioesters in a2M. The present results confirm our previous findings thatdisruption of the internal thioesters, is not in itself sufficient to cause the conformationalchange of a2M typical of a2M-proteinase complexes; the electrophoretically slow formof a2M with [14C]methylamine incorporated was isolated. Moreover, this group isstabilized by derivatization of the exposed cysteine thiol groups. Cyanylation with2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effectiveprocedure, yielding essentially only slow-form a2M. Other thiol-specific reagents were

less effective. A different reactivity was observed for half of the cysteine residues in a2Mcomplexes. When allowed to react with trypsin the cyanylated derivative (slow-forma2M with thioesters broken) produced anomalous complexes; only half the expectedamount of trypsin was bound, whereas the complexes were fully inhibited by soya-beantrypsin inhibitor and were proteolytically active. Despite this, the anomalous complexeswere recognized by two highly specific probes: the fibroblast a2M-complex receptor andthe monoclonal antibody (F2B2) directed against the receptor-recognition site on a2Mcomplexes. The results show that the internal thioesters in a2M are necessary for theconformational change producing sterically inhibited endoproteinase complexes, but donot participate as such in receptor-mediated endocytosis of these complexes.

a2-Macroglobulin is a unique proteinase inhibitorin the circulation of mammals because of its stericmode of inhibition of virtually all endoproteinases,preserving the catalytic site of the enzymes in thecomplexes (Starkey & Barrett, 1977). Resultinga2M-proteinase complexes are rapidly cleared fromblood and interstitial fluids by receptor-mediatedendocytosis by fibroblasts (Van Leuven et al., 1979),macrophages (Debanne et al., 1975; Kaplan &Nielsen, 1979) and probably Kiipffer cells (Ohlsson& Skude, 1976). The receptor is highly discrimi-native and recognizes specifically a2M-proteinasecomplexes (Van Leuven et al., 1979; Kaplan &

Abbreviations used: a2M, a2-macroglobulin;Nph,SCN, 2,4-dinitrophenyl thiocyanate; Nbs2, 5,5'-dithiobis-(2-nitrobenzoic acid); iPr2P-F, di-isopropylfluorophosphate; Tos-Phe-CH2Cl, tosylphenylalanyl-chloromethane ('TPCK'); Bz-Arg-pNA, benzoylargininep-nitroanilide; a2M.MA.CN, a2M methylamine-inacti-vated in the presence of Nph2SCN; a2M.MA, methyl-amine-inactivated a2M; Tos-Lys-CH2Cl, tosyl-lysyl-chloromethane ('TLCK').

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Nielsen, 1979). We have previously shown that a2M,after inactivation by primary amines (methylamine;Steinbuch et al., 1968), is recognized and internalizedby fibroblasts in culture by the same mechanism asa2M-trypsin complexes (Van Leuven et al., 198 la).Furthermore, the methylamine derivative proved, bycriteria of rate electrophoresis and isoelectric focus-ing, to be in a conformation indistinguishable fromproteinase complexes (Van Leuven etal., 198 la).To examine this structure of complexed a2M

recognized by the cellular receptor, we have pre-pared monoclonal antibodies towards methylamine-inactivated a2M. One hybridoma was found toproduce an antibody specifically reactive withcomplexed a2M (either proteinase- or methylamine-generated) and thereby inhibiting receptor-mediatedendocytosis of the complex. Extensive characteri-zation and further evidence that this antibody bindsat the receptor-recognition site on a2M-complexeswas obtained (Marynen et al., 1981). Evidence hasbeen presented that methylamine, as well as otherprimary amines, reacts covalently with a2M at a

0306-3275/82/050405-07$01.50/1 © 1982 The Biochemical Society

F. Van Leuven, P. Marynen, J.-J. Cassiman and H. Van den Berghe

reactive internal thioester (Swenson & Howard,1979; Tack et al., 1980; Sottrup-Jensen et al., 1980).

In the present work we examined the reaction ofmethylamine with a2M, resulting in the exposure ofthe cysteine thiol groups, in relation to the con-formational change and the exposure or assembly ofthe receptor recognition site, both typical forproteinase complexes. The results indicate that thecysteine thiol group, liberated by methylaminereaction, participates in the conformational changeof a2M, but not in the recognition of the complex bythe cellular receptor. Fortuitously the results allowedus to examine in more detail the formation of'anomalous' a2M-trypsin complexes previouslydescribed (Van Leuven et al., 1982).

ExperimentalMaterials

Iodoacetamide, p-chloromercuribenzoate and N-ethylmaleimide were from Sigma. Nph2SCN andNbs2 were from Aldrich. Amersham Internationalsupplied Na251I (carrier-free), iodo[1-14C]acetamide(sp. radioactivity 53mCi/mmol), [(4C]methylamine(sp. radioactivity 56mCi/mmol) and [1,3-3H]iPr2P-F (sp. radioactivity 6.5Ci/mmol). Tos-Phe-CH2Cl-treated trypsin was from Worthington.

Methodsa2M was isolated from human citrated plasma as

described previously (Van Leuven et al., 1980).Labelling with Na125I and chloramine-T, cell cultureof normal human skin fibroblasts and measurementsof receptor-mediated endocytosis were also asdescribed previously (Van Leuven et al., 1979,1980).Rate electrophoresis. Electrophoretic mobility of

native proteins was examined by rate electro-phoresis in 5% (w/v)-acrylamide gels. A discon-tinuous buffer system was used. The reservoir bufferwas 0.041 M-Tris/0.040 M-borate, pH 8.6; the stack-ing gel was 4% acrylamide in 0.054M-Tris/0.020M-H2SO4, pH 6.1; the separating gel was 5% acryl-amide in 0.095 M-Tris/HCl, pH 5.7. Samples werediluted in reservoir buffer and contained 10% (v/v)glycerol. Gels were cast in cassettes (Pharmacia,Uppsala, Sweden) of dimensions 8cm x 8 cm x 0.27cm and run for 3 h at 125 V. Gels were subsequentlyfixed in 10% (w/v) trichloroacetic acid, stained withCoomassie Brilliant Blue (at 600C) and destained bydiffusion overnight. Photographs were taken with aPolaroid camera using positive/negative type 665ifim. Quantification of radioactivity was done byexcision of Coomassie Blue-stained bands with arazor blade. The pieces of gel were dissolved in 1 ml

of aq. 30% (v/v) H202 at 560C overnight. Afteraddition of 15 ml of scintillation cocktail (Instagel II;Packard) radioactivity was counted in a PackardTri-Carb liquid-scintillation counter. Counting effi-ciency was determined with an internal standard(['4C]toluene; Packard). This procedure was shownto be accurate for determination of specific radio-activity of radiolabelled proteins (Van Leuven et al.,1981a,b, 1982).

Trypsin activity towards Bz-Arg-pNA. This wasmeasured at 370C as described previously (VanLeuven et al., 1981a); the assay mixture containedTris/HCl buffer (0.1 M, pH 8.2), 20 mM-CaCl2, 2mM-Bz-Arg-pNA and enzyme in a final volume of 2.5 ml.A410 was continuously recorded. Activities areexpressed as AA410/min at 370C. When indicated,soya-bean trypsin inhibitor was added, equivalent tothe amount of trypsin present on a weight basis.

Proteolytic activity towards [3Hlacetylatedhaemoglobin was measured. Bovine haemoglobin(type I, Sigma) was labelled with [3Hlacetic an-hydride (3 Ci/mmol; Amersham); 600mg of haemo-globin was allowed to react with 25 mCi of[3H]acetic anhydride in 0.5 M-borate/1.4 M-sodiumacetate buffer, pH 9.0, at 00C for 24h. The labelledprotein was collected and freed from unchangedanhydride by repeated precipitation with trichloro-acetic acid and finally dialysed against saline(0.15 M-NaCl). The final preparation contained0.62mg of protein/ml and was stored at -200C. Itcontained 4500c.p.m./,ug of protein, of which 0.4%was not precipitated by trichloroacetic acid.

The assay mixture contained, in a final volume of0.5 ml, the following: Tris/HCl (50mM, pH 8.2),lOO,l of the labelled haemoglobin preparation andeither trypsin (1,ug) or a2M (200,g). Incubation at370C for 1 h was followed by addition of 0.5 ml ofoutdated newborn-calf serum (Gibco) and 0.4ml of50% (w/v) trichloroacetic acid. After 30min in icethe solution was cleared by centrifugation and 1 mlof supernatant was added to 15 ml of Instagel II.Radioactivity was counted in a Tri-Carb liquid-scintillation counter with external standardization.

Active-site titration of trypsin in a2M-trypsincomplexes was done with [3H]iPr2P-F; the proteinwas made to react with [3H]iPr2P-F (0.77mM) in50 mM-Tris/HCl, pH 8.0 for 1 h. After separation byrate electrophoresis, radioactivity was determinedas described above.

Determination of thiol residues. Thiol residues ona2M were determined either spectrophotometricallywith Nbs2 essentially as described by Sottrup-Jensenet al. (1980) or by derivatization with iodo[14C1-acetamide. For the latter, a2M-complexes weremade to react with 0.5mM-iodo[14C]acetamide for2h or for the time periods indicated (Table 1). Afterseparation by rate electrophoresis, radioactivity wasdetermined as described above.

1982

406

Internal thioesters in a2-macroglobulin

Results and discussion

Thiol residues on a2M complexesTo determine the exposed thiol (cysteine) resi-

dues on %2M-proteinase and on a2M-methyl-amine complexes, the complexes were carboxy-methylated with iodo[ 4Clacetamide. Radioactivityin a2M was determined after separation by rateelectrophoresis on 5% polyacrylamide gels (VanLeuven et al., 1981a). Negligible incorporation wasfound in native a2M (Table 1). On complexesprepared either with trypsin or with methylamine,apparently two thiol groups reacted (Table 1). Asfour were expected (one per monomer), thiolresidues were therefore also determined spectro-photometrically with Nbs2 essentially as describedby Sottrup-Jensen et al. (1980). By this procedure,four thiol groups were measured, at least when thea2M-trypsin complexes were measured immediately(Table 1). When the a2M-trypsin complexes wereleft for 30min at room temperature before reactionwith Nbs2, only about half of the original thiol

groups could be detected (Table 1). This indicatesthat two out of the four thiol groups on a2M-trypsincomplexes are rapidly oxidized under these con-ditions, corroborating the results obtained withiodo[ 4C]acetamide. With a2M-methylamine com-plexes also, only two thiol groups could be detectedby the Nbs2 reaction (Table 1).To circumvent the rapid oxidation of thiol groups,

the reaction with methylamine was done withiodo[14C]acetamide present during incubation withmethylamine. Subsequent separation by rate elec-trophoresis showed that, under these conditions,treatment with methylamine did result only partiallyin fast-form a2M (Fig. 1), although quantification oflabel indicated that nearly four thiol groups wereexposed (Table 1).

Other thiol-specific reagents were tested undersimilar conditions. Nbs2 (1 mM) and p-chloro-mercuribenzoate (0.1 mM) did not appreciably inter-fere with the conformational change (generation offast-form a2M) by treatment with methylamine (Fig.1). N-Ethylmaleimide was about equally effective as

Table 1. Determination of thiol residues on a2Mcomplexes

Quantification of thiol residues was performed byreaction with iodo['4C]acetamide and separation byrate electrophoresis or spectrophotometrically withNbs2 (see under 'Methods'). In Expt. (A) a2M-trypsin complexes were prepared by reaction of a3:1 molar excess of trypsin (Tos-Phe-CH2CI-treated,65% active) with a2M. Excess trypsin was removedby gel filtration. The a2M-methylamine derivativewas obtained by reaction of a2M with 0.2M-methyl-amine at 250C in Tris/HCl (50mM, pH8.0) for6 h. In Expt. (B) the same conditions were used toprepare the a2M-trypsin complex, but determinationwas done either immediately (2min) or after thecomplexes were left for 30min at room temperature.Excess trypsin was not removed, as the enzymepreparation was found not to contain free thiolgroups. In Expt. (C) a2M was allowed to react with25 mM-methylamine (Tris/HCI, 50mM, pH 8.0,25 IC) but overnight and in the presence of 0.5 mm-iodo[ '4C lacetamide. Results are the means of two orthree determinations.

Thiol groups (mol/mol ofa2M) reactive with:

Expt. Treatment(A) a2M

a2M-trypsina2M-methylamine

(B) a2M-trypsin2 min30min

(C) a2M-methylamine

iodo[14Clacetamide0.21.901.84

Nbs20.31.751.68

4.102.25

3.76

(1) (2) (3) (4) (5) (6)

-Slow_ _ gIUI~ -

Fast

Dye

Fig. 1. Effect of thiol-specific reagents on conformationalchange ofa2M by reaction with methylamine

a2M (2 mg/ml) was allowed to react with 25mM-methylamine in Tris/HCl (50mM, pH8.0) at 250Covernight. The reaction mixture contained no furtheradditions (1) or 1 mM-iodoacetamide (2), 1 mM-N-ethylmaleimide (3), 1 mM-Nbs2 (4), 1 mM-Nph2SCN (5) or 0.1 mM-p-chloromercuribenzoate(6). Rate electrophoresis was done on 5% polyacryl-amide gels and lOpg of protein was loaded per lane.Slow- and fast-form a2M and the position of the dyefront are indicated on the right-hand side.

Vol. 203

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F. Van Leuven, P. Marynen, J.-J. Cassiman and H. Van den Berghe

iodoacetamide (both at 1 mM). The most powerfulagent was Nph2SCN: practically no fast-form a2Mwas generated when Nph2SCN was present duringmethylamine treatment (Fig. 1). Incorporation of[14C]methylamine was' not inhibited, neither byiodoacetamide nor by Nph2SCN; only the distri-bution of label in slow- and fast-form a2M was

Table 2. Effect of iodoacetamide and Nph2SCN on[14C]methylamine incorporation into a2M

a2M was allowed to react with [14CJmethylamine(25mM) in Tris/HCl (50mM, pH8.0) at 250C for3h, without further additions, or in the presence of1 mM-iodoacetamide or 1 mM-Nph2SCN. After rateelectrophoresis (see Fig. 1), slow- and fast-forma2M were excised separately from the gels and radio-activity determined in both. Total incorporation isthe mathematical sum of these two determinations.Although after 3 h incubation methylamine incor-poration is not complete, this experiment is repre-sentative for the extent of incorporation of [14CI-methylamine into the two forms of a2M for longerperiods of incubation.

AdditionsNoneIodoacetamide (1 mM)Nph2SCN (1 mM)

[14C]Methylamine incorporation(c.p.m./lO,ug) into:

r ~ ~ A

Slow form Fast form Total468 2735 32031737 1385 31222743 591 3334

affected (Table 2). The difference between thesethiol-reactive probes might be related to the rate atwhich they react or to steric factors.Nph2SCN, which has been shown to act as a

specific cyanylating agent for thiol groups (Degani& Degani, 1980), introduces the least bulky groupon to a2M. This in itself can influence the rate ofreaction with the thiol group, but apparently doesnot affect the change in conformation, since fourthiol groups can react with Nbs2 on a2M-complexes(Table 1), whereas Nbs2 is the least effective inblocking the conformational change (Fig. 1). Fur-ther experimental work will have to be done to revealwhether the microenvironments of these thiol groupson a2M complexes are different. This would addanother argument to our previous findings indi-cating that the subunits in a2M are asymmetricallyorganized in the tetramer (Van Leuven et al.,198 la,b).

Soya-bean-trypsin-inhibitor-inhibited complexesApart from these considerations, the present

findings allowed us to examine further the mech-anism of conformational change by which a2Minhibits proteinases. As Nph2SCN did not inhibit['4C]methylamine incorporation but affected onlythe distribution of label between slow- and fast-forma2M (Table 2), this was reminiscent of our previousfindings that [14Clmethylamine incorporation pro-ceeds via labelled slow-form a2M, which to someextent proved stable to isolation by polyacryl-amide-gel electrophoresis (Van Leuven et al.,

Table 3. Enzymic activity oftrypsin and a2M-trypsin complexesEnzymic activity towards [3Hlacetylated haemoglobin and Bz-Arg-pNA was measured as detailed in the Experimentalsection. Complexes were prepared and isolated as described in the text. In the haemoglobin assay, activity is ex-pressed as radioactivity (c.p.m.) not precipitated by trichloroacetic acid, liberated by 1pg of trypsin or by theequivalent of 200,g of a2M as trypsin complexes. In the Bz-Arg-pNA assay, activity is expressed as increase inabsorbance at 410nm/min with lO,g of trypsin or the equivalent of 125,pg of a2M as trypsin complexes. Tos-Lys-CH2Cl was used at a final concentration of 2mM; 20pg of soya-bean trypsin inhibitor (STI) was used per assay.Results are means of duplicate determinations. Results from the haemoglobin assay are corrected for a blank valueof 1169 c.p.m. (no trypsin or a2M added, but otherwise identical conditions).

Enzymic activityA

Conditionsa2M (native)Trypsin

+ STI+ Tos-Lys-CH2Cl

a2M-trypsin+STI+Tos-Lys-CH2Cl

a2M.MA.CN-trypsin+ STI+ Tos-Lys-CH2Cl

Substrate . . .Bz-Arg-pNA(AA/min)0.0010.0410.0010.0010.0300.0280.0020.0150.0020.001

PH]Haemoglobin(c.p.m.)

2210417

221234326618621259982515111036

1982

408

Internal thioesters in a2-macroglobulin

1981a). We hypothesized that the presence of thisform might have led to our finding of 'abnormal'a2M-trypsin complexes that were proteolyticallyactive and were inhibited by soya-bean trypsininhibitor (Van Leuven et al. 198 lb).We prepared several batches of a2M.MA.CN as

follows. a2M (10mg) was allowed to react withmethylamine (25mM) in the presence of Nph2SCN(1 mM) at pH 8.0 (Tris/HCl, 50mM) at roomtemperature overnight and the derivative was iso-lated by gel filtration (Ultrogel AcA-22; LKB).Control preparations of a,M.MA (no Nph2SCNpresent) were obtained under otherwise identi-cal conditions. Rate electrophoresis showeda2M.MA.CN to be about 90% slow form, whereasa2M.MA contained about 90% fast form (judged byvisual inspection of the gels).

Native a2M, a2M.MA and a2M.MA.CN weremade to react with a 3:1 molar excess of trypsin(65% active trypsin as determined by active-sitetitration). On rate electrophoresis, all preparationswere shown to be transformed into fast-form a2M.Thus trypsin overrides the block in conformationalchange imposed by derivatization of the thiol groupsin a2M.MA.CN. The a2M complexes were sub-sequently separated from excess trypsin by gelfiltration and examined for trypsin activity (Table 3).

Amidase activity, measured spectrophotometri-cally towards Bz-Arg-pNA, showed less than 10%of the total activity of a2M-trypsin to be inhibited bysoya-bean trypsin inhibitor, whereas Tos-Lys-CH2Clinhibited completely. This is the classical behaviourof a2M-proteinase complexes. An equivalentamount of a2M.MA.CN-trypsin complexes showedonly 50% of the activity of the control preparation,and this activity was completely inhibited bysoya-bean trypsin inhibitor. Under the same con-ditions, a2M.MA (fast form) did bind less than 10%of the amount of trypsin bound by native a2M.

Proteolytic activity was measured towards

Table 4. [3H]iPr2P-F incorporation in L2M-trypsincomplexes

a2M derivatives and complexes with trypsin wereprepared as described in the text. They were madeto react with [3HIiPr2P-F (0.77mM) for 1h. Afterseparation by rate electrophoresis, radioactivity ina2M was determined (see under 'Methods'). In thiscase counting efficiency was not determined, andthe results are given in c.p.m./lO,g of a2M.

Incorporation

a2M-trypsina2M.MA.CN-trypsina2M

Vol. 203

Relative (%)100453

[3Hlacetylated haemoglobin as previously described(Van Leuven et al., 198 lb). Although some activitywas found associated with a2M-trypsin complexes,the proteolytic activity of a2M.MA.CN-trypsin wasmuch higher (Table 3). All activity was inhibited bysoya-bean trypsin inhibitor.As a third approach we measured the in-

corporation of [ 3H]iPr2P-F into the complexes;radioactivity was quantified after separation byrate electrophoresis. Relative to a2M-trypsin,a2M.MA.CN-trypsin complexes incorporated about45% of [3H]iPr2P-F, whereas incorporation in nativea2M was negligible (Table 4).From these findings it is clear that trypsin

complexes are formed with a2M.MA.CN, as judgedfrom the incorporation of 13HIiPr2P-F. However,only about half as much trypsin forms a complexwith a2M.MA.CN as compared with a2M, as shownby both amidase activity towards Bz-Arg-pNA(Table 3) and by active-site labelling with [3HIiPr2P-F (Table 4). Moreover, essentially all trypsin boundto a2M.MA.CN is inhibited by soya-bean trypsininhibitor, concomitant with a high proteolyticactivity in the haemoglobin assay.

These data strengthened our previous hypothesis,namely that slow-form a2M, in which the thioester isdestroyed by primary amines, can be generated (VanLeuven et al., 1981a). The stability of this form isincreased when the cysteine thiol groups are deriva-tized, especially by cyanylation. However, trypsincomplexes derived from this form of a2M containonly half as much proteinase as normal complexesand, more importantly, must be designated 'ab-normal' or 'open' inasmuch that soya-bean trypsininhibitor inhibits all the trypsin and that the com-plexes are proteolytically active (Van Leuven et al.,1982).

Receptor-mediated endocytosis and binding ofmonoclonal antibody (F2B2)As stated previously (Van Leuven et al., 1982), it

seemed worthwhile to determine whether generationof 'abnormal' complexes would have biologicalimplications. Therefore we examined the behaviourof the 'open' complexes in receptor-mediated endo-cytosis by the cellular assay (Van Leuven et al.,1979, 1980) and the receptor recognition site on thea2M complexes, with the monoclonal antibody,F2B2. The latter was shown to be totally specific fora2M complexes generated either by proteinase or bymethylamine treatment (Marynen et al., 1981).Moreover, this antibody, and Fab portions derivedfrom it, blocked the receptor-recognition site on thea2M complexes (Marynen et al., 1981). The bindingof F2B2 to a2M complexes is fast and stable and canbe detected by rate electrophoresis on 5% poly-acrylamide gels (Marynen et al., 1981). Thisprocedure was applied to the complexes generated in

c.p.m./lO,g17 2347747510

409

F. Van Leuven, P. Marynen, J.-J. Cassiman and H. Van den Berghe( 1) (2) (3)

'-..---{.---2 . 3 (4)the experiments described above. As judged from

t +t 4- + Fig. 2, binding of the monoclonal antibody to the

anomalous complexes was of the normal extent; thepattern of bands obtained was identical with thatobtained with classical a2M-trypsin complexes(Marynen et al., 1981). Surprisingly, however,whereas F2B2 did not react with native a2M,slow-form a2M.MA.CN did bind the monoclonalantibody (Fig. 2, lane 4).We have previously shown that fibroblasts in

_ __ wY _ Slow culture carry a receptor at their plasma membrane...... -._ *' jFast which discriminates a2M complexes from native

a2M; as a consequence, only a2M-proteinaseplexes are internalized by receptor-mediated endo-cytosis (Van Leuven et al., 1978, 1979; Marynen

- F21B2 etal., 1981). To examine whether the receptor would

- Dye also recognize the anomalous complexes described,we measured inhibition of endocytosis of i25I-labelled a2M-trypsin complexes by the derivatives

Fig. monoclonal obtained. It was found that a2M.MA.CN-trypsina2M-dermnivaves complexes were nearly as potent as a2M-trypsin inDerivativesof a2M (20,g), prepared as described in inhibiting endocytosis, indicating their effectivethe text, wereallowed to react with the monoclonal recognition by the receptor. Moreover, slow-formantibody F2B2 (20,ug of protein) for 30min at a2M.MA.CN was also inhibitory for endocytosis,250C. electrophoresis polyacrylamide whereas native a2M was not (Table 5), confirminggels and the untreated our finding with the monoclonal antibody described

derivatives. (1) Nativea2M; (2) a2M.MA.CN- above.trypsin; (3)a2M.MA; (4)a2M.MA.CN; in each case Taken together, these results indicate that, despiteuntreated (-) or treated with F2B2 (+). The position the proteolytic nature of the anomalous complexes,of slow- and fast-forma2M, of the free antibody their interactions with the highly specific probes(F2B2) and the dye front are marked. Note theabsence of reaction with native a2M (Marynen et al., (cellular receptor an dmonoclonalantibody) are not1981) and the shift in mobility of the band when impaired or even markedly altered. The data furtherreaction has occurred with other derivatives. Some indicate that cyanylation or carboxymethylation ofslow-form ;M,still present in

a2M.MA (3) (Van the cysteine thiol group does not interfere in the twoal., la) reacted with FM2 , systems mentioned; therefore it does not appear that

indicating were disrupted. the thiol groups as such participate in receptor-

Table 5. Receptor-mediated endocytosis of12II-labelledaXM-trypsin complexes by fibroblasts in culture: inhibition by

a2M derivativesfibroblast monolayers were examined for receptor-mediated endocytosis for30mn at,370C with

1251-Habelled a2M-trypsin complexes (50Opg/ml), without further additions or with the derivatives indicated at 20O0pg/otherwise indicated. Results are expressed relative to control (no addition, 5412 c.p.m. perl0o

endocytosed). General procedures were as described previously (Van Leuven et al., 1980). Derivatives of a2Mprepared described in the text. Results are means of duplicate cell layers in two experiments.

Receptor-mediated endocytosis

Additions relative to control(%)a2M 96

a2M-trypsin 36a2M-methylamine 44a2M-trypsin/carboxymethylated 4 1a2M-methylamine/carboxymethylated 46

a2M.MA.CN50ug gmml 782002 ug/ml 49a2M.MA.CN-trypsin505g g/ml 60

2002ug/ml 33

1982

410

Internal thioesters in a2-macroglobulin 411

mediated endocytosis (Table 5). Thus our resultsindicate that the structure making up the receptor-recognition site on a2M complexes does not includethe thioester residues and is not affected by theanomaly of the complex. This is substantiated bothby the cellular assay (Van Leuven et al., 1979) andby the normal binding of the complex-specificmonoclonal antibody (Marynen et al., 1981). Thisindicates that if the conformational change of a2M inthe complex were to be incomplete, so as not to'wrap' the proteinase completely (as can be en-visaged in a normal complex), the difference wouldbe only minute and would not affect the receptor-recognition site on a2M complexes. This obviously isadvantageous for the situation in vivo: even theformation of proteolytically active a2M-proteinasecomplexes will lead to their removal and degra-dation by cellular mechanisms before they canbecome harmful in the circulation or in the extra-cellular space. Apparently this mechanism extendseven further. Our data indicate that slow-form a2M,in which the active thioesters are destroyed(a2M.MA.CN), is also internalized by receptor-mediated endocytosis (Table 5) and binds themonoclonal antibody (Fig. 2). These findings indi-cate that the receptor-recognition site on a2M isalready accessible to both the receptor and themonoclonal antibody before the dramatic change inconformation occurs. The only necessary conditionfor revealing the receptor-recognition site seems tobe the disruption of the covalent internal thioester,which is of course expected to produce minorchanges in conformation locally. Therefore it seemslikely that the receptor-recognition site is located inthe vicinity of the internal thioester.

Conclusions

Our data indicate that disruption of the internalthioesters in a2M by primary amines is sufficient toexpose the receptor recognition site and does notnecessarily lead to the conformational changetypical of proteinase complexes. Moreover, thesequence of events, namely breaking the thioester,followed by proteinase complex-formation, leads toequimolar complexes instead of the usual 2:1binding of trypsin to a2M. The proteinase in theresulting complex is fully accessible to soya-beantrypsin inhibitor and has proteolytic activity. Thismight explain the disparate data in the literature on

the exact stoichiometry of proteinase-a2M com-plex-formation and on the proteolytic character ofthe complexes produced. Clearly such studiesrequire fully active a2M, in which activity is bestassessed not only by rate electrophoresis butpreferably by titration of the active thioesters with['4Clmethylamine. A perfect probe appears themonoclonal antibody F2B2 (Marynen et al., 1981),which distinguishes in slow-form a2M, two speciesthat are dependent on the state of the thioesters.

This work was supported by grant no. 3.0043.79(Fonds voor Geneeskundig Wetenschappelijk Onder-zoek). P. M. is a Research Assistant of the National Fundfor Scientific Research, Belgium. The expert technicalassistance of Ms. M. Caems and Ms. L. Stas is gratefullyacknowledged.

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Degani, C. & Degani, Y. (1980) J. Biol. Chem. 255,8221-8228

Kaplan, J. & Nielsen, M. L. (1979) J. Biol. Chem. 254,7329-7335

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