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erization of different in Trypanosoma br Guillemette Huet, Colette Richet, Dominique Domir D Daniel Tetaert, Kia Ki I~ Han ~

lytic activities tcei Ierv6 Bisiau, Benoit Degand

Unitd INSERM No. 16, Lil,

(Receive Received 30 May 1 (Revised manuscript received 2

Key words: Proteinase; Proteinas, Glycoprc

t)

:ei )

variant surface glyeoprotein of African trypanoson mnosomes is reh overnight incubation of p tariffed m n t l e a by Corn uoncanavalm A Se ~epha ~harose affinity chrorr ~H 5.5 phosphate glucose buffer and may be purifier addition of proteinase inhibitors during the para~ mrasite incubation is necessaryy to prevent the pr, released proteinases. Using this procedure without iant surface glycoprotein by the trypanosomal relea~ variant teinase inhibitors, the proteolytic activities, releas released from the bloodstream forms Trypanosom, proteinase iant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatq romatography. The unre! variant mthetic substrates Z-Phe-Arg-AM -AMC and Z-Arg-Arg-. (F1)) shows hydrolytic activity against the two synthetiq nulated by dithiothreitol, but not inhibited by E-~ E-64, and characterized by an a alkaline pH op stimulated imated molecular mass of 80-100 kDa. The Mi Michaelis constant for the substrates Z.Argestimated 'he-Arg-AMC was, respectively, 2.8 and 6.7 p M . IThe retained material eluted elute by addition of ] Z-Phe-Ar nnopyranoside (F2) shows hydrolytic activity again ainst the synthetic substrate Z-Phe-Arg-AMC, v mann0 fibited by E-64, active between pH 6.0 and 8.0, and could cou be separated inl lated by dithiothreitol, inhibited )eak of lower molecul~ activity by HPLC, one peakk of high molecular mass ( > 70 kDa) and the other pea] on contains several kDa). By electrophoresis in gels containing gelatin as substrate, this fractior 'eas the unretained fraction F1 did not have any gelatin gelatinolytic activity, whereas gelatinolytie activity.

Introduction ~ied according to analogous Proteinases are classified ities to a group of specific mechanisms and sensitivities inhibitors [2]. Parasite protetnase ,teinases EIses have been shown to

Trypanozoon antigenic type; VSG, Abbreviations: AnTat, Autwerpenn Try I )S-PAGE, sodium dodecyl sulfatevariant surface glycoprotein; SDS-PAGF sis; Con-A, concanavalin-A; TLCK, polyacrylamide gel electrophoresis; N-a-p-tosyl-L-lysine-chloromethyl-ketone; Z-Arg-Arg-AMC, N-~benzyloxycarbonyI-L-arginyl-L-argiginine-7-amido-4-methyl coumarin; Z-Phe-Arg-AMC, N-be nzyloxycarbonyl-t :arbonyI-L-phenylalanyl-L-arginine-7amido-4-methyl coumarin; E-64,, L-trans-epoxysuccinyl-leucylamidohiothreitol; M~, molecular weight; (4-guanidino)-butane; DTT, dithiothreit( :etate; Dl DFP, diisopropyl fluorophosEDTA, ethylene diamine tetraacetate; phate; GP-HPLC, gel permeation-high matography; IE-HPLC, ionic exchange chromatography.

play an important role rol in survival of the host and in pathog~ )athogenicity of parasitic viewed in Ref. 3). As / examples, a m~ proteinase is require~ uired for hemoglobin the food vacuole of Plasmodium falc~ zoites [4], and the major proteinase histolytica is involved in the cytopathic lent trophozoites [5]. Trypanosomes arc group of parasites due d~ to their potent pathophysiological mechanisms m of prot and therefore the pr )roteolytic enzymes sites have come under und increasing scrut last decade. Cazzulo et q al. [6,7] have ch~ acid sequence t provided partial ammo amil cysteine oroteinase from epimastig i. The enzyme is a mon, aowing homology in its n and cathepsin L. oma brucei brucei Lonsd

n g a Mr of 27 kDa by ike activity. Also, Mothe e D N A sequence of a hat shows a high degree Moreover, their study lusual C-terminal extenddition, Ashall [10] has alkaline cysteine peptie of molecular mass > 200 kDa in Trypanosorna zi and other trypanosomatids including T.b. brucei Ne have previously isolated the VSG forms from variant AnTat 1.1 of T. b. brucei [11] using a htly modified procedure described by Baltz et al. Release of VSG was obtained by overnight incuba1 of trypanosomes at 4°C in a phosphate glucose fer (pH 5.5) in the presence of proteinase initors, and the VSG was isolated by Con-A Sepharose omatography. The addition of proteinase inhibitors ing parasite suspension was necessary to prevent degradation of the VSG forms by the trypanoso[-released proteinases. We have previously studied proteolytic activities released when the same procedure'e was applied to the trypanosomes, without prolase inhibitors [12]. In this study we have characteinase zed the released proteinases using Con-A Sepharose terized chromatography and HPLC. Materials and Methods

Gelatin, DTT, leupeptin, T L C K and E-64 were ducts of Sigma, U.S.A. The synthetic substrates products Arg-Arg-AMC and all the Z - P h e - A r g - A M C and Z-Ar ed peptides were obtained methylcoumarin derivatized d. Acrylamide for P A G E of from Bachem, Switzerland. "chased from BDH, U.S.A. analytical grade was purchas Con-A Sepharose was a product of Pharmacia, Sweden. Source of Trypanosoma brucei brucei. Stabilized tryed A n T a t 1.1 of T. b. brucei panosomes from the cloned sor were provided by Professor N. Van Meirvenne (Incine, Antwerpen, Belgium). stitute of Tropical Medicine. The parasites were grown in Sprague-Dawley rats and isolated from the infected animals as described [13]. ~nes. A n T a t 1.1 trypanosomes Incubation of trypanosomes. were suspended and incuubated overnight at 4°C in 0.125 M sodium phosphate:e buffer (pH 5.5) containing Lrasite suspension was cen1% glucose [1]. The parasite trifuged (3000 X g for 15 min). The supernatant was ltrifugation (165 000 × g for 1 then submitted to ultracentrifu h). ~atography. The material obCon-A Sepharose chromate tained in the previous steep was submitted to affinity chromatography on Con-A Sepk brated with 0.01 M phosphate bu ing 0.5 M NaC1, 1 mM CaCl 2

Sep met hau cha t sea~ usir AM pro det{ tio~ and pies lyo[ M,,

lmn after buffer enrichl anopyranoside. The frac ~zed and lyophilyzed befl FI.

the fluorogenic substrate

thiol proteolytic activity i~ rates Z-Phe-Arg-AMC a proteinase activator DT" bitor E-64. The hydrolytic ~'ixed incubation time und sence of DTT, in the pre sence of D T T plus E-64 f lyophilized fraction FI •tion F2) were diluted in sphate bufl'er or by Britt lsted to different pH vah son ~. After the addition of 1 2.5 (w/ the mixture was preincu a 125 /,tl of 400 # M sub for Brij ¢as added and the incuba at 37°C for 10 rain. The reaction was addition of 1 vol. of a solution of 0.1 acid/0.1 M sodium a, acetate. The release was measured with e: excitation at 380 nn at 480 nm on a spec )ectrofluorometer us methyl-coumarin for calibration. The a pressed in m U / l (1 mU of activity corr release of 1 nmol oof amino methyl-o The extent of hydroly tic reactions was a the presence of D T T (final concentratic and in the presence of 1.25 mM D T T 1: concentration 2 /~M or 1 raM). Diffe were also tested for ttheir effect upon th Z-Phe-Arg-AMC and Z-~ the substrates Z-Pheleupeptin (0.1 raM), T L C K (0.1 raM) #M). The fractions F FI and F2 were th against other synth hydrolytic activity a~ conditions. using the same condil Gelatin substrate SDS-PAGE. Gel were carried out using S D S - P A G E [15] wer( ent polyacrylamide gels (Laemmli 19 0.1% gelatin• After migration, gels w X-100, and then incl 2.5% ( v / v ) Triton Xsodium phosphate 1: for 16 h in 0.l M sc hiothreitol. Gels were sta with 1 mM dithiothre omassie brilliant blue R-~ 0.3% ( w / v ) Coomassi 3 6 / 8 / 7 6 , v / v / v ) a~ acetic a c i d / w a t e r ((3( cid/water (20/7/73, m e t h a n o l / a c e t i c aacid Hydrolysis assay af~".ter SDS-PAGE. Ea ;DS-PAGE according to submitted to SDS-P/ electrophoresi,, of Laemmli [16]. After Af ( v / v ) Triton X-100 and wizontal strips. Each strip 1 h, at 37°C in 0.1 M ph ! 8.3) containing 2 mM nM

7-Phe-Ar~-AMC

¢~r [

scence was measured as mino-4-methyi-coumarin

nance liquid chromatog)rmed on a column ul× 300 mm) (Beckman) ograph. The system was tilibrated in 0.1 M sodium phosphate, 1 M sodium 3ride buffer (pH 5.8) at a flow rate of 0.4 ml/min. ~ounts of 500 tzg of fraction FI and 1 mg of fraction were applied to the column, and eluates were lected in 0.4 ml fractions. The enzymatic analyses "e carried out immediately after the fractions were lected, using samples of 50 Ix l according to the tditions previously described. The column calibra1 was obtained using phosphorylase b (M~ 94 000), ,ine serum albumin (M~ 67 000), ovalbumin (M~ 43 0, carbonic anhydrase (M~ 30 000), soybean trypsin ibitor (M, 20 000) and a lactalbumin (M~ 14 000) molecular weight calibration.

tonic exchange-high performance liquid chromatog-

t t]l

ttt~

raphy. hy. Pooled fractions from the previous step were diablyzed overnight, concentrated and applied to a columnn AX-300 (4.6 × 30 mm) (Touzart and Matignon). The system was equilibrated in 0.01 M potassium phos-

pha Elu nM ml fort the pre phc ED AI~ was

in 1 for at 1 mlV tai~ SE

pH 8.0) at a flow rate c rried out with a gradien ae buffer. Eluates were c nd the enzymatic analy iously described using say es. Enzyme from fraction steps (GP-HPLC and I 'or 5 min at 30°C in 0 !er containing 1.25 mM I 25% Brij-35. The substra a.rg-AMC or Tosyl-Gly:1 and the reaction was fo Fluorometer. The reactio in. Initial velocities (t,) w( bstrate concentrations [S] ~M. Michae!is constants )ts of l~/[S] against t~, usi]

Res

The fractions F1 and F2, obtainec Sepharose chromato~ 3graphy of the supe centrifuged parasite suspension, wer( Coomassie blue stain stained SDS-PAGE wil previous treatment by 2-mercaptoeth

A KD

B i

94

o r

67 r,

43 30

i

20.1 14.4

iii!:ii

S

S

1

ions. S, standard proteins with

:

y heterogeneous set of contained several pro105 kDa without reduct an approximate molecer reduction. The visible were identified by imntiserum raised against } and corresponded to tct or proteolysed A n T a t 1.1 VSG. I'he proteinases contained in the fractions F1 and were analysed by gelatin substrate S D S - P A G E (Fig. 50 p.g of each lyophilyzed fraction was used. Using "tion F1, gelatin digestion was very faint. Using :tion F2, gelatin digestion revealed several bands, h intense destaining between 28 and 40 kDa. =ractions F1 and F2 were examined for their hydro= activity against both the substrates Z-Phe-ArgIC and Z - A r g - A r g - A M C in the presence of D T T at values ranging from 3 to 10. The activities at the ious pH values of fraction F1 against the two syntic substrates (e.g. = Z - P h e - A r g - A M C and Z-Arg;-AMC) showed a similar profile: Fig. 3A shows the profile file obtained with the substrate Z-Arg-Arg-AMC. The p H optimum was observed around pH 8.0. Fractiont F2 showed a higher hydrolytic activity against the substrate strate Z - P h e - A r g - A M C in comparison to the subae Z-Arg-Arg-AMC. Fig. 3B demonstrates the enstrat,

kDa

94 67 43 30

20.1 14.4

S Fig. 2. Detection of enzymatic activities

F 1

-~ t~

= E sE 2 o

--~" >

N uJ

I

I

I

4

6

8

1

F2 c



.=_ E o .o

g

.-/ lO

= us

0 2

I

I

I

4

6

8

Fig. 3. Hydrolytic activity of the fractions measm Robinson buffer in the pre ~resence of DTT and EDq values ranging from pH 3 t~ to t0. (A) Hydrolytic acti FI against the substrate Z Z-Arg-Arg-AMC; (B) hy ainst the substrate Z-Phe the fraction F2 agai

various p H values of frac zyme activity at variou '~-Phe-Arg-AMC. A brom the substrate Z-Phe-~ etween p H 6.0 and 8.0. was observed betweer For each fraction, we have examined the thiol proteinase activator D T T (] inhibitor E-64 (2 /xl~ • M) upon the hydr~ ydrolytic activities of fra (Table I). The hydrol3 ainst both substrates ined at p H 8.3 agains similarly by D T T (rr mean fold-stimulati didn't inhibit enzyme the inhibitor E-64 did either substrate. In ffraction F2, Z-Phe was more highly acti drolysis at p H 8.3 w~ nulation = 7.7), and sign (mean fold-stimulatic ited by inhibitor E-64 (70%); Z-Phe-Arg. stimulated 5.5-fold by sis at p H 6.0 was stim D T F and was strongl gly inhibited by the (92%). rmine the enzymati In order to deterr the fractions F1 and ]F2, different inhibi nd 1 mM), leupeptin (0. (12 /xM). Z-Arg-Arg-A tion FI could be signific ~, 0.1 mM T L C K and 0.1 ~nmo

nh~or'vntic~n ,oun~

m

ydrolytic activities o f fractions FI and F2 ivity DTT g D.

Hydrolytic activity in presence of D T T p . m o l m i n ~g fraction Con A i -+S.D.

Hy~ in I +[ mir Col

, T

D T T activating factor a + S.D.

[ i r

1

7tton 1~l he-Arg-AMC ,ssay at pH 8.3 ~rg-Arg-AMC ,ssay at pH 8.3

410_+ 16

1190+ 58

10z

2.9_+0.4

440 + 25

1187_+116

11~

2.7+0.6

~tion F2 he-Arg-AMC ,ssay at pH 8.3 he-Arg-AMC ,ssay at pH 6.0

8-+ 1

63 +

7

7.7_+0.3

9-+ 1

49+

2

5.1 _+0.6

he D T T activating factor represents the multiplying factor upon up~ the hyd fference to the hydrolytic activity measured without DTT. he E-64 (2 /zM) inhibition percentage represents the percentage of the hydl =e addition of E-64.

T A B L E II

~ct of inhibitors upon the hydrolytic activity of fractions F1 andt F2 Effect Inhibitor

Z-Arg-Arg-AMC hydrolytic activity of fraction F1 at pH 8.3

Z-Phe-Arg-AMC hydrolytic activity of fraction F2 at pH 8.3

at pH 6.0

DFP 0.012 m M T L C K 0.1 m M Leupeptin 0.1 m M E-64 0.002 m M E-64 1 m M

99% 88% 98% 2% 38%

O% 79% 92% 70% 89%

0% 92% 92% 89% 93%

Arg-AMC hydrolytic activit~ ivity of this fraction. Z-Phevity of the fraction F2 at pH Arg-AMC hydrolytic activtt nificantly 6.0 (Table II) could be significant antly inhibited by E-64 at both concentrations, 0.1 mM TLCK and 0.1 mM leupeptin. At pH 8.3, Z-Phe-Ar -Arg-AMC hydrolytic activity

f

generated by the addition of measured in presence of DTT,

by 0.1 mM ! was also strongly inhibited inhi inhibited appr( mM E-64. 2 /zM E-64 Eactivity. At pH 7.0, 0.1 0. mM TLCK inhibi 9ition was obtained at activity. No inhibitio~ 8.3 with 12 /zM DFP. DFP We have extended the hydrolytic ass~ in the prese synthetic peptide substrates sub (Table III). F proteinase activator DTT l different su able to hydrolyse several se carboxyl side of an arginyl al residue and at the carboxyl side of a lysyl residue. primarily hy~ pH 6.0 and 8.3, showed shc against the substratees Z-Phe-Arg-AM£ Arg-AMC. in the fract The enzymatic activities act lyzed from gel strips incubated with an substrate (e.g. = Z-Arg-Arg-AMC Z-~ that the appT AMC). We could estimate esti

T A B L E III

Hydrolytic activities of fractions F1 a n d F 2 ~ Substrate

~ m o l m i n ~gF1 Con A ~ _-t-S.D. assay at pH 8.3

/~molmin l g F 2 Con A - J + S.D. assay at pH 8.3

/xmol rain 1 g F2 Con A - i _+S. assay at pH 6.0

Z-Gly-GIy-Arg-AMC Bz-Arg-AMC Pro-Phe-Arg-AMC Z-Arg-Arg-AMC Z-Phe-Arg-AMC Tosyl-Gly-Pro-Arg-AMC

2587_+ 75 1704_+ 84

/ /

/ /

Tc~vl -GIv-Prt~-I

'u~- A ~ l C '

45_+2

/ 49_+2

/ /

> _u o

fl

~xx

i

= 4.'~" 10

\

20 retention tlme (rain)

RR pH 8.3 D'l-r FR pH 8.3 DTT

2O 0 .

.

.

~ .

.

17"A . . .

0,5 1 1,5 2 2,5

.

.

.

.

.

.

.

.

.

.

rT] . .

.

.

.

.

_::J_ 30

~ -

FR p FR p

.

3,5 4 4,5 5 5,5 6 6,5 7 7,5 8 8,5 9 9,510

distance

migrated ( c m )

B 120 80-100 77 /j

100

Z 7 ,7~

80

// vJ

-

Kda

/

z; 40

/I

0:

o

z1 t ; / .

.

.

.

0

20

10

30

RR pH8.3 D'l-r

.

.

.

.

.

.

.

.

.

.

0 , 5 1 1 , 5 2 2 , 5 3 3 , 5 4 4 , 5 S S,5 6 6 , 5 7 7,S 8 8 , 5 9 9 , 5 1 0

distance

-'~

retention time (mln)

/

.

/"\~-

migrated ( c m )

Fig. 4. Hydrolysis assays of the fraction F1 after migration by SDS-PAGE. (A) Incubation at pH H 8.3 with the substrate Z-Arg-ArgAMC; (B) incubation at pH 8.3 with ~¢ith the substrate Z-Phe-Arg-AMC.

ent M~ of both Z-Phe-Arg;-AMC and Z-Arg-Arg-AMC nt in fraction F1, were behydrolytic activities present tween 80 and 100 kDa (Fiig. 4). In fraction F2, Z-Phe'ity at pH 6.0 and at pH 8.3 Arg-AMC hydrolytic activit' ka appeared diffusely through the gel (data not shown), )f fraction The hydrolytic activity of fracti ction F1 was purified by (Fig. 5). By GP-HPLC, ZGP-HPLC and IE-HPLC -Arg-AMC hydrolytic activity Phe-Arg-AMC and Z-Arg-Arg on F1 gave a single peak of profile at pH 8.3 of fraction activity showing a maximum ~m at a retention time of 16 rain, which was insensitive to E-64 (2 ~ M ) and sensitive to DFP (12 /zM). The pooled fractions containing the hydrolytic activity were re further purified by anion exchange chromatography. The hydrolytic activity eluted as a single peak of ~f activity and the fractions tctivity were pooled and concontaining the hydrolytic activit, es performed with the puricentrated. Inhibitor studies fied fraction showed that the h completely inhibited by DFP (12 mM) and T L C K (0.1 mM), but w~

Fig. 5. (A) Analysis of Z-Phe-Arg-AMC Z-P[ and Z-Ar~ lytic activities in presence of DTT after GP-HPL of fraction F1. (B) Analysis csis of Z-Arg-Arg-AMC h! presence of DTT after IE-HPLC I chromatogral isolated from step pooled fractions frac

for different substrat abstrates. The K m value strates Z-Phe-Arg-A~ g-AMC, Z-Arg-Arg-A~ respectively, Gly-Pro-Lys-AMC were w~ #M. In order to identif2y the different pro1 lysing the substrate Z-Phe-Arg-AMC i the hydrolytic activityy against that subs1 ysed after GP-HPLC (Fig. 6). Z-Phe-Ar lytic activity of fraction fractic F2 at pH 8.3 wa 200oo

~ ~ E

,/

~oooo 15000 5000

_ ......... 10

20 30 retention time (mln)

FR pH 8,3 DTT

4 RR p

Z-Phe-Arg-AMC and Z-Arg-A ace of DTT after GP-HPLC c

~pective retention times owing a maximum at a aad significant, but less E-Arg-Arg-AMC at pH rg-Arg-AMC hydrolytic ae-Arg-AMC hydrolytic

2.0

6O00

A

A

5oo0

E 1.0 ~

3O00

2000 1000 10

20

30

retention time



FR pH 8.3 DTr FR pH 8.3 DTT+E-64

~o

40

(mln) •

Discussion

FR pH 6.0 D'I-I"

FR pH 6.0 DTT+E-64

2.0

~o

4000

v

3000

o

2000 1000

o 1.0 ~\ 1 l

|

0 0

\ 10

30

20 retention

40

50

time (rain)



FR pH 8.3 DTT FR pH 8.3 DTT+E-64

FR pH 6.0 DTT

FR pH 6.0 DTr+E-64

6000

2.o

C

5000 4000

1.o

3000

20001000 ~i~\ lt ~tt\ ,

/ ~ t / ~~

:

I0

20 retention

~,d to be shared between erefore this profile was (data not shown). Cons )lytic activities were sep~ high molecular mass ( > ecular mass (30-70 kDa alyzed by anion-exchan~ and B). The activity at showed a main peak wi! a time 21 min with a st t 6 the activity includes ,~r (at 18 min) (Fig. 7A). F rolysis activity profile a he one at pH 8.3 (Fig. 713 ytic activity were signific; 4) except, Z-Arg-Arg-A] .3 (Fig. 7C) in the low M, single peak of activity th;

50

B

>,

activ note one grou HPI one was raph high at tl mini anot grou diffc peal~ by I acti~ elutl tive

-

]

30

t

1

40

~0

time (rain)

RR pH 8.3 DTT

Fig. 7. Analysis of Z - P h e - A r g - A M C and activities in presence of D T T after IE-H high M r group of proteinase activity is substrate Z-Phe-Arg-AMC. The low M.

RR pH 8.3 D'I-I'+E-64

1.1 of T. b. b From the variant AnTat A pro separated different thiol-dependent tl: )harose chromatograph tics by Con-A Sephar~ characterized them in respect to their h' ity against different st;ubstrates, their sen ous inhibitors, their behaviour by SDSHPLC. These proteolytic activities were overnight incubation of AnTat 1.1 tr} and were ol phosphate glucose buffer, bt supernatant of the centrifuged parasi This supernatant has been previously sh, stimulated by ] two proteolytic activities activit only one is sensitive tto 2 / x M E-64 [12]. of a cathepsin L-like activity has been brucei-brucei [8], and since cathepsin L J Sepharose columns retained by Con-A S ed the supernatal therefore submitted Sepharose chromatogIraphy. fraction was chara( The unretained fr hydrolytic activity ag;ainst N-blocked pt carboxyl side of argi ginyl or lysyl residt substrates Z-Arg-Arg -AMC and Z-Phe. proteolytic activity is stir show that the proteo reducing agent DTT,, is characterized pH optimum (around pH 8.0) and is sigr ited by TLCK, leuppeptin and DFP. ' activity against the tw, two substrates (i.e. Zand Z-Ar~-Ar~,-AM( g-AMC) migrated simil g an approximate mole( 00 kDa. Kinetic studies ! :d by G P - H P L C and IE ; Michaelis constants for

6.7, 2.8 and 7 /.~M. This sualized on gelatin subecent papers have deeith some similar characlkaline serine proteinase residues in the primary by Lonsdale-Eccles and ty was inhibited by DFP, CK, leupeptin, HgCI 2 and p - c h l o r o m e r c u r i lzoate. The high molecular mass ( > 200 kDa) alkacysteine peptidase described by Ashall [10] in T. zi and other trypanosomatids, shows similarities in ard to substrate specificity (cleavage of bonds on carboxyl side of arginine and to a lesser extent ne residues, a Michaelis constant of about 1 # M for krg-Arg-AMC [17], and a lack of detection by elecphoresis in gelatin containing P A G E ) and inhibitor sitivity (strong inhibition by TLCK, leupeptin, ZJ-Lys-CHN2 and D F P [17]). In the amastigotes of shmania mexicana mexicana a group of proteinases higher molecular weight compared to three other ups of cysteine proteinases at around 30 kDa described bed in this paper, is characterized by a high activity toward ,ards fluorogenic substrates in particular Z-ArgArg-g-AMC and Z-GIy-Gly-Arg-AMC. One of these proteinases lases, which is highly active against the fluorogenic substrates strates, has very low activity against gelatin [18]. Am{long the two groups of T. b. brucei enzymes described bed by Robertson et al. [19], the group of higher molecular lecular weight, comprising three enzymes, shows high activity against the substrate bstrate Z-Gly-Gly-Arg-AMC, is insensitive to diazomethanes runes, E-64 and cystatin, and is sensitive to PMSF and leupeptin. The authors have suggested that these enz3lmes are of the serine type [19]. The retained fraction F2 ~2 was found to have hydrolytic activity primarily ag~ainst the substrates Z-PheA r g - A M C and Pro-Phe-Arrg-AMC. After separation by SDS-PAGE, Z - P h e - A r g - AkMC M C hydrolytic activity appeared at multiple molecular llar weights. Also, on gelatin substrate SDS-PAGE, several eral proteinases were visualized, in particular a 28 kDa Da protein and a protein of higher molecular mass (around round 40 kDa). Z-Phe-ArgA M C hydrolytic activity ofF fractio fraction F2 showed a broad p H optimum between p H 6.0 and 8.0. The hydrolytic activity was significantly inhibited by the thiol proteinase inhibitors such as E-64 (lmM), leupeptin (0.1 mM), but was not inhibited ted by D F P (12 /zM). The various characteristics of this :his fraction suggest that this fraction contains several cathe athepsin-like activities. Lonsdale-Eccles and Grab [8] have previously found a predominant cathepsin L-like activit,. ganelles having a p H optimum a lytic activity of fraction F2 at

obt feral fractions. Fraction I hyd vity at pH 8.3 and sew gra Jere used in order to def in J ) the others. This activit! fro~ faction Fl: it was sensitix ins¢ DFP and it did not cot acti t the substrate Z-Arg-A acti htained in several fracti chr, y. The I E - H P L C activity 8.3 ~erimpose on the HPLC at ing that the activities at pH ~me from different enzyrr to ~ydrolytic activity against Z-F IC, fraction F2 showed h ity Arg-Arg-AMC that was E-6 ary, using G P - H P L C ant ideJ action F2 several cathepsi ties d differ (i) in M r (ii) in i,, and imal pH (acidic or alkali Ar~ :olytic activity. The low h ity detected against the tt substrate Z-Arg not appear to belong to an enzyme of th, The existence of a group gr~ of several cyst( resembling (probably four in number) nui nhibitor specificities in 7 substrate and inhibit( also been described by Robertson et to the previous, SDS-PAGE: in addition additi kDa enzyme [8], three other enzymes we molecular weights using t slightly higher molecu enzyme activity with fluorogenic substr~ SDS-PAG PAGE. By gelatin substrate su enzyme was detected whereas the three displayed low activit!y or no activity t~ [19]. The enzymes could cou come from the ~, post-transcriptional modification t or c, scribed from differer lifferent members of the ;enes [19]. The trypan~ teine proteinase gene contains more than 20 copies of cyste genes [9]. Several cathepsin L-like proteolytic the Con-A Sep therefore, coeluted from fr with the VSG and VSG V5 fragments prod ~roteinases from some olysis. Cysteine prot¢ characterized by a tbinding to Con-A cysteine proteinase of o T. cruzi is a gl} binds to Con-A Seph ~harose [6]. The am m. rnexicana contain :ontam three groups of teinases with molecular molect masses aroun enzymes which one group comprises co to Con-A Sel: characterized by binding binc [18]. m, we characterize seve b. brucei. The Con-A S~ pears to be a very user Lwo major groups of tryl

81_ 1

catis for r e a d i n g of the Iathon and C l a u d e V a n , and Fran~oise Roussez ?'his w o r k was partially of La Fondation pour la • 'erences 3altz, T., Baltz, D. and Pautrizel, R. (1976) Ann. Immunol. Institut Pasteur) 127C, 761-774. ~Iartlcy, B.S. (1960) Annu. Rev. Biochem. 29, 45-72. VicKerrow, J.H. (1989) Exp. Parasitol. 68, 111-115. Rosenthal, P.J., McKcrrow, J•H., Aikawa, M., Nagasawa, H. and Leech, J.H. (1988)J. Clin. Invest. 82, 1560-1566. Keene, W.E., Petitt, M.G., Allen, S. and McKerrow, J.H. (1986) I. Exp. Med. 163, 536-549. ~azzulo, J.J., Couso, R., Raimondi, A., Wernstedt, C. and Hellnan, U. (1989) Mol. Biochem. Parasitol• 33, 33-42. ~azzulo, J.J., Hellman, U. Couso, R. and Parodi, A.J.A. (199(I) Viol. Biochem. Parasitol. 38, 41-48.

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s, J.D. and Grab, D.J. (1987) North, M.J., Barry, J.D. and Cc ,211-215. )) Mol. Biochem. Parasitol. 38, Hublart, M., Gomes, V., Mere Jet-Duvillier, G. and Degand 1: Huet, G., Demeyer D., Richet, Biophys. Acta 1035, 369-377. and Godfrey, D.G. (1970) F nd Kirschke, H, (1981) Meth d Dowdle, E.B. (1980) Anal. E (1970) Nature 227, 680-685. ealy, N., Shaw, E. and Ashall, ] 865-866. ). and Coombs, G.H. (1990) M~ 76. 3 , North, M.J., Lockwood, [ Gen. Microbiol. 136, 921-925.

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucos...
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