Cloning and expression of chitinases of Entamoebae

E L S E V I E R Molecular and Biochemical Parasitology 85 (1997) 139-147

MOLF_EULAR AND BIOCHEMICAL PARASITOLOGY

Cloning and expression of chitinases of E n t a m o e b a e 1

Humberto de la Vega ~, Charles A. Specht ~, Carlos E. Semino ", Phillips W. Robbins a, Daniel Eichinger b, Daniel Caplivski c, Sudip Ghosh c,

John Samuelson c,,

a CenterJor Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA b Department of Medical and Molecular Parasitology, New York University Medical Center, New York, USA

c Department of Tropical Public" Health, Harvard School (~[" Public" Health, 665 Huntington Avenue, Boston, MA 02115, USA

Received 12 September 1996; accepted 3 December 1996

Abstract

Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cyst formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht l ) predicts a 507-amino acid (aa) enzyme, which has 93 and 74°/,, positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Munduca sexta (29%). Recombinant Entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h. © 1997 Elsevier Science B.V.

Keywords : Entamoeba histolytica ; Entamoeba dispar ; Entamoeba invadens ; Cysts; Chitinase; Hydrophilic repeats

Abbreviations: 4MU-(GIcNAc)3, 4-methylumbelliferyl fl-D-N,N',N"-triacetylchitotriose; aa, amino acid(s); Ed, Entamoeba dispar; Eh, Entamoeba histolytica; Ei, Entamoeba invadens; Eh chtl, Eh chitinase 1 gene; GlcNAc, N-acetylglucosamine; nt, nucelotide; ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reverse-transcriptase PCR; SREHP, Ser-rich Eh protein; TLC, thin layer chromatography; UTR, untranslated region.

* Corresponding author. Tel.: + 1 617 4324671; fax: + 1 617 7384914; e-mail: [email protected] Note: Nucleotide sequence data reported in this paper are available from the GenBank T M data base with the accession numbers

U78318, U78319, and U78320.

I)166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S01 66-6851 (96)028 1 7-4

140 H. de la Vega et al. / Molecular and Biochemical Parasitology 85 (1997) 139 147

1. Introduction

Biochemical, immunological, and molecular biological techniques have been used to distinguish trophozoites of Entamoeba histolytica (Eh ), which are associated with dysentery, from those of Enta- moeba dispar (Ed), which are not [1]. Within the colonic lumen, trophozoites of both Eh and Ed transform into cysts, which are the infectious form of parasite because chitin-walled cysts are resistant to stomach acids [2-4]. As Eh does not encyst easily in axenic culture, cyst formation has been studied using the reptilian pathogen Ei [5]. During Entamoeba invadens (Ei ) cyst formation, chitin synthase and chitinase are both expressed, and cyst formation is inhibited by allosamidin, a substrate analog inhibitor of chitinases isolated from Streptomyces sp. [6,7].

Chitinases are glycohydrolases that degrade the fl(1, 4) linkage between N-acetylglucosamine residues of chitin [8-21]. Exochitinases remove diacetylchitobiose from the nonreducing end of the chitin fibril, while endochitinases release small multimers of N-acetylglucosamine from internal cleavage sites distributed randomly along the chitin fibril. Genes encoding chitinases have been isolated from a broad range of organisms including fungi, insects and nematodes that use chitin as a structural polysaccharide, bacteria that use chitin as a food source, and plants and mammals that may defend themselves against fungal infec- tions. The chitinases of bacteria, nematodes, insects, vertebrates, and some fungi and plants are similar, while chitinases of most fungi and plants have somewhat different structures [8-10,13 19]. The structure of the chitinase A of Serratia marcescens, which is a potent anti-fungal agent, has recently been solved, confirming certain active-site amino acids (aa) suggested by site-directed mutagenesis [15,16]. In addition to the catalytic portion, chitinases often have a chitin- binding sequences, which vary in their structure from one species to the next [20,21]. These may include a lectin-like sequence (nettle), a fibronectin 3 sequence (bacteria), acidic repeats (nematodes), or other sequences (yeasts) [8 10,16 21].

Here molecular biological techniques were used

to clone and express genes encoding Eh, Ed, and Ei chitinases.

2. Materials and methods

2. I. Isolation and sequencing of the ameba chitinase genes

The ameba chitinase genes were isolated in two steps. First, ~ 380-nucleotide (nt) segments of the Eh cht l and Ei eht l genes were isolated from Eh HM-I:IMSS and Ei IP-1 strains genomic DNAs using the polymerase chain reaction (PCR), a degenerate sense primer GCTA(CT)T(AT)(CT) (AT)CI(AT)(AC)ITGGGC to the peptide CY (YF)(ST)(NS)WA (degenerative nt and aa are in parentheses) and an anti-sense primer (AG)(AT) A(CT)TCCCA(AG)TCIA(AGT)(AG)TC to the peptide D(LI)DWE(YF), which are conserved in chitinases of bacteria, nematodes, insects, and vertebrates (de la Vega, Specht, Liu, and Robbins, unpublished data) [22]. Other degenerate PCR primers to identify a fungal-type chitinase (sense = T(AT)TA(TC)TGGGG(AT)CA(AG)AA to the peptide (VI)YWGQN, sense= GG(AT) (ATG)T(AT)GA(TC)TT(CT)GA(TC)AT(AT)GA to the peptide G(FVI)DFDIE, anti-sense = (AT)GG(AG)CATTG(AT)GG(ATG)GC(ATG) GC to the peptide AAPQIP, and anti-sense = (AT)GG(AG)TT(AG)TT(AG)TA(AG)AA(TC) TG to the peptide QFYNNP) failed to produce PCR products of the appropriate size. Second, Eh ehtl and Ei chtl PCR products, which had been sequenced, were radiolabeled by random oligomer-priming and hybridized to genomic DNA and cDNA libraries of Eh HM-I:IMSS strain (Eh chtl) and a genomic DNA library an Ei IP-1 strain (Ei chtl) [23,24]. Two genomic clones contained the entire open reading frame (ORF) of the Eh chtl gene, while a single phage contained the entire Ei chtl ORF. A 1400 nt segment of Ed chtl gene was isolated from DNA of an Ed SAW 1734 strain cDNA library using the PCR, a sense primer G A A G G T C T T T C T A A C G G A to the 5' end of Eh chtl gene, and an anti-sense primer G T A A T A C G A C T C A C T A T A G G G C to the T7 promoter in the vector 2ZAP II.

H. de la Vega et a l . / Molecular and Biochemical Parasitology 85 (1997) 139 147 141

Each ameba chitinase gene was sequenced on both strands using vector primers and some newly synthesized primers. Hydrophilicity plots were drawn; signal sequences identified; amebic chitinases compared with protein sequences in the National Center for Biotechnology Information (NCBI), Bethesda, MD; and the progressive alignment constructed, using software available at the BioMolecular Engineering Computing Center at Boston University, Boston, MA [25 28].

2.2. Characterization of recombinant amebic chitinases

The Eh and Ed chitinases were expressed in bacteria as polyHis-fusion proteins and purified by affinity chromatography [29]. Chitinase activities of the recombinant enzymes were measured by a microtiter method employing 4-methylumbelliferyl []-D-N,N',N"-triacetylchitotriose (4MU- (GlcNAc)3) as a substrate [30]. Alternatively, recombinant amebic chitinases were identified in 10% SDS-PAGE cast with glycol chitin [31]. After electrophoresis, proteins were renatured in 100 mM sodium acetate pH 5 containing 1% Triton X-100, and dark bands of digested chitin were visualized against a fluorescent background after staining with Calcoflour White M2R. Chitin-binding was determined by centrifuging recombinant chitinases, which had been incubated for 15 h at 4°C with powdered chitin suspended in 20 mM sodium phosphate buffer, pH 6.0 [21]. Chitin used in binding experiments was prepared from purified chitin (Sigma No. C-3387), which had been boiled 10 min in 1% SDS and 1% fi-mercap- toethanol and then extensively washed with water [181.

Chitopentaoses of N-acetylglucosamine (Glc- NAe; Seikagaku) were labeled on all five sugars with N-acetyl [C 14] (GlcNAe)5-[~4C]) or labeled on a single reducing sugar with sodium borohydride [3H] (GlcNAc)5-ol-[3H]) [32]. Radiolabeled oligosaccharides were purified on Bio-Gel P2 (Bio-Rad) and incubated with recombinant amebic enzymes in 50 mM NaH2PO4 (pH 6.5) at 37°C for 1 h. Digested sugars were separated by thin layer chromatography (TLC) using silica gel 60 (EM Science) and n-butanol:ethanol:water (5:4:3).

Radiolabeled sugars were visualized by exposing TLC plates to X-ray films at - 7 0 ° C with a surface autoradiography enhancer (EN3HANCE; Dupont) for 24 h.

2.3. Measurement of chitinase m R N A in Ei trophozoites and cysts

Axenic Ei trophozoites were induced to encyst in vitro by concentration of trophozoites to 2 × 105 ml ~ and transfer to induction medium [24,33,34]. After 0 -72 h in encystment medium, amebae were washed, lysed in saturated guani- dinium, and centrifuged through a 5.7 M cesium chloride layer. Pelleted RNA was resuspended in DEPC-treated water and reverse transcription (RT) was performed with an anti-sense primer G T G A C A T C G T C C C A A G A A to the peptide TVDDWS of the Ei chtl mRNA. Thirty-five cy- cles of PCR was performed with TaqI polymerase using with the same anti-sense primer and a sense primer G G G C A C A G T A C A G A C A A A to the peptide WAQYR. For each PCR cycle, target DNA was denatured for 30 s at 94°C, primers were annealed for 30 s at 55°C, and DNA was synthesized for 30 sec at 72°C. Negative controls included all steps but RT, while positive controls included RT-PCR with Ei small sub- unit ribosomal RNA gene primers (forward = A A T A A C A G G T C T G T G T G and reverse = GG- C G C C G T G T G T A C A A A G ) (Ghosh and Samuel- son, unpublished data) [35].

3. Results and discussion

3.1. The Eh chitinase gene (Eh chtl) encodes an enzyme that includes a set o f hydrophilic and acidic 7-aa repeats

The Eh chtl ORF is 1521 nt long, encoding a putative 507-aa enzyme with a calculated M r =

57298 and pI 5.68 (Fig. 1). The A TG chosen as a start codon of Eh chitinase is probably correct because: (1) an in-frame stop codon is present 7 9 nt from the start codon in the 5' untranslated region (UTR); (2) the next potential start codon (Met211) is well into the overlap with other chiti-

142 H. de la Vega el al. Molecular and Biochemical Parasitology 85 (1997) 1.39 147

signal s ec~uences

F~ [MS. LLAL IYLIY~JA}~CEGLSNGFYC I DNKNYLWCYGRTEGTPGKC IGETVCKCGRTQYNPCVWNFLDLP 71

Ed -- -T ........ H-T- - -L ............. S ..... 52

Ei I--AMAFVCFVLAL--SQ~-- D ..... DTS .... H-Q QIT--SSGL .... K A-T .... SWT--- 72

Eh DCEKKPGDFIEKSPDS~D~KHES~_IKPDS .J~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 2

Ed .......... L- -PD- S-HE- KPDS S~IKPDS~IKPDS~IKPDS ~IKPDS ~IKPDS~ 125

E d - - S L . . . . Y~D- ~EEPLPES~EPPEPE--~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

E h . . . . . . . . . . . . . . . . . . . . . . . . . . . .IESKHESdEVKPDSdESKHES~IKPDS~SKHES ~PEVS I PKKT 147

Ed IEIKPDS~?CKLDS~EIKPDS~CKPDS~-V-PD-J ..... J ...... J-V- -J- ..... J- .... V- - 198

E d . . . . . . . . . . . . . . . . . . . . . . . I E V K P E P P A E S - ~ - E - - K P E - - S ~ E Q P K P E E S S ~ E E K P - P - E S S ~ - E - G - - - V 1 5 4

E~b V A Y Y T N W A Q Y R F N S I DGI, T I ~ K Y T A D N I D P T I V D V I N Y A F V V F D S T Y T L K E Y E W N D D Q . . . . . . M I P K I V A N N S R N P N L K V L ~ F N F Y D S 2 3 5

E d . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . - ] ~ ' ~ ] - ~ - 2 8 6

HS C S S . . . . . . . E D G . S C F P - A L R F L C T H - I - S - A N I S N D H I - T N . . . . VT . . . . . . LYGNLNTL ~ . . . . . . ~- f f -H l - r -o . . . . =o2 i i i i i

Bm GC ..... .... EG.-FLPG--PNGLCTH-L---AKV ELGDS-PF-- -EDTEWSKG-YSAVTKLRET--G .... ~ - ~ - G .... ii0

Ms C FS---V ...... P VG.R-GIED PVEKCTH-I-S-IGVTE, .GNS-VLII PELDVDKNGFRNFTSLR-SH-SV-FM~A~J-~EG .... 108

Sm -L-QAH- D-KI- ~:~h_~rLS . . . . 278

Eh TKHLYSQMAEKQATRATFIKSAMSFARKyNL.DG~P.. .ANEDQGGRPVDTQSFTLLLKEFREAIDKEAGNG.KSKLLLTIAAPAGPW 324

Ed ............. N ......... . -~-~-~[- . . . - ....................... V--- . ............. 375

Ei .... F-E --TS--A ............ "--l--~II-'''- K ........ V ........... A-KLS-GR-R ......... K 302

Hs .SQRF KI-SNTQS-R- VPP-L-THGF. -...GRR-KQHF... TLIKEMKA--IKEAQPGK .... -QL- SAALSAGKVT 184

Bm . SAIFTGI-KSAQKTER ..... IA-L N-F.--~-~-~[ .... VGVAEEHA... KLVEAMKTAFVE--KTSGK .... QRL--TAAVSAGKGT 192

MS .SSK- H V A Q K S - - M S - R V V - - L K - - D F . - - ~ - ~ - ~ [ . . . . G A A - R - - S F S K D K - L Y - V Q - L R - F I R V G . . . . . GWE-TAAVPL-NFR 193

Nt . STA GZ- RQPNS-KS - ~ - S I ~ - - Q L G F . H - ~ - H - ~ H . . . I ~ S a ~ G . - L a , rm~Tamr- -~SGR . . . . ~ - - T a a V S N S P R V 186 Sm . .DPFFF-GD-V.K-DR-VG-VKE LQTWKFF--%[J~-GGKGANpNL-S-Q-GETYV--M--L-ANL QLSAET.GR-YE- SAIS--KD 367

Eh NIKNIEVSKFHQY~I~WDAVTGSHTALYVTDGI ....... SVDDAVTAYLNAGVPSTKINLG~WTLKS SSDH .... E 407

Ed ..... I YK-~-~-- J-~J-~ J-~--S I-I -l-~-~J-~ -p ..... A-- . ................... A ........ ll~I ............. 458

~s D s s ~ I a - ~ s - H ~ - r ~ - r 1 - r 1 - ~ ~ o ~ - - ~ - ~ - ~ A ~ P ~ - ~ ' - - ~ ' ~ - ~ - A - ~ - ~ w - ~ I ~ I - [ ~ - - A - ' - ~ . . . . ~ ~ ~o, ~ , ~ - ~ , ~ - p ~ 1 - P 1 - r l - r - ~ G K - H P - K - E V S G I G I F N T E F - A D Y W A S K - M - K E - - I I - I P ~ I ~ - - DNP-~P . . . . A 276

. s ~ o ~ . - ~ - ~ - ~ q - [ 1 - r ~ - P A ~ v - ~ ~ . ~ - , - ~ , . ~ = ~ - ~ - - , - ~ v v i P ~ 1 1 - l ~ - - s a o ~ m ~ m ~ ~ 7 .,~: - ~ i ~ - ~ ~ l - r 1 - M - H - p s ~ s e ~ s ~ ~ , ~ - v . . . . ~ s - ~ i . - ~ i ~ - - - ~ - ~ v - - i ~ - i p ? - ~ - w ~ i . . . . . . ~ o Sm K DKVAYNVAQNS~-F~JS~_~FDLKNLG-QTALNAPAWKPDTAY.TTVNG N-L-AQ--KPG- W-T-~JJ~ GVGNGYQNNI. P 460

Eh MGSAATGASKSGTCTGENGYLSKYEI DAL I PQENI KFDSKSKTM .... yGYKNDQWF S FDTKETFKYKADYLCKKKL~ LD~ 491

N . . . . . . . . . . . . . . . . . . . . . . .... - ........ Q . . . . . . . . . . . . . . 542

Ei P S---T- K ....... S---AA--Qy A--M--....F--SG ..... D-D--EH VQ V-DN . . . . 469

Hs V APIS-PGIP RF K-A-T-AY CDFLRGATVHRTLGQQVP .... -AT-GN--VGY DQ-SV-S-VQ--KDRQ-A-A-~ ~ -J-I 357

Bm I-A--SRP SASKTNPAG TA YW--CKYLKEGGKETVHQEGVGA...-MV-G -YGY-NE IRI-MKW-KE-GY- AF~-~--~- I 367

Ms FINKEA GGDPAPY-NAT FWAY---CTEVDKDDSGWTK-WDEQGKCP-A GT--VGYEDPRSVEI-MNWIKQ-GYL-A-~-~I-~J 375

N t . - L R PA-G--NVGAV]gD-SMTYNR R D y - V E S R A T T V S ~ N A T I V G D . . - C - S G S N - I - Y DTQ-VRN-VN W ~ - I ~ - ~ I T W q - I ~

Sm FTGT- -PV-GTWEN IVD-RQIAGQFNSGEWQYTYDATAEAPYVF...KPSTGDLIT- DARSVQA-GK-VLD Q--~LFShFI-;~J 544

E27 NYVN KYIKSLIEKC* 507

Ed ..... E * 558

Ei ...... F--KELDQ-* 485

Fig. 1. Alignment of the aa sequences in single letter code of the Eh, Ed, and Ei chitinases with each other and with catalytic domains of chitinases of H. sapiens (Hs; GenBank accession number - M80927), B. malayi (Bin; No. M73689), M. sexta (Ms: No. U02270), N. tabacum (Nt; No. X77111), and S. marcescens chiA (Sm; No. Z36294) enzymes [27,28]. It is important to note that the Ed c D N A clone did not include a start codon, and non-homologous aa at the NH2-end of the S. marcescens chiA catalytic domain have been omitted. Dashes mark positional identities with the Eh chitinase, and periods mark gaps. Arrows mark locations of conserved aa used to design degenerative primers for the PCR. The shaded vertical boxes mark residues homologous to those of the active site of S. marcesens chiA, while the unshaded vertical boxes identify residues conserved in at least 90% of bacterial chitinases (both identified in ref. [16]). Also marked are putative Eh and Ei chitinase signal sequences (shaded horizontal boxes), Eh and Ed 7-aa repeats (unshaded horizontal boxes), and Ei repeats that have variable lengths (unshaded horizontal boxes) [22,26].

H. de la Vega et al. ,/Molecular and Biochemical Parasitology 85 (1997) 139-147 143

nase sequences; and (3) there is a putative signal sequence with a cleavage site between Alal5 and Hisl6, which obeys the -1, -3 rule of von Heijne (Fig. 1) [26].

Like most other Eh genes, the Eh ch t l O R F is not interrupted by introns, and 86% of the third positions in the codons are A or T (not counting Met or Trp residues) [36]. A putative ribosome binding sequence ATCA is present 4 - 7 nt prior to the start codon in the 5' U T R of Eh cht l , while a TATA-like sequence TATTTAA, which is found in the 5' U T R of many Eh genes, is absent. Sequences of the PCR product that overlapped the genomic clone are identical. The Eh cht l sequence was not identified in the Eh c D N A library. Attempts to use degenerate primers and PCR to identify a segment of an Eh gene similar to fungal chitinases were unsuccessful (see Section 2).

Conserved aa used to design the PCR primers are located within the chitinase catalytic domain at the COOH-ha l f of the Eh chitinase (Fig. 1). Between the Eh chitinase signal sequence and the catalytic domain are eight tandem copies of a degenerate 7-aa sequence, which is rich in hydrophilic amino acids including Ser, Glu, Asp, Lys, and Pro (Figs. 1 and 2) (Table 1) [25,26]. These 7-aa sequences are absent in other chitinases in GenBank, although multiple 14-aa acidic sequences are present at the COOH-termini of filarial chitinases [8,9]. Similar acidic and hydrophilic aa are also present in the 8- and 12-aa repeats of the Ser-rich Eh protein (SREhP) [37]. However, the aa sequence and the codon usage of the aa present in the chitinase repeats are not the same as those of SREHP repeats. SREHP is an antigenic amebic surface protein, which has been used to immunize gerbils against amebic liver challenge [38].

3.2. Similari ty o f the Ed chitinase gene (Ed ch t l ) to that o f Eh

i leader repeats catalytic domain :

q' : I !

i ~ : t , , i~ ~jtl I 4 ] ~ , '4 j i I ] 'l i ] r I ! :l

t i P I

- ~-i- E. histolytica

catalyt ic domain i

repeats

!;1' i

:,i I ! I i I ' , ~ '

leader repeats

E. dispar J i I ~ P I , ' , , ~ ' ' i

catalytic domain

- ' E. invadens

I ~'~' ' z ~ . . . . 24Q ' ' ' 3 ~ ~

Fig. 2. Hydrophilicity plots of Eh, Ed and Ei chitinases [25]. A 6-aa window was used.

library was made from a xenic culture of Ed in which cysts as well as trophozoites are present. About 19-aa, which likely include the signal sequence, are absent from the 5' end of the Ed cht l c D N A PCR product when compared to that of

Table l Amino acids (percent of total) present in repeat sequences of Entamoeba chitinases (EhCH1, EdChl and EiChl) and serine rich Eh protin (SREHP) [25]

aa EhChl (%) EdChl (%) EiChl (%) SREHP (%)

The largest O R F of the Ed ch t l c D N A PCR product is 558-aa long, encoding an enzyme with a calculated M,. = 62 545 and pI 4.81 (Fig. 1). The identification of the chitinase sequences within the Ed c D N A library is not surprising, because this

Ser 38 31 24 30 Glu 21 13 32 12 Asp 7 15 2 12 Lys 14 14 24 14 Pro 7 11 2l 11

144 H. de la Vega et a l . / Molecular and Biochemical Parasitology 85 (1997) 139-147

Eh cht l. The Ed cht l O R F differs from that of Eh chtl in 33 aa over a 498-aa overlap, with an additional 48 nt changes that are silent. The 7% aa and 6% nt differences between Eh chtl and Ed chtl genes are similar to those found for other homologous genes of Eh and Ed [23].

The Ed chitinase contains at its NH2 terminus 18 tandem repeats of a degenerate 7-aa sequence, composed of the same hydrophilic aa present in Eh chitinase and SREHP (Figs. 1 and 2) (Table 1). One 21-nt (7-aa) sequence is repeated without modificat ion 'seven times, while two other 21-nt (7-aa) sequences are repeated without modifica- tion three times.

3.3. Unique features of the Ei chitinase gene (Ei chtl)

The Ei chtl ORF is 514-aa long, encoding a putative enzyme with a calculated Mr = 54 300 and pI 4.6 (Fig. 1). There is an in-frame stop codon in the 5' UTR of Ei chtl, while conserved ribosome-binding site ATCA and TATA-like box are absent. Further, a homology matrix alignment revealed only rare instances of 6-nt identities between 300-nt segments of the 5' U T R of Eh chtl and Ei chtl (data not shown). This result suggests that Eh chtl and Ei chtl genes may not share common promoter/enhancer sequences or that these sequences are difficult to identify.

Like the ElF chitinase, the Ei chitinase includes a signal sequence, a series of acidic and hydrophilic aa, and a catalytic domain at the COOH-half . However, the Ei chitinase acidic and hydrophilic sequences are made up of seven degenerate repeats, which vary in length from 4- to 12-aa (Figs. 1 and 2) (Table 1). This made it impossible to align these sequences with those of EIF and Ed. The remaining portions of Ei chtl showed a 74% aa and 68% nt identity with those of the Eh chtl. The low nt identity is caused in part by the fact that Ei chtl coding region contains only 58% A or T in the third codon position, while the Eh ehtl coding region contained 86% A or T in third codon position. That Ei chtl is different from Eh chtl and Ed chtl is not surprising, since Ei grow at 25°C rather than 37°C and infect reptiles rather than man.

before ppt. after ppt. Ei Eh Ed Ei Eh Ed

Fig. 3. In gel localization of recombinant Entamoeba chitinases activities before or after precipitation with powdered chitin [311.

3.4. Similarity of the Eh chitinase to other eukaEvotic and prokaryotic chitinases

The catalytic domain of the predicted Eh chitinase shows extensive positional identities with those of Ed (95%), Ei (76%), man (34%), B. malayi (33%), Manduca sexta (29%), and N. tabacum (25%; Fig. 1) [8,13,14,16,17]. For com- parison, the catalytic domains of three nematode chitinases (B. malayi, Onchocerca volvulus, and Acanthocheilonema viteae) show 77 79% positional identities [8 10]. The Entamoeba catalytic domains include six amino acids homologous to residues W275, E315, M388, D391, Y444, and R446 in the active site of S. marcescens chiA and nine other aa conserved across bacterial chitinases [16]. In contrast, the ameba chitinases showed little similarity to most fungal and plant chitinases.

3.5. Chitinase and chitin-b#Ming activity of recombinant amebic chitinases

Gels containing glycol-chitin were used to demonstrate the chitinase activity of recombinant Eh, Ed and Ei chitinases (Fig. 3). An additional lower tool wt. band of chitinase activity in the Ei lanes may represent a degradation product of the recombinant chitinase during preparation from Escherichia coli. Further, 70-90°/,, of the activity of the recombinant Entamoeba chitinases (mea-

H. de la Vega et a l . / Molecular and Biochemical Parasitology 85 (1997) 139 147 145

G/cNAc ---I,,-

(GIcNAc)2~

( G[c NA c)3,---I~

(G Ic NA c)4"~1~

(GIcNA C)S----~"

(GIcNA ¢)6--l~

(GIcNAc]z-OI

(GIcNAc)3"ol

(GIcNAc)4"OI

(GIcNAc)s'Ol

1 2 3 4 5 6 7 8 9 10 11 lZ 13

Fig. 4. TLC of radiolabeled chitooligosacharides digested by recombinant Entamoeba chitinases. Lanes: 1, GIcNAc and chitobiose iGlcNAc)2 standards; 2, chitotetraose (GIcNAc)4, chitopentaose (GlcNAc) 5, and chitohexose (GIcNAc) 6 standards; 3 6, products generated using chitopentaose (GlcNAc)5-[~4C] as a substrate in presence of E. coli extracts expressing: 3, Ed chtl; 4, Eh chtl; 5, Ei chtl; 6, no amebic genes (negative control); 7, chitobiositol (GlcNAc)2-ol and chitotriositol (GlcNAc)3-ol standards; 8, chitopentaositol (GlcNAc)5-ol standard; 9, chitotetraositol (GlcNAc)4-ol standard; 10 13, products generated using chitopentaositol (GIcNAc) 5- ol-[3H] as a substrate in presence of E. coli extracts expressing: I0, Ed chtl; 11, Eh ~htl; 12, Ei chtl; 13, no amebic genes (negative control).

sured with 4 M U - ( G l c N A c ) 3 ) is p rec ip i t a tab le with chitin. Whe the r non-ca ta ly t i c repeats at the NH2- terminus o f the amebic chi t inases are responsible lbr chi t in-binding, as is the case for bacter ia l and fungal chit inases, remains to be de te rmined [18

211. Ch i too l igosacchar ides were used to charac ter ize

the r ecombinan t E n t a m o e b a chi t inases (Fig. 4). Each r ecombinan t amebic chi t inase degrades (GlcNAc)5-[~ac] into (GlcNAc)2-[~4C] and (Glc- NAc)3-[~4C]. Fu r the rmore , each r ecombinan t amebic chi t inase degrades (GlcNAc)5-ol-[3H], which is labeled only on the reducing end with [~H], to (GlcNAc)3-oI-[3H] but not (GlcNAc)2-ol - [~H]. It appea r s then that a d iace ty lch i tob iose has been removed f rom the nonreduc ing end o f each ch i topentaose , consis tent with an exochi t inase ac- t ivi ty by each r ecombinan t amebic enzyme. These results, however , do no t rule out an endochi t inase act ivi ty by the amebic enzymes.

0 12 24 48 72

chtl

rRNA

Fig. 5. Changes in chitinase mRNA and small sub-unit ribosomal RNA (detected by RT-PCR) by Ei encysting in vitro. In each lane, four times as much of the chitinase RT-PCR product as that of ss-rRNA was loaded. No products were detected in control PCR from which RT was omitted.

146 H. de la Vega et al./ Molecular and Biochemical Parasitology 85 (1997) 139 147

3.6. Changes in Ei chitinase gene expression with

encystation in vitro

R T - P C R showed that Ei cht l m R N A is absent f rom cultured trophozoites, bu t is present in parasites encysting for 24 72 h with a max- imum level at 48 h (Fig. 5). It is no t clear what the two chitinase bands at 48 h means. The a m o u n t of amebic r R N A decreases dur ing encystat ion. Previously, polyadenyla ted m R N A encoding histone H2B and a basic peptide of u n k n o w n funct ion were shown to increase in Ei dur ing encystat ion, while Ei actin m R N A was shown to decrease dur ing encystat ion [24,33,39]. Excystat ion was not studied because we do not know how to induce Ei cysts to ex- cyst.

In summary, we identified chitinase genes from three species of Entamoeba, showed that the r ecombinan t amebic enzymes have chitinase activity, and demons t ra ted cyst-specific expression of the chitinase m R N A by Ei in vitro. As the chitinase inhibi tor blocks encysta t ion of amebae, we presume that the amebic chitinase is involved in mold ing the amebic cyst wall, as has been demonst ra ted for the fungal wall of Saccharomyces cerevisiae [7,18]. It is possible that chitinase inhibi tors may be used in con- junc t ion with other ant i -amebic drugs to pre- vent cyst fo rmat ion and t ransmiss ion of the parasite.

Acknowledgements

We thank Dr Egbert Tann ich of the Bernard Nocht Inst i tute for Tropical Medicine for the Eh and Ed c D N A libraries and Chitra Mishra for making initial experiments with chi- tooligosaccharides. This work was supported in par t by grants from the N I H (AI33492 to JS, GM31318 to PWR, AI33716 to DE and CA14051 to Richard Hynes), US-Mexico Foun - da t ion for Science, and John D. and Cather ine T. MacAr thu r Founda t i on . HV received a pre- doctoral fellowship from the Na t iona l Counci l for Science and Technology (Mexico) and CIN- VESTAV.

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Cloning and expression of chitinases of Entamoebae