Homologous Cysteine Proteinases of Pathogenic and Nonpathogenic ...

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ative agent of human amoebiasis. Half a billion people are infected, but only 10% of the infected individuals develop diseases such as hemorrhagic colitis or ...

T H EJOURNALOF BIOLOGICAL CHEMISTRY (0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 8, Issue of March 15, pp. 4798-4803,1991 Printed in U.S.A.

Homologous CysteineProteinases of Pathogenic and Nonpathogenic Entamoeba histolytica DIFFERENCESINSTRUCTURE

AND EXPRESSION* (Received for publication, November 5, 1990)

Egbert Tannichz, Henning Scholzes, Rose Nickel, and Rolf D. Horstmann From the Bernhard Nocht Institute forTropical Medicine, 2000 Hamburg 36 and the §University of Osnabriick, 4500 Osnabriick, Federal Republic of Germany

A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%.Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii)the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.

amoebae and suggest an important role for the enzymes in pathogenicity (14). To characterize the cysteine proteinasesof E. histolytica in greater detail,we have cloned the corresponding cDNAs from pathogenicandnonpathogenicforms using anantiserum raised against a 27-kDa enzyme purified previously (15, 16). We foundevidence for differencesin structure and expression between homologous cysteineproteinases frompathogenic and nonpathogenic E. histolytica. MATERIALS AND METHODS

E. histolytica Isolates and Culture Conditions-The E. histolytica iso1at.e HM-1:IMSS was cultivated in the medium TYI-S-33 in the absence of bacteria (axenically). The isolates SAW 142, SAW 755, SAW 760, SAW 891, SAW 1734, and SAW 1798 provided by P. Sargeaunt (London) were grown in TYI-S-33 or TYSGM-9 in the presence of a mixed bacterial flora (xenically) (17, 18). All isolates were characterized by three criteria as being pathogenic or nonpathogenic, i.e. by (i) the clinical histories of the infected individuals, (ii) isoenzyme classification (19), and (iii)genetic analysis (5). Construction of cDNA Libraries-TotalcellularRNAfrom E. histolytica HM-1:IMSS and SAW 1734 was isolated from exponentially growing trophozoites by extraction with 4 M guanidine isothiThe protozoan parasite Entamoeba histolytica is the caus- ocyanate, followed by centrifugation through a 5.7 M CsCl cushion (20). Subsequently, poly(A) RNA was purified bychromatography on ative agent of human amoebiasis. Half a billion people are oligo(dT)-cellulose (21). Double-stranded cDNA was prepared by the infected, but only 10% of the infectedindividuals develop method of Gubler and Hoffman (22). ThecDNAs were ligated with diseasessuch as hemorrhagic colitis or extraintestinal abEcoRI linkers into the bacteriophage vector h gtll and packaged in scesses (1).The discrepancy between prevelance and morbid- vitro. Recombinant phages were plated onEscherichia coli Y1090and ity may be due to theoccurrence of genetically distinct forms, further maintained asliquid stocks. Identification and Isolation of cDNA Clones-A rabbit antiserum nonpathogenic and potentially pathogenic E. histolytica (2- raised against a 27-kDa cysteine proteinase from E. histolytica HM5). Studies are in progress to determine which factors enable 1:IMSS (16) was used to screen approximately 2 X lo5recombinant pathogenic forms to destroy host tissues, i.e. to invade the phages of the HM-1:IMSS cDNA library (23). Duplicate filter lifts intestinal mucosa, enter theblood vessels, and disseminate to were analyzed with a 1:lOOO dilution of the antiserum. Horseradish peroxidase conjugated to anti-rabbit IgG antibody was used to visuother organs. Current data indicate that two functions of the amoebae alize posit,ive plaques with chloronaphthol. Positive clones were purified to homogeneity by serial dilutions, and thecDNA inserts were are predominantlyinvolved in tissue destruction:lysis of cells released from the recombinant phagesby EcoRI digests. by pore-forming proteins (6-8) and proteolytic activities, To isolate homologous clones from nonpathogenic E. histolytica, which may damage both thecells and the extracellular matrix approximately 1 X IO5recombinant phagesof a cDNA library derived of the host (9-12). Several reports have focused on the role of from the nonpathogenic isolate SAW 1734 were hybridized to the cysteine proteinases. Higher activities of the enzymes were radioactively labeled insert of a clone isolated from the HM-1:IMSS measured in pathogenic compared to nonpathogenic E. his- cDNA library (specific activity of 1 X 10’ cpm/pg) under low strinconditions (2 X SSC (1 X SSC: 0.15 M NaCI, 0.015 M sodium tolytica (13). Cysteine proteinases were found to abolish adhe- gency citrate, pH 7) a t 50 “C). Hybridizingphages were purified as described

sion of tissue culture cells and to degrade matrix proteins such as collagens, laminin, and fibronectin. These activities correlate well with the invasive properties of pathogenic

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely t.o indicate this fact. $ To whom correspondence should be addressed: Bernhard-NochtInstitut, Bernhard-Nocht-Str. 74,2000 Hamburg 36, Federal Republic of Germany.

above. DNA Sequencing-E. histolytica cDNA insertswere subcloned into the plasmidvector pBS or the phage vectors M13 mp18or M13 mp19 (24). Sequence analysis was performed by the dideoxy chain termination method (25).Sequence data were analyzed using the DNASIS program (Pharmacia LKB Biotechnology Inc.). Isolation of Genomic DNA and Southern Blot An.alysis-E. histolytica was harvested in late logarithmic growth phase by chilling on ice for 10 min andlow speed centrifugation at 4 ”C for 5 min. Nuclei were obtained from washed cell pellets by lysis in 1%Nonidet P-40 and centrifugation at 500 X g a t 4 ”C for 5 min. The nuclear pellet

4798

Proteinase Cysteine

of Entamoeba histolytica

was resuspended, and DNA was released by treatment with proteinase K (1 mg/ml) in a buffer containing 100 mM NaC1, 10 mM Tris, 10 mM EDTA, and 0.5% N-lauroylsarcosine at 60 "C for 2 h. The DNA was extracted twice with phenol/chloroform,1:l (v/v), and once with chloroform andwas precipitated with ethanol. High molecular weight genomic DNA was digested with different restriction endonucleases

4799

cEh-CPp, suggesting that the full-lengthcodingsequence comprises a few additional amino acids only. From the deduced amino acid sequence (Eh-CPp) itmay be predicted that its gene product is a preproenzyme containing a hydrophobic leader and an additional part of about 80 amino acids that under conditions recommended by the supplier (Boehringer Mann- seems to becleaved off to release the active, mature enzyme heim).The DNA was separated on agarose gels, transferred to a nylon of 222 amino acids. From themolecular weights of the amino membrane (Pall Biodyne A, 0.2 pm) and hybridized to the radioac- acids, the molecular weight of the mature enzyme was calcutively labeled probes (26). Probes were prepared by isolating the cDNA insert and labeling it by random priming with[32P]dCTPusing lated to be24.1 kDa. Sequence alignments indicatedhomologies to othercysteine Multiprime Labeling Kit (Amersham Corp.). Filters were hybridized at 42 "C for 4 h in 50% (v/v) formamide, 5 X Denhardt's solution proteinases such as papain (34%) (28) (Fig. 1) and chicken (27), 5 x SSPE (1 x SSPE: 0.18 M NaCI, 10 mM phosphate, pH 7.4, cathepsin L (40%) (29). The highest degree of homology was I mM EDTA),0.2% sodium dodecyl sulfate, denatured herring sperm found to thecysteine proteinase2 of Dictyostelium discoideum DNA (200 pg/ml). Hybridizationswere carried out in the same buffer (45%) (30)and to anothercysteine proteinase of E. histolytica at 42 "C overnight. The probes were usually appliedat a concentration (42%) (31)(Fig. 1). The percentage homology was calculated of 1-3 X lo6 cpm/ml with the specific radioactivity being 1 X 10' from the ratio of identical to total amino acids; the calculation cpm/pg. Filters were washed either at low stringency in 2 X SSC at was based upon optimal alignments, which were achieved by 50 "C or at high stringency in 0.1 X SSC at 65 "C. Northern Blot Analysis-Total cellular RNA from various isolates introducing gaps into the sequences. A number of residues of E. histolytica was isolated as described above. RNA (5 pg/lane) have been described to essentially contribute to the active site was separated by electrophoresisin formaldehyde-agarose gels, blotted onto a nylon membrane, and hybridizedto a 32P-labeled purified of cysteine proteinases (32); all of them were found in EhcDNA insert as described for Southern blot analysis. The 28-mer CPp at the predictedpositions. Isolation and Characterization of cDNA Clones Coding for oligonucleotidecEh-CP AS28 (5'-CATTTGATCCAAGTCCTCCATTACATCC) was synthesized on an Applied Biosystems DNAsynthe- the Homologous Cysteine Proteinase of Nonpathogenic E. hissizer and labeled by kinasing with y-ATP. The filter was hybridized tolytica-Approximately 100,000 recombinant phages of a in 2 X lo6 cpm/ml "P-labeled oligonucleotide, 6 X NET (1X NET: cDNA library constructed from thenonpathogenic isolate 0.15 M NaC1, 0.015 M Tris, pH 8.3, 1 mM EDTA) containing 0.5% Nonidet P-40, and 100 pg/ml yeast tRNA, at 40 "C overnight. The SAW 1734 were screened with cEh-CPp underrelaxed stringency. Three clones were detected. Sequence analysis of these filter was washed in 6 X SSC at 50 "C. Primer Extension-Anoligonucleotide(5"GTTGAAGATTGCcDNA clones indicated that theywere derived from the same TCTTCTTCGGAGTG) complementary to nucleotides94-119 of the mRNA species. The longest one, designated cEh-CPnp, was cDNA sequence cEh-CPp (Fig. 2) was synthesized on a DNA synthe- calculated tohave 84% homology to cEh-CPp innucleic acid sizer (Applied Biosystems) and labeledby kinasing with y-ATP. One picomole of the labeled primer was incubated together with 3 pg of sequences and 83% homology in the deduced amino acid poly(A) RNA derived fromHM-1:IMSS in100 pl of a 10 mM PIPES' sequences (Fig. 2). The amino acid substitutions were found buffer, pH 6.4, containing 80% formamide, 0.4 M NaC1,l mM EDTA, t o be distributed over the entire sequence, but all residues 1 mM dithiothreitol, and 25 units of RNAsin (Pharmacia LKB) at considered important for the activity of cysteine proteinases 42 "C for 16 h. After ethanol precipitation,the RNA/primer hybrids were conserved. In the parts representing the mature enzymes, were redissolved in 100 p1 of a 50 mM Tris-HC1buffer,pH 8.3, sequence homology was found to be85% compared with 75% containing 60 mM NaC1, 6 mM magnesium acetate, 10 mM dithiothreitol, 5 mM dNTP, and 25 units of RNAsin. Twenty units of M- calculated for the N-terminalregions. Southern Blotting-Genomic DNA from a pathogenic and MuLVreverse transcriptase (BoehringerMannheim) were added, and the sample was incubated at 42 "C for 3 h. After ethanol precip- a nonpathogenic isolate were digestedwithtwo restriction itation, the length of the extension product was determined by com- enzymes, and a Southern blotwas performed underlow strinparison to a knownDNAsequence after electrophoresis in a 6% gency conditions usingas a probe cEh-CPp, the cDNA derived polyacrylamide-urea gel. from pathogenicE. histolytica. Multiple hybridizing fragments were observed in the DNA digests of the pathogenic isolate. RESULTS In contrast, only one and two hybridizing fragments, respecIsolation and Characterization of cDNA Clones Coding for a tively, were found in DNA from the nonpathogenic isolate Cysteine Proteinase of Pathogenic E. histolytica-An anti- (Fig. 3A). When genomic DNA from five additional isolates, serum raised against the 27-kDa cysteine proteinase purified two pathogenic and three nonpathogenic ones, were hybridfrom E. histolytica HM-1:IMSS was used to screen a X g t l l ized under the same conditions, it became evident that the cDNA libraryderived fromthesame isolate; five out of pattern of hybridizing fragments was conserved within each 200,000 recombinant phages were found to react. Sequencing group of amoebae (Fig. 3B). Washing of the blot under high of the cDNAs and analysis of the deduced amino acid sestringency conditions selectively abolished the hybridization quences showed that one of them contained the stretch of with DNA from nonpathogenic isolates (data not shown). amino acids that had been determined to be the N terminus Analogous results were obtained when cEh-CPnp, theprobe of the 27-kDa enzyme of E. histolytica. This sequence was also the only one with overt homology to known cysteine derived from nonpathogenic E. histolytica, was used. Under proteinases. Rescreening of the library with this cDNA under low stringency conditions, it showed the same hybridization relaxed stringency revealed 28 cross-reacting clones out of patterns as cEh-CPp; under high stringency, it contrasted 100,000 recombinant phages. Cross-hybridization under low cEh-CPp in that it hybridized to DNA from nonpathogenic and high stringency conditions and subsequent sequencing of isolates only (data not shown). Taken together, these findings indicate that (i) the pathosome of them suggested that all were derived from the same genic isolates containmore genomic fragments hybridizing to mRNA species as the originalone. the probe than the nonpathogenic isolates, (ii) the genomic The longest cDNA clone, designatedcEh-CPp,didnot contain the complete coding region. Primer extension exper- organization is conserved within each group of amoebae, and iments revealed an extension product of 14 nucleotides 5' to (iii) none of the corresponding genes is found in both the pathogenic and the nonpathogenicE. histolytica. comparethe levels of mRNA The abbreviation used is: PIPES, 1,4-piperazinediethanesulfonic Northern Blotting-To acid. E. histolytica, expression in pathogenic and nonpathogenic

4800 FIG. 1. Amino acid sequence of a cysteine proteinase from pathogenic E. histolytica HM-1:IMSS (EhCPp) compared with another cysteine proteinase of E. histolytica (Eh-CPpcr) and to papain. The sequence of Eh-CPp is deduced from a cDNA clone isolated from an expression library of the pathogenic isolate HM1:IMSS. Identical residues are indicated by verticaldashes. Foroptimal alignments, gaps (. . .) were introduced into the sequences. Underlined are those residuesmatchingtheN-terminalamino acids determined by protein sequencing of the enzyme as it was purified from E. histolytica HM-1:IMSS. Residues known 140 AAGKDFQLYRGG..IFVGPCGNK to essentially contribute to the active sitePapain of cysteine proteinases areboned. aa nos., Eh-CPp amino acid numbers. aa nos. CEh-CPp

-90

-00

I

L

W

200

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F

Y

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G

Y

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-70

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D

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N

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C GC.....C.A...CT...................C...T....................T........AG.... L A . A N A A . . . . . . . . . . .

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-60 -50 -40 COMGMGAOCMTCTTCMCATOM~CMGM~~CAG~CMTAG~GAMCA~AM~ATCAGTAGAT00ACCA~

R Em-cmp

.FAKIKSYNRVARNNEVE.LKAAISQGLVDVSID

GTCAmTCATOTTTTATATXATAXiOOATTCAmCMTACA~TTDCCMTMCMTAMCACTTCACAGCAGTTOAGTCA~ V

cCh-CPp

120

100

80

A

R

R

A

I

F

N

W

N

A

R

I

V

A

E

N

N

R

K

E

T

F

K

L

S

V

.......... . . . .T........T........T...T.......T...~....A.....GTT.............T..T.....T...... . . . . . . . F . . . F . K . G S . . . . . .

G

P

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. . .

-10

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CEh-CPp

OCTaCTATOACAMTDMGMTATMTAGT~CTOAMCT~CGMG~~MG~ffiAGMG~AGATh~MTA~ A A W T N E E Y N S L L K L K R S G E E K G C V R Y L N I Q

cLh-CPnp

........... . . . .T ............. . . . . GA.C......T...TC....A...C..T.........T...A......C......A.....T... R T . . . S . . T V . . N . K . T . 1

. . . .

CEh-CPp

20 30 O C A C C C A M O C A G T A G A T T O O A G ~ ~ A M G T M C A C ~ ~ G A G A T ~ ~ M ~ ~ T C A T D ~ A T A C A ~ A ~ A P K A V D Y R K K G K V T P I R D Q G N C G S C Y T F G 8

Ch-CPnp

.....~.. C...........A........T......A.........CCC.A.....A..............C..T.. . . C T8....... . . . . . . . . . . .

cEh-CPp CEh-CPnp

Cch-CPp Cm-cPnp

CEh-CPp cm-cmp

CEh-CPp

FIG. 2. Sequence differences between homologous cysteine protein40 so 60 ases from pathogenic and nonpathA ~ O C A C T T O M ~ ~ ~ M G A T T T M T T O A G ~ ~ ~ ~ X ~ A T A G T D A G A C A C T T O A ~ ~ C A G M G M C A T A T ~ ~ ~ M ~ T ~ ~ ogenic E. histolytica. Shown are the I A A L E G R L L I E K G G D 8 E T L D L S C C H W V Q C T cDNA sequence and cDNA-derived C ............................... A.........A..OC.A.T........................C.............. amino acid sequence of the enzyme deL . . . . . . . . . . . . . N A N . . . . . . . . . . . . . rived from the pathogenic E. histolytica 70 80 90 (cEh-CPp). For thehomolA Q O O M G A T 0 0 ~ T M T 0 0 A m T M T 0 0 A ~ A C T T O O A T C A M HM-1:IMSS T ~ R E D G N N G C N G C L G S N V Y N Y I W E N G I A K E ~ D ogous enzymefrom the nonpathogenic E. histoolytica SAW 1734, only differing ..A TA.......C....................................G....C.....AC........G................. . D N . . . . . . . . . . . . . . O . . I Q . . V . . . . . nucleotides andamino acids arepresented (cEh-CPnp); conserved residues 100 110 110 TATCCATACACAffiMGTOA~CMCATOTAGMGTOATOTOAMOCA~~T~TCMGAGTTATMTCOAG~~~~T~T are indicated by dots. Numbering of Y P Y T G S D 8 T C R S D V K A F A K I K S Y N R V h R U N amino acids (aa nos.) relates to the N........ T..T. ...c.. ....T ..T....AG.C.A....T..G................CTO.A..C...M....C.......C.. c terminal amino acid determined by pro. . . . . T . . . . K T N . . . . . . . T G . . K . P . . . tein sequencing of the enzyme as it was 130 140 150 G M G ~ M C T T A M O C A O C M m C A C M ~ T ~ T T O A T D T ~ C M T T O A ~ A T C A T C m ~ M ~ C C A G ~ A T A C Mpurified G A G ~ A from E. histolytica HM-1:IMSS. ,

.

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160 170 180 O C A T A T A C A G A C A C A C M M C M G M T M ~ A ~ C A T T O M T C A T D M G ~ ~ m ~ A T A T 0 0 m ~ T O A ~ A M G M

A CEh-CPnp

Q

....c.. ....... . . .G ...... . . CL. ... . ... . ... . .. . ... . ... . ... . .. . ... . ... . ... ... . ... . .A. . .K. C. . A. . .. . ... . . A . . . . . ... . A. . ..T. . . .

Ch-CFQ

A

..

E

cm-cmp

10

Y

T

D

T

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C

K

N

N

Y

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N

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..... . .C.GT8 ...T ...TAK..... T......C.....TC.....F.T..........C.......C............................T........A........ 190

Y

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zoo

210

CSh-CPp

TOTTOOATAG~AGAMCTCA~MCAffiA~AGAGAM~ATATATCMCAT00~ATTOMffiAMTA~m~~~ C W I V R N 8 Y G T G W G E K G Y I N W V I E G N T C G V A

ash-cmp

........ . . .T..C. ................. . . . . . .T ........... . . . D................................................. . . . . . . . . . . . . . . . .

~Eh-Cpp

A C m A T C C A C T T T A K C M C T O ~ M T A T C T ~ M T A T 1 I A ) n T D P L Y P T G V E Y L .

CEh-cmp

.............. . . . . .C ............ . . . . C.......T...G..A......T...OCCACT.A....... 9 . . *

220

Northern blot analyses were performed. Total RNA fromfive different isolates were probed with the cDNA clones cEh-CPp and cEh-CPnp. Under high stringency conditions, cEh-CPp, the probe derived from pathogenic E. histolytica, exclusively

MlA)n

hybridized to RNA from pathogenic amoebae, whereas cEhCPnp only hybridized to RNA from nonpathogenic isolates (Fig. 4). Because of the known differences between the two cDNA sequences contributing to the intensities of the hybrid-

Proteinase Cysteine

of Entamoeba histolytica

4801 DISCUSSION

A variety of proteinases have been attributed toE. histolytica, several of whichhavebeen characterized as cysteine proteinases (10, 11, 12, 15). Three laboratories reported on the purification of such an enzyme from E. histolytica; the source was in each case the pathogenic isolate HM-1:IMSS. The purified enzymes resembled each other in substratespecificities and pH optima. Their molecular weights were estimated tobe 56, 27, and 26 kDa, respectively. The N-terminal amino acid sequences were determined for the 26- and the27kDa enzymes; they showed a high degree of similarity, i.e. 12 out of 15 residues were identical. The results presented here relate to the 27-kDa enzyme because (i) an antiserum raised against this protein was used wto isolatethe cDNAclones, (ii)the deduced amino acid sequence contained a stretch of residues identical with those Xmn I Xmnl Taql found by sequencing the27-kDa protein, and (iii) themolecular weight of the matureenzyme determined from the cDNAFIG. 3. Southern blot analyses of pathogenic ( p ) and nonpathogenic ( n p ) isolates of E. histolytica hybridized with the deduced amino acids was in sufficient agreementwith the one cDNA corresponding to a cysteine proteinase of the patho- obtained studying the purified protein. genic E. histolytica HM-1:IMSS (cEh-CPp).Genomic DNA was Recently,Eakin et al. (31) sequenceda genomic DNA digested with restriction enzymes as indicated. A, hybridization of fragment encoding approximately 150 amino acids of a cysDNA fromthe pathogenic isolate HM-1:IMSS and the nonpathogenic isolate SAW 1734, both digested with different enzymes. R, hybridi- teine proteinase from E. histolytica. The fragment was obtained by gene amplificationusingthe polymerase chain zation of DNA from seven different isolates, three pathogenic ones and four nonpathogenic ones, all digested with the same enzyme. reaction and generic probes as primers. The sequence data Both blotswere hybridized under low stringency conditions(2 X SSC, cannot be correlated with any of the purified enzymes because 50 "C). Size markers are indicated in base pairs. it does not include the N terminus of the encoded protein. The sequencediffers substantially from the one described v) sgzj ;? here. The degree of homology cannot be assessed adequately because the amplified gene fragment comprises less than 50% s z 3 8 3 2;: ;: $ 5 of the total proteinsequence. For the partsequenced, homolP P "P P nP ogy does not exceed 42%. Thus, it islower than thehomology l.l cEh-CP, between the enzyme described here and a cysteine proteinase -~ that discoideum from D.discoideum. It is interesting to note D. 1.1 cEh-CP,, was also found to contain two cysteine proteinases with a sequence homology of not higher than 43% (30). The reason 1.1 CEh-CP AS 28 for the existenceof a t least two such enzymes in D.discoideum and in E. histolytica is not known a t present. The clone presentedhere did not encodeafull-length 1.4- i ) I) 1)r) cEh-Actin protein sequence. However, it covers the entire sequence of FIG. 4. Northern blot analysis of pathogenic ( p ) and nonthe active proteinase and additionalregions characteristic of pathogenic ( n p ) isolates of E. histolytica. Five micrograms of a preproenzyme. Fromprimerextensionexperimentsand total RNA from the pathogenic isolates HM-l:IMSS, SAW755, and comparisonstorelated genes of other species, it may be SAW 891 and the nonpathogenic isolates SAW 1734 and SAW 760 concluded that theclone is lacking only a few base pairs. The were submitted to electrophoresis, blotted, and sequentially hybridized under high stringency with (i) cEh-CPp, thecDNA corresponding presence of a hydrophobic leader sequence indicates that the E. histolytica HM- enzyme is not cytosolic. From the sequence data alone, no t o acysteine proteinasefromthepathogenic conclusions canbe drawn as to whether it may be lysosomal/ 1:IMSS; (ii)cEh-CPnp,the homologous cDNA derived fromthe nonpathogenic E. histolytica SAW 1734; (iii) cEh-CPp AS28, a 28- vacuolar,associated withthe cell membrane,or secreted. mer anti-sense oligonucleotide probe representing a sequence identi- However, electron microscopic studies indicated that the encal in cEh-CPp and cEh-CPnp; and (iv) cEh-Actin, an actin cDNA zyme is located inside the vacuoles/lysosomes of the amoeof E. histolytica. Size markers are indicated in kilobase pairs. bae.' It should be noted that the protein sequence does not contain N-linked glycosylation sites. This isof interest since, ization signals, neither oneof the probes was suitedreliably to in animal cells, transport into lysosomes is mediated by a compare the quantities of specific mRNA expressedby patho- carbohydrate moiety, whereas plants and fungi use a carbogenic and nonpathogenic isolates. Therefore, a 28-mer anti- hydrate-independent mechanismfor transport into vacuoles, sense oligonucleotide probe was synthesized, which repre- their functional equivalentof lysosomes (32). The same structural motifs found in Eh-CPp were present sented a stretch of sequence identical in cEh-CPp and cEhCPnp (corresponding to aminoacid numbers 67-75 in Fig. 2) in the cDNA-deduced amino acid sequenceof the homologous enzyme from nonpathogenic E. histolytica. The overallseand thus was supposed to bind with equal affinity to the mRNAs of both pathogenic and nonpathogenic E. histolytica. quence homology between the clones derived from pathogenic was 84% on theDNA level and 83% Hybridization with this probe disclosed that pathogenic iso- and nonpathogenic forms on the protein level. In a previous report, we have described lates expressed the mRNA in much larger amounts than did nonpathogenic ones (Fig. 4). As a control, the blot was re- a similar degree of divergence between corresponding coding probed with a fragment of actin cDNA of E. histolytica, and sequences from the two forms of E. histolytica (5). In that the intensities of the hybridization signalsshowed that comU. Lohden-Bendinger, H. Scholze, C. A. Baigent, G. Muller, and parable quantitiesof total RNA were present in all samples. T. Bakker-Grunewald, manuscript in preparation.

8

-'a# "'

''-3

4802

Cysteine Proteinase of Entamoeba histolytica

FIG. 5. Approach to thedivergence in tertiary structure of the homologous cysteine proteinases of pathogenic and nonpathogenic E. histolytica. The figure is derived from the tertiary structure of papain with only the cu-carbonof each amino acid being shown (32). Solid circles, amino acid identities between papain and either one or both of the E. hktolytica proteinases; open circles, amino acid differences between papain and both E. histolytica proteinases; asterisks, amino acid differences between the two E. histolyticu proteinases; hatched burs, disulfide bonds in papain; shaded area, putative catalytic site of papain.

study, however, the clones had been selected solely on the basis of antigenic, e.g. structural differences between the two gene products, regardless of functional aspects. In thecase of cysteine proteinases, however, the gene product is considered a major virulence factor for the pathogenic forms, whereas it evidently lacks such a function in the nonpathogenic ones. Here, functionaldifferences would therefore be of great interest. So far, enzymological studies were limited to theproteinases from pathogenic isolates so that comparisons are lacking. The relationship between the structure and the function of cysteine proteinases has been studied in great detail withthe plant enzyme papain (28,33). X-ray crystallographyrevealed that theenzyme consists of two domains separated by a deep cleft. A number of residues facing the cleft were identified as being essential for the proteolytic function. All of them were found to be conserved in the enzymes from pathogenic and nonpathogenic E. histolytica. Moreover, alignments of the sequences with the one of papain presented in tertiary structure indicate that all amino acids forming and neighboring the putative catalytic site areconserved (Fig. 5). This finding suggests that the structural divergence of the homologous enzymes from pathogenic and nonpathogenic E. histolytica did not result in functionaldifferences. In contrast, the quantitativedifference in the expression of these two genes was striking. Northern blot analyses indicated that theexpression of the respective mRNA was consistently 10-100-fold higher in pathogenic than in nonpathogenic isolates. This estimate is in good agreementwithfunctional studies described previously (13,14). In light of these findings, recent data on the expression and sorting of the yeast proteinase A are of interest (34). This enzyme is transported into vacuoles. When it was expressed in excessive amounts, however, it was secreted into the medium, suggesting that the intracellular sorting system had been saturated. It may be hypothesized that, assuming that similar sorting mechanisms exist in E. histolytica, overexpression of the cysteine proteinase in pathogenic forms causes an overflow of the enzyme from vacuolar/lysosomal to secretory pathways. Thus, a quantitative difference in expression might result in a profound alteration of cellular properties, in this case the de nouo secretion of a proteolytic enzyme. It cannotbe excluded that

such an event is crucial for the expression of pathogenicity by E. histolytica. The regulatory basis for the difference in expression has not yet been examined. The Southern blots with DNA from the pathogenic isolates showed a more complex hybridization pattern than thosewith DNA from the nonpathogenic ones. The complexity could be due to intervening genomic sequences or to gene multiplicity. Sincenointerveningsequences have so far been found in the genome of pathogenic E. histolytica (35-37), the results of the Southern blots more likely suggest that the pathogenic amoebae contain multiple genes or pseudogenes corresponding to thecysteine proteinase or closely related proteins.Whether gene multiplicity contributes to theincreased expression remains to be determined. The difference in the genomic organization and itsconsistency within the groups of pathogenic and nonpathogenic E. histolytica again support the notion that the two groups are genetically distinct. Acknowledgments-We thank R. Huber and W. Bode for advice,

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