BIOCHIMIE, l!h'.lo~ 62, 497-5~12.

Human 2-macroglobulin : time under denaturing condition .

Jean-Pier re FRI~NOY * and Roland BOURRHJ,ON.

(23-11-1979).

dependent degradation

Labora to i re de B i o c h i m i e , U.E.R. Biom~;dicale des Sainls-Pi 'res . 75006 Paris , France .

Introduction.

Conflict ing results [1-4] were publ ished concer- n ing the subuni t s t ruc ture of ¢~2-macroglobulin (a2-MG), one of the p r inc ipa l plasma glycopro- reins [5]. Harpel [6] ind ica ted that the i nh ib i t i on of the activity of var ious serum proteases by com- plex format ion wi th tc2-MG , is accompanied by

u

. . . . . . S _ . ........ ,:_

Fro. 1. - - Purification of human o~-MG. 5 per cent polyacrylamide ~el.s were prepared at pH 10.2 according to Rodbard ~ Chrambaeh [13]. S : serum, a : after ammonium sulfate precipitation between 40 and 48 per cent, pH 6, b : aster Porath column electrophoresis, c : purified o~-~G after gel-filtration.

Abbreviations :

o~-MG : u,-Macroglobulin, SDS : sodium dodecyl sulfate, ~-ME : B-mercaptoethanol.

(*) Attachd de Recherche au C.N.R.S. This study is a part of the << Th~se de Doctorat d'Etat >> of J. P. Frd- noy at the Unioersity Paris, 7, June 1976.

proteolysis of the polypept ide chains of a2-MG and, presently, authors general ly agree that a2-MG is a te t ramer of ident ica l subuni ts (Mr. 180,000-190,000) linlced in pairs by d i su l fde bridges and then assembled non-covalent ly [3, 6-9].

W or k i ng wi th thyroglobul in , a pro te in presen- t ing some s t ruc tura l analogies wi th a2-MG [1], Ambesi- Impiobato & Pitt-Rivers [10] c la imed that du r ing prepara t ion of the porc ine 19S-protein, a proteolyt ic enzyme becomes i r revers ib ly bound to the thyrogIobul in and is responsible for its subse- quent b reakdown, either in the reduced or unre- duced state, into components of molecular weights smal ler than 100,000. Similar results were obtained wi th the same prote in from various species by Rol land a Lissitzky [11], but another exp lana t ion was proposed in relat ion to the complex subuni t s t ruc ture of thyroglobul in , the existence of var ious k inds of monomer ic subuni ts of the same molecu- lar weight but of different aminoac id composit ion.

The present study reports modif icat ions in the subun i t chain size of h u m a n a2-M~G which depends on the condi t ions of incubat ion . We propose that a2-MG binds protease (s) which are responsible for the subsequent hydrolysis . Moreover these pro- teases are active in the presence of sodium dodecyl sulfate (SDS) and ~-mercaptoethanol (i[3-ME).

Materials and Methods.

Human ~-MG :

Human ~-MG was prepared at 0-4 ° from pooled serum or plasma (C.N.T.S. Paris) by (NH4)~SO~ preci- pitation at pH 6, followed by cellulose column electro- phoresis and gel-filtration [12]. The homogeneity of the preparation was checked by immunoelectropho- rests, using horse anti-human a~tiserum (Institut Pas- teur, Paris), polyacryl~amide gel eleetrophoresis and analytical ultracentxifugation. In polyacrylamide gel at pH 7.5 and 10.2 [13] one protein hand is noticed (fig. 1) with a second faint hand already described [12, 14]. A single sedimenting boundary with an S~o of 18 S was observed by analytical ultracentrifugation.

498 J.-P. Frdno U and R. Bourrillon.

Chemicals :

Acrylamide, m e t h y l e n e b i s a c r y l a m i d e and N, N, N', N ' - t e t r am e th y l ened i amin¢ were pu rchased f rom East - m an . Sod ium dodecyl su l fa te was f r o m Touzar t et Mat ignon and ~-mercap toe thanol f r om Serva.

Bovine s e ru m a l b u m i n was a S igma product and h u m a n tran, s fer r in was prepared in the l abora to ry [15].

France) equipped wi th a yel low filter Kodak n u m b e r 9 (dvns i tomet ry at 580 nm) .

Incubations of armacroglobulins : A solu t ion of p ro te in (concentrat ion 3.25 m g /

ml) was incuba ted a t 37 ° in 0.01 M sod ium phospha t e buffer, pH 7.0, a lone or wi th ~-ME and SDS, and in

190 0 0 0

83 0 0 0

+ ~ m i d 96h

3 0 r a i n

. . . . 2 4 h

. . . . . . 9 6 h

1 9 0 0 0 0

1 2 0 0 0 0 / - / . . .:t-- 83000

.~ ": ~ e o o o o

,: ,:." , : : / , :

- ,....,.'-....

M O B I L t T Y

Fro. 2. - - SDS-Polgacrglamide gel electrophoregrams and densitometer scans of gels of u2-MG at various times of incubation wi th I per cent SDS, 1 per cent

ME.

SDS polyacrylamide gel electrophorcsis :

Elect rophorcs is ,and s t a in ing of the geSs were carr ied out according to Webe r ~ Osborn [16], except t h a t 5 per cent po lyac ry l amide gels were used.

Pho tomet r i c t rac ing of the ~stained gels was per for - med in a Phi 5 Vernon a u t o m a t i c dens i t ome te r (Paris,

BIOCHIMIE, 1980, 62, n ° 7.

the presence of s o d i u m azide to avoid bacter ia l conta- mina t ion . 20 I~1 a l iquo ts were t aken a t 30 min , 4 h, 24 h, 48 h, 72 h and 96 h, and stored a t - - 4 0 ° . The samples incuba ted w i th eRher SDS or ~-ME were re incuba ted 10 m i n w i t h the o ther reagent . The m i x t u r e con ta in ing buffer, b romophe no l blue, glycerol and ~-ME, ~s described by W~ber • Osborn [ 1 6 ] , was added to each und ia lyzed aliquo~ j u s t before e lectrophoresis .

H u m a n % - m a c r o g l o b u l i n degradat ion . 499

R e s u l t s .

Incubations o[ %-MG with 1 per cent SDS and 1 per cent g-ME.

As a l r eady noted, w h e n serum %-MG is incu- ba ted w i t h 1 pe r cent SDS and 1 per cent ~-ME, a complex pa t t e rn of bands is observed by gel e l ec t rophores i s (fig. 2). Whereas non- reduced %-MG shows two bands of molecu la r weight 300,000-400,000 in the p resence of SDS 11, 6], the componen t s desc r ibed as subuni ts of the p ro te in [1, 3, 4, 6] are m a i n l y observed af ter 30 min of incuba t ion at 37 °. The subuni t of 180,000-190,000 (one four th of the nat ive molecule) is the most impor t an t component . The second most dense band was the one w i t h a molecu la r weight of 83,000 High molecu la r we igh t componen t s (Mr 300.

These f indings are summar i zed quan t i t a t ive ly in f igure 3 w h e r e the r e l a t ive pe rcen tage of each c o m p o n e n t is p lo t ted versus the t ime of incuba- t ion. However , the resul ts have to be cons ide red carefu l ly , due to the non- l inea r r e sponse of the dens i tomete r to h igh ly s ta ined p ro te in bands .

The same dependence of the gel pa t t e rns on the t ime of incuba t ion was observed, wi th s l ight dif- ferences , for severa l %-MG p r e p a r e d f rom serum.

It has been suggested that the 83,000 componen t , i so la ted by reduc t ion-a [ky la t ion of se rum %-MG, was due to a pro teo lys i s o c c u r r i n g du r ing coagu- la t ion [6] ; whert the same expe r imen t s were done w i th the p ro te in p r e p a r e d f rom human p lasma, a rough ly s imi l a r pa t t e rn was observed by gel elec- t rophores i s , w i th even a s l ight ly grea ter degrada- t ion of the 190,000 component .

40

' - 3{ ~ x x "x 190,000

I I i I I

4 24 48 72 96 Time (hours)

Fro. 3 . - Dependence of the breakdown of polypeptides chains of a2-MG on incubation time in presence of I per cent SDS, 1 per cent (~-ME. The relative amounts were obtained by densitometry at 580 nm of the Coomassie blue-stained gels.

400,000) were also p resen t among the r ema in ing bands , cons t i tu t ing .a m i n o r f rac t ion of the total pro te in .

W h e n incuba t ion t ime was leng thened f rom 30 min to 96 h, two bands appea red (molecular weights of 60,000 and 120,000), w k i c h inc reased w i th t ime, whi l e the concen t ra t ion of the 190,000 and 300-400,000 species decreased . S imi l a r ly an en la rgement of the low molecu la r weight bands (Mr < 50,000) can also be no t iced (fig. 2).

Incubation of serum %-MG alone, or wi th either 1 per cent SDS or 1 per cent Z-ME.

The respec t ive inf luences of the d i s soc ia t ing and r e d u c i n g agents on %-M~ deg rada t ion we re c he c ke d by incuba t ion of samples of a~-MG alone or ~vith e i ther only SDS or [~-ME. Inc rease of c o m p o n e n t s w i th molect t la r weights of 60,000 and 120,000 to the de t r imen t of the h igh molecu la r we igh t subuni ts was observed. The re was also an inc rease in the minor bands .

r31OCHIMIE, 1980, 62, n ° 7. 34

500

Incubations of transferrin and albumin with 1 per cent SDS, I per cent ~-ME.

In order to test the procedures used to evaluate %-MG, various other p lasma proteins , i.e. t rans- fe r r in and albumin, were treated in the same

J.-P. Frdnoy and R. Bourr i l lon .

Incubation of serum e~-MG with 1 per cent SDS, 1 per cent B-ME after 10 min at 100%

The evolut ion of eleetrophoresis pat terns as a funot ion of time, in the presence or absence of reduct ive or dissociat ing reagents is typical of

30 win

...... 96 h

1 9 0 0 0 0

1 2 0 0 0 0 .

~ ~ - - 8 3 0 0 0

MOBILITY

FIG. 4. - - SDS-Polyacrylamide gel electrophoregrams and densitometer scans o[ gels o[ a2-MG at various times of incubation with 1 per cent SDS, 1 per cent ~-ME, after treatment 10 win at 100%

m a n n e r as az-MG. No significant changes of the geI pat terns were noted d,uring incubat ion , even after 96 hours, i nd ica t ing that the phenomenon is typical of a2-MG.

BIOCHIMIE, 1980, 62, n ° 7.

a2-MG, and could be due to the presence of pro- teases bound to the protein. If these results repre- sented enzymat ic proteolysis, then dena tura t ion of the enzymes should result in the e l iminat ion of the

Human %-macroglobulin degradation. 501

fo rma t ion of the smal le r components . When %-MG was t rea ted for 10 min at 100 °, before add i t i on of SDS and ~-ME and, after addi t ion , i ncuba t ed as desc r ibed in Mater ia l and Methods, the electro- phore t i c pa t t e rn s we re not s ignif icant ly modi f ied wi,th t ime of incuba t ion (fig. 4), con t ras t ing wi th the results ob ta ined f rom the exper imen t s wi thou t p rev ious heat ing.

Moreover, these resul ts exclude the t ime-depen- dent d i ssoc ia t ion of the subuni ls as poss ible expla- nat ion for the evolut ion of gel pa t te rns as a func- t ion of t ime, since, in that case, heat ing 10 min a,t t00 °, p a r t i c u l a r l y in the p resence of SDS, wou ld increase the d i s soc ia t ion p rocedu re y i e ld ing pa t t e rns of ful ly dissoci 'ated p ro t e in [17].

Thus, the p h o t o m e t r i c t rac ing of these gels is used as zero t ime in figure 3, all possible over incu- barton of the p roduc t s dur ing e lec t rophores i s being avoided.

Discussion.

In vitro studies of the in t e rac t ion be tween a2-MG and pro teases ind ica te tha t fo rmat ion of the com- plex is a c c o m p a n i e d by p ro teo ly t i c modi f ica t ions in the subuni t cha in s t ruc ture of au-MG [6]. In the p resen t s tudy t ime-dependen t degrada t ion of the subuni ts of ae-MG is observed after incuba t ion under va r ious dena tu r ing condi t ions . Tak ing into account the expe r imen t s pe r fo rmed w i t h a2-1VlG t rea ted 10 rain at I00 ° before incubat ion , these obser~cations cannot be asc r ibed to t ime-dependen t d i ssoc ia t ion of the subuni ts or incomple te reduc- t ion of the molecule at shor t t imes.

It is unli~kely that SDS .is respons ib le for the hyd ro ly s i s of pep t ide bonds since no changes we re observed for a lbumin and t r ans fe r r in af ter incuba- t ion under the same condi t ions . Moreover, e lectro- phores i s in SDS is a common p r o c e d u r e and does not appea r to sp l i t pep t ide bonds. The above results suggest the exis tence of pro teases bound to a2-MG. This b i n d i n g would very wel l p ro tec t the enzyme aga ins t S~)S dena tura t ion . The pro- teolysis could howeve r be e l imina ted by p r i o r hea t ing of %o-MG sol.ution at 100 ° for 10 rain.

Another poss ib i l i ty is the exis tence of p ro tease ac t iv i ty i n t r i n s i c to a~-MG. This cannot be for- matly d i s t inguished from bound proteases , s ince the affini ty cons tant of c¢,,-MG for proteases is very high, 10 lo [18~ ; moreove r it has been demons t ra - ted that when pro teases are bound to %-MG, the i r p ro teo ly t i c ac l iv i ty is genera l ly inh ib i ted (except

t o w a r d s the macrog lobul in ) but the i r es te ro ly t ic ac t iv i ty against low molecu la r weight subs t ra tes is m a i n t a i n e d [19-20] and even p ro t ec t ed agains t i nh ib i to r s ( soybean t r y p s i n inh ib i to r , for exam- pie), p r o b a b l y for s ter ic h i n d r a n c e [21].

The re ten t ion of p ro teo ly t i c ac t iv i ty in sod ium dodecy l sulfate and reduc t ive agent con ta in ing so lu t ions has been d iscussed [22-25]. A communi - ca t ion of Po r t e r ~ Pres ton [26] demons t r a t ed it for t r yps in and c h y m o t r y p s i n s tudy ing the a l te ra t ions of SDS-po lyac ry lamide gel profiIes, and it is now cons ide red as a poss ible p r o d u c e r of a r t i fac t s in SD S-po lya c ry l a mide gel e lec t rophores i s .

If a s t rongly bound p ro t eo ly t i c enzyme is pre- sent in a2-MG p r e p a r a t i o n s ,then the quest ion ar ises as to w h y smal ler po lype p t i de s of cons tan t molecu- la r weights are p roduced . This wou ld i m p l y e i ther a very h igh speci f ic i ty of the bound enzyme, or the p resence in the a2-]Y[G molecule of exposed area of p e p t i d e chains , p r o b a b l y be tween its s t ruc tu ra l domains [27], w h i c h are r ead i ly a t t acked by pro- t eo ly t ic enzyme (s). The l a t t e r suggest ion seems to be a l ike ly exp lana t ion s ince it agrees w i t h a me- chan i sm a l r eady p roposed for the i n t e r ac t ion of ~e-MG wi th pro te inases [283. This mechan i sm con- sists in the cleavage of a pe p l i de bond in the sen- s i t ive region of the macrog lobu l in as a first s tep for the in te rac t ion .

Although it is poss ible tha t a f rac t ion of %-MG may have been complexed wi th p ro t eo ly t i c enzymes de r ived from the in vitro clot t ing of the blood, this seems improbab le , s.ince the same e l ec t rophore t i c pa t te rns of .¢t2-MG were ob ta ined af ter incuba t ion of e i ther p l a sma .or serum. Moreover , a po r t ion of .c i rculat ing a2-MG may be com:plexed w i t h a p lasmin- l ike enzyme [29].

The na ture of the enzyme (s) bound to ¢%-MG is st i l l unknown. Pro teases p rev ious ly shown able to b ind to %,-MG ( t ryps in , t h rombin , p lasmin , ka l l ik re in , for ex~ample) a re p r o b a b l y not involved in this mechan i sm since they a p p a r e n t l y do not p roduc e 120,000 and 60,000 fragments . They spl i t a pept.ide bond y i e ld ing only subuni ts of 83-85,000 wi,thout any components .of i n t e rmed ia t e molecu- lar weights [6, 7].

A s imi la r s i tuat ion seems to occur w i th thy- rog lobul in where many small species have been found after reduc t ion of the disulf ide bonds. It now appears that all the smal le r molecules result essent ia l ly from the p ro teo ly t i c ac t iv i ty of enzymes 1.iberated when the t hy ro id glands are frozen af ter removal from the animals [301, and that the l imi t molecu la r weight for the po lypep-

BIOCttlMIE, 1980, 62, n ° 7.

5 0 2 J . -P . F r g n o y a n d R. Bourr i l l on .

t i d e c h a i n of the t h y r o g l o b u l i n is 330,000, n a m e l y o n e h a l f of t he n a t i v e p r o t e i n [30, 31~.

U n t i l n o w , no p o l y p e p t i d e c h a i n s of m o l e c u l a r w e i g h t s a b o v e 190,000 w e r e o b s e r v e d f o r a2-MG a f t e r r e d u c t i o n . T h e e x i s t e n c e of c h a i n s w i t h 300- 400,000 Mr a f t e r r educL ion , is s h o w n in o u r w o r k (fig. 1 a n d p a r t i c u l a r l y fig. 3). T h e 300-400,000 c h a i n s c o u l d be a g g r e g a t i o n p r o d u c t s c r e a t e d b y b o i l i n g , ye t h e a t i n g ,in t h e p r e s e n c e of SDS w o u l d m o r e p r o b a b l y i n c r e a s e t h e d i s s o c i a t i o n p r o c e - d u r e . A l t e r n a t i v e l y , b u t in c o n t r a d i c t i o n w i t h l i t e r a t u r e , t h e s e c h a i n s c o u l d r e p r e s e n t the h a l f m o l e c u l e (11S) of ct2-MG (18-19S, M r : 780,000 [27]) a n d t h e cons t i t t~ t ive p o l y p e p t i d e c h a i n s of t h e p r o - t e i n , as f o u n d f o r t h y r o g l o b u l i n . I n t h a t c a se t h e 190,000 w o u l d r e s u l t .of a p r o t e o l y t i c c l e a v a g e d i s p l a y e d o n l y a f t e r r e d u c t i o n .

Acknowledgements .

This work was supported by a grunt from the C.N.R.S. (L.A. ~93).

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BIOCHIMIE, 1980, 62, n ° 7.