EXPERIMENTAL

PARASITOLOGY

75,

308-322 (1992)

Schistosoma mansoni: Cloning of a Complementary DNA Encoding a Cytosolic Cu/Zn Superoxide Dismutase and High-Yield Expression of the Enzymatically Active Gene Product in Escherichia co/i’ ZHI HoNG,*,~ PHILIP T. LOVERDE,~ MARIE-LOUISE HAMMARSKJ~LD,~~. AND DAVID REKOSH*+$.~ Departments of *Biochemistry,

fhficrobiology, and #Oral Biology, State University of New York at Bu#alo, Buffalo, New York 14214, U.S.A.

HONG, Z., LOVERDE, P. T., HAMMARSKJ~LD, M.-L., AND REKOSH, D. 1992. Schistosoma mnnsoni: Cloning of a complementary DNA encoding a cytosolic Cu/Zn superoxide dismutase and high-yield expression of the enzymatically active gene product in Escherichia coii. Experimental Parasitology 75308-322. We recently purified a 16-kDa cytosolic Cu/Zn superoxide dismutase (CT CulZn-SOD) from Schistosoma mansoni, a human parasite. Three peptide sequences were obtained, one from the unblocked N-terminal and two from internal peptides which were generated by digestions with trypsin and cyanogen bromide. These sequences were aligned to the corresponding sequences of 19 cytosolic Cu/Zn-SODS from various species. Degenerate oligonucleotides were then designed according to the sequence and the position of each peptide. The oligonucleotides were used to amplify a complete cDNA using the polymerase chain reaction with either adult schistosome total RNA or a cercariae Xgtll phage cDNA library as the template. The protein encoded by the cDNA has 153 amino acids with a calculated molecular weight of 15,693. It also has 6&65% homology to 19 cytosolic Cu/Zn-SOD from various species. All of the copper/zinc binding sites and SOD activity sites are conserved. Computer analysis predicts that the Cu/Zn-SOD has a p1 value of 6.6, which is very close to the experimental results of IEF analysis (6.0 and 6.3). The entire coding sequence from the cDNA was cloned into a bacterial alkaline phosphatase cytosolic expression vector and a large amount of soluble product was expressed and purified to homogeneity. We compared the bacterially expressed Cu/Zn-SOD with the native enzyme derived from schistosomes and found that they are identical by the following criteria: (1) They focus at the same positions on IEF gels; (2) they form dimers in solution as measured by gel filtration; (3) they have the same unblocked N-terminal sequence; (4) they both are enzymatically active with comparable specific activities. The specific activity of the bacterially derived enzyme was increased somewhat (-10%) by incubation with copper and zinc ions. 0 1992AcademicPress,Inc. INDEX DESCRIPTORSAND ABBREVIATIONS: Schisrosoma mansoni; Superoxide dismutase; Bacterial expression vector; cDNA clones; Cytosolic superoxide dismutase (CT-SOD); Signal peptide containing superoxide dismutase (SP-SOD); Isoelectricfocusing (IEF); Polymerase chain reaction (PCR); High pressure liquid chromatography (HPLC); Bacterial alkaline phosphatase cytosolic expression vector (pBAce); Polyethylenimine (PEI).

i Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M97298. ’ Present address: Molecular Genetics Department, Schering-Plough Research, 68 Orange Street, Bloomfield, NJ 07003. 3 To whom correspondence should be addressed at Department of Biochemistry, c/o Oral Biology, State University of New York at Buffalo, 304 Foster Hall, Buffalo, NY 14214.

1NTRoDucT10~

Superoxide dismutase is an ubiquitous metalloenzyme (EC. 1.15.1.1) in aerobes. It catalyzes the dismutation of deleterious radicals (McCord and Fridovich 02 1969). Three distinct types of SODS have been found so far. They can be grouped into two unrelated families with respect to the 308

0014-4894/92 $5.00 Copyright0 1992by AcademicPress,Inc. All rightsof reproductionin anyformreserved.

,khiStOSOt?lU

mUnSOni:

metal ion bound by the enzyme (Fridovich 1989). Mn-SOD and Fe-SOD form one family. These enzymes are usually found in prokaryotes and subcellular organelles. The other family consists of the Cu/ZnSODS that occur primarily in the cytosol of eukaryotic cells and the chloroplasts of plants. An extracellular form of Cu/Zn SOD has been recently characterized in mammalian body fluids (Marklund 1980). Both forms of CulZn-SOD are believed to have evolved from the same ancestor. Schistosoma munsoni, an intravascular human parasite, lives in a hostile environment closely contacted by the host cytotoxic humoral and cellular factors. To circumvent the immune responses from the host, a number of evasion mechanisms seem to have developed. These fall into three general categories which have been previously reviewed (Smithers and Doenhoff 1982): (1) Acquisition of host antigens, (2) outer membrane modulation, (3) suppression of host immune responses. It has recently been suggested that antioxidant suppression of host oxidative killing may also play a protective role in the parasite life cycle (Callahan et al. 1988). Oxidative killing of the parasites would be expected to occur when host effector cells release toxic oxygen species through a respiratory burst (Rossi 1986). O2 * - radicals are a major part of the toxic oxidants which are massively produced by eosinophils, neutrophils, and macrophages (Badwey and Karnovsky 1980). Many superoxide dismutases, mainly Cu/ Zn-SODS, have been characterized and/or purified from various parasites of different species (Paul and Barrett 1980; Fairfield et al. 1983; Rhoads 1983; Kazura and Meshnick 1984; Leid and Suquet 1986; Sanchez et al. 1988; Callahan et al. 1991; Henkle et al. 1991; Michalski and Prowse 1991). In Trichinellu spiralis, CulZn SOD activity was even detected in the culture medium and is believed to be excreted by the para-

CYTOSOLIC

CU/Zll

SOD

309

sites (Rhoads 1983). In addition, some parasites or microorganisms like Plasmodium berghei and Treponemu pullidum appear to acquire host Cu/Zn-SODS during their infections (Austin et al. 1981; Fairfield et al. 1983). Our laboratories recently identified two forms of Cu/Zn-SOD from S. munsoni: a cytosolic Cu/Zn-SOD (CT Cu/Zn-SOD) (Hong et al., 1992) and a signal peptidecontaining Cu/Zn-SOD (SP Cu/Zn-SOD) (Simurda et al. 1988). The SP CulZn-SOD gene product contains a hydrophobic N-terminal (20-30 a.a.> and a N-linked glycosylation site, suggesting an extracellular or membrane-associated form of the enzyme. The presence of different forms of Cu/Zn superoxide dismutase in schistosomes and also in other parasites (Rhoads 1983; Henkle et al. 1991) suggests a distinct role for the antioxidant enzymes in the parasite life cycle. We recently characterized the SOD activity present throughout all the developmental stages of the life cycle of S. munsoni (Hong et al., 1992). It was found that adult worms had the highest SOD specific activity, which was about five times as high as that found in eggs and miracidia and twice as high as that found in cercariae and schistosomula. Over 95% of the total SOD activity was sensitive to cyanide which is a potent inhibitor of Cu/Zn SOD. This indicates that CulZn SOD is the major species of the SOD enzymes present in the parasite. Furthermore, the SOD activity was found to be enriched in a Triton X-100 extract that contained outer surface components (Hong et al., in press). We report here the isolation, characterization, and expression of a cDNA that encodes a schistosome CT Cu/Zn-SOD. The cDNA is about 600 bp long, and contains 22 bp of the 5’ untranslated region, a coding region (462 bp), and a poly(A) signal in the 3’ untranslated region (121 bp). The gene product of this cDNA was expressed in E. coli and purified to homogeneity. The en-

310

HONG

zyme was then characterized in comparison with the native enzyme derived from adult parasites. MATERIALS

AND

METHODS

Plasmid, bacteria, chemicals, and enzymes. The bacterial cytosolic expression vector, pBAce, was generously provided by Dr. Sydney P. Craig III of the University of California at San Francisco. E. coli strain DHSa was obtained from BRL. Enzymes for cloning were purchased from BRL, Promega, or New England BioLabs. Chemicals were from Sigma Chemical Co. or other common commercial sources. Protein concentration assay. Protein content was determined by a BCA assay as described previously (Smith et al. 1985). Bovine serum albumin (1 mg/ml) was used as the standard. SOD nitrate assay. SOD activities were determined by their inhibition of nitrite formation. One unit of SOD activity was defined as the amount of enzyme that inhibited 50% nitrite formation under the assay conditions specified (Oyanagui 1984). It has better sensitivity and less interference compared to the standard cytochrome c assay, and 8.5 nitrate units are equivalent to 1 unit of cytochrome c unit. SOD IEF gel assay. IEF analysis was carried out as described previously (Kaars and Kosman 1983). The SOD activities on the gel were stained by riboflavin photooxidation and Oz . - reduction of nitroblue tetrazolium (Beauchamp and Fridovich 1971). The MnSOD can be distinguished from Cu/Zn-SOD by its appearance on the gel when stained in the presence of 2 m&f KCN. Oligodeoxynucleorides. The following synthetic oligonucleotides used in these studies were purchased from a commercial source (Oligos Etc.) or the Center for Advanced Molecular Biology and Immunology at the University at Buffalo: PB-dT,,, ATCTGCAGGATCCTTTTTTTTTTTTTTTTT; NT- 1, ATGAA(AG)GC(AGTC)GT(AGTC)TG(TC)GT(AGTC)ATGAC(AGTC)GG;

NT-2,

ATGGT(AGT-

C)AT(ATC)CA(TC)GA(AG)AA(TC)GA(AG)GA(TC) GA; NT-3, TT(TC)CA(TC)GT(AGTC)CA(TC) GA(AG)TT(TC)GG(AGTC)GA; CT-l, (TC)TC(AG)TT(TC)TC(AG)TG(GTA)AT(AGTC)AC-

TC(AG)TC-

CAT; CT-3, TCCATTTGTTGTATCACCAAATTC; CT-4,

TTCTTGAGTAAATITGACAACACC;

Xgtl 1

forward primer, GGTGGCGACGACTCCTGGAGCCCG; Agtl 1 reverse primer, TTGACACCAGACCAACTGGTAATG. Those bases in the parentheses represent randomly mixed bases at the wobble positions. lsolarion PCR. (1)

of schisrosoma

CT CulZn-SOD

cDNA

by

Amplification of specific cDNA fragments from schistosome total RNA using degenerate oligonucleotides (Ferre and Garduno 1989). Single-stranded

ET AL.

cDNAs were synthesized by reverse transcription from adult schistosome total RNA, prepared as described (Chirgwin et al. 1979), using PB-dT1, (oligodT,, with a PsrI and BamHI extension) as the primer. The single-stranded cDNA was then used as a template for PCR with the degenerate primer NT-1 as the upstream primer and PB-dT,, as the downstream one (Fig. 1). The PCR products were separated on a 3% NuSieve agarose gel and several amplified DNA fragments of different sizes (450,500,550,600, and 650 bp) were cut out and purified. A second round of PCR was carried out with each fragment as the template, using three different pairs of degenerate oligonucleotides: NT-l/CT-l, NT-Z/PB-dT,,, and NT-3/PB-dT1,. Only those PCRs which used the 600 and 650 bp templates gave the same three dominant amplifications. These were a 360-bp fragment with NT-l/CT-l, a 260-bp fragment with NT-2/PB-dT,,, and a 450-bp fragment with NT-3/PB-dT,,. The size of these fragments correlated with the predicted distance between the corresponding peptide sequences. These predictions were based on alignments of the peptides with SOD sequences from the other species. All three PCR products were subcloned into pGEM3Zf( - ) and sequenced according to the directions provided by the manufacturer (Sequenase-Version 2.0, United States Biochemical). (2) Amplification of the 5’ untranslated region using a cercarial Xgtl 1 cDNA library as template (Young and Davis 1983). The phage DNA template was prepared by heating 5 &l of phage library (5 x 10’ pfu/ml LB Broth) at 70°C for 5 min, followed by immediate cooling on ice. Specific phage forward or reverse oligonucleotides (Promega) were used as the upstream primers. Ten PCR-derived DNA fragments with different sizes were amplified, subcloned into pGEM3Zf( -), and sequenced. A 5’ untranslated region containing a 22-bp sequence was identified and the sequence under the 5’ degenerate oligonucleotides (NT-l) was also confirmed. (3) isolation of the intact cDNA. Finally, a SOD cDNA containing the entire coding region was isolated from adult schistosome total RNA by PCR, coupled with reverse transcription (PCR-RT) using specific 5’ end and 3’ end primers. Its sequence was confirmed in clones from three independent PCR-RTs to rule out mistakes generated during the PCR process (Saiki er al. 1988). Cloning and expression. The bacterial expression vector, pBAce, was described previously (Craig et al. 1991). It contains a unique NdeI site at the start codon and a strong bacterial transcription terminator (trp A). It is derived from a previously described bacterial PhoA vector, pBSprts (Yuan et al. 1990). The alkaline phosphatase promoter (PhoA) can be induced in low phosphate medium and expresses foreign cDNA at a relatively high level (Oka et al. 1985). For expression in pBAce, the complete schistosome CT Cu/Zn-SOD coding region was amplified by PCR so that a unique

Schistosoma Schistosome CT Cu/zn-SOD with three peptides sequenced

mansoni:

Pl

P2

m

I

Protein

P3 I

-

NT-I L

NT-l L

Mixture of SOD cDNA from the primary nonspecific PCR

1

Design degenerate.primers basedon the peptide sequences

1

Adult schistosome total RNA template

311

Cu/Zn SOD

CYTOSOLIC

T

mRNA

PBxt, Synthesisof singlestranded cDNA by RT and doublestranded cDNA by PCR NT-2 A

i NT-3

-L

?Fl

PBTT,,

Second-round

F’CR for

(1)

more.specificcDNA fragments

360bp

260

bp

Specific SOD cDNA fragments from second-round PCRs

Forward ReKBe

J

or

Subcloned into PGEM3Zf(-) for double-strand sequencing

cDNA Insert

Cercariaehgtll Phage _ * cDNA library templates - - - CT-4

CT3

PCR amplification of 5’ end specific

1I

220-2fiO bp

primers

sequence with of both phage and cDNA

---

PCR products containing - - 130-160 bp 5’ end of SOD cDNA

---

Adult schistosome total RNA template

5’ -L -

(2)

J

Double-strand sequencing

DNA of 5’ end

mRNA

Three independent PCR-RT amplificationsof the cDNA to eliminate PCR mistakes

Intact

cDNA

Schistosome CT Cu/Zn-SOD ..,,,,+ cDNA with confirmed sequent?“” FIG. 1. Schematic diagram describing the isolation of schistosome CT Cu/Zn-SOD cDNA. The full-length protein is represented by the open box at the top and the three sequenced peptides are indicated by the black bars. mRNA and cDNA are drawn as a single line and a double line, respectively. The wavy line represents 5’ and 3’ untranslated regions. The dashed line represents poly(A) or phage DNA. The positions and orientation of the degenerate primers (NT-l, NT-2, NT-3, and CT-l) are indicated by the arrows. The corresponding peptide sequences are: Pl , MKAVCVM (N-terminal); P2, FHVHEFGD (trypsin digestion); and P3, MVIHENED (CNBr digestion). CT-3 and CT-4 are specific primers derived from the cDNA fragments. The numbers in the parentheses represent the three major steps in isolating the SOD cDNA. The sequences of all of the primers are listed under Material and Methods.

(3)

312

HONG ET AL.

NdeI site was created at the start codon and a PstI site was placed right after the stop codon. This fragment was then inserted into pBAce between the NdeI and PstI sites (Fig. 2). The final expression plasmid, pBAce-CTSOD, was transformed into bacterial DH5a cells and l/1000 vol of an overnight culture of bacterial cells in LB Broth was inoculated into low phosphate medium (Craig et al. 1991) containing 100 pM Cu’+/ Znz+ ions. The culture was then incubated at 37°C overnight for about 15 hr. The promoter is turned on when the phosphate concentration becomes limiting during the incubation.

ATG

S.

mansoni

CT

RESULTS

Isolation of a schistosome CT CulZnSOD cDNA using the polymerase chain reaction. Our laboratories previously isolated

a cDNA clone from adult schistosomes which encoded a protein that had many features in common with the extracellular form of Cu/Zn-SOD found in human cells, including a hydrophobic leader sequence and an N-linked glycosylation site (Simurda

Cu/Zn

SOD

cDNA

TAG

-I

Pstl

Ndel

PhoA + TATAGTC

GGAGAAAATCATATGGCAAmFATATCmCTGCAG Ndel Clal SD

EcoRV

Sal1

Pstl

CCC TrpA

TTTTTCTAGA Term

Xbal

pBAce (3.26

kb)

FIG. 2. pBAce-CTSOD construction. The entire coding region (open box) of schistosome CT Cu/ Zn-SOD cDNA was reamplified by PCR so that a unique NdeI site was created at the start codon and another PstI site was created several nucleotides downstream of the stop codon. The cDNA fragment was gel purified and then digested with both NdeI and Pd. The digested cDNA was inserted into the pBAce vector between the PhoA promoter and the TrpA terminator (Craig et al. 1991).

Schistosoma mansoni: CYTosoLxc Cu/Zn SOD et al. 1988). We refer to this protein as SP Cu/Zn-SOD, since studies on its expression in mammalian cells demonstrated that the hydrophobic leader functions as a signal peptide (Hong, et al., submitted for publication). The isolation of this clone prompted us to undertake a study of the SOD activity present throughout the schistosome life cycle (Hong et al., 1992). In this study we purified the protein giving the major SOD activity to apparent homogeneity and compared a partial sequence of this protein with the sequence of protein pre-

dieted from the cDNA clone. To our surprise, the data revealed that the major SOD activity isolated from the soluble fraction of adult worms was derived from a different protein that then encoded by the originally isolated clone (Simurda et al. 1988; Hong et al., 1992). The HPLC profile of the purified protein representing the major SOD activity present in soluble extracts from adult worms is shown in Fig. 3A. As shown in Fig. 3B, this SOD enzyme has a different immunoreactivity from that of SP Cu/Zn-

(A) S. mansonf

_-- J,,

CT Cu/Zn

SOD

w_ _ --.*-.. J _....- ‘..-

(B)

’ “%

0 1% TFA80% CHJCN

69 kd+ 44 kd+

- 50% 0% Oml

30ml

,____.A.--- -----

313

60ml

0 1% TFA

29 kd+

___ -..-

18 kd+ 14 kd+ ____./.-.* -FIG. 3. Differential immunoreactivity between CT Cu/Zn-SOD and SP Cu/Zn-SOD. (A) A preparation of purified schistosome-derived CT Cu/Zn-SOD was injected onto an HPLC reverse-phase column (Waters, p,Bondapak C,, column). It was eluted with a linear gradient of acetonitrile from 0 to 75% in 0.1% trifluoroacetic acid as indicated. The protein was detected at a wavelength of 214 nm. (B) Purified schistosome CT Cu/Zn-SOD was analyzed by 15% SDS-PAGE and Western blot using both anti-SP Cu/Zn-SOD mouse monoclonal antibodies (lane 1, l/5008 dilutions) and anti-SP Cu/Zn-SOD rabbit polyclonal antiserum (lane 2, l/l000 dilutions). The Western blots stained by alkaline phosphatase conjugated second antibodies are shown.

314

HONG

SOD. Western blot analysis demonstrates that the new enzyme reacts with polyclonal antiserum directed against SP SOD (lane 2) but not with anti-SP SOD monoclonal antibodies (lane 1). This indicates that the newly purified enzyme contains different epitopes from those of SP Cu/Zn-SOD and may represent a distinct polypeptide. The reason that multiple bands are seen in lane 2 is not entirely clear, but studies by other workers have indicated that SODS often form oligomers which cannot be easily dissociated on SDS-PAGE (D. Kosman, personal communication). Three stretches of peptide sequence were obtained from the purified protein either by directly sequencing its amino terminus, or by sequencing peptides derived from it after trypsin or cyanogen bromide cleavage. The three peptide sequences obtained were: (1) MKAVCVMTGTAGVKGVVKFTQE (at N-terminal); (2) HGFHVHEFGDTT (from trypsin-cleaved protein) and (3) MVIHENEDDLGRGG (from CNBrcleaved protein). The sequences underlined were used to design degenerate oligonucleotides as described below. All of the three peptide sequences could be aligned to the known sequences of cytosolic SODS from 19 different species, so that their orientation with respect to each other (N to C terminal) could be determined (see Table I). Only limited homology was observed between these sequences and those of the SPSOD which was previously cloned. A procedure involving reverse transcription, PCR amplification, and cloning was employed to obtain a cDNA encoding the purified SOD. In this procedure, the primer PB-dT,, (see Materials and Methods) was first used to reverse transcribe total adult schistosome mRNA into single-stranded cDNA. This DNA was then amplified by PCR using families of degenerate oligonucleotides corresponding to the underlined portion of each of the above peptides (designated NT-l, NT-2, NT-3, CT-2). The positions and orientations of these oligonucle-

ET AL.

otide families are illustrated in Fig. 1. Their sequence as well as the details of this procedure are given under Materials and Methods. The cDNA was then sequenced. Its sequence showed that the mRNA from which it was derived contains a 5’ untranslated region of at least 22 bp, a 462-bp coding sequence, and a 121-bp 3’ untranslated region followed by a poly (A) tail. A large open reading frame in this cDNA has the capacity to encode a protein of 153 amino acids with a predicted molecular mass of 15,693 Da (Table II). This is very close to the observed molecular mass of the purified protein on an SDS gel (Fig. 5, lane 2). Computer homology analysis showed that the protein has 6065% homology to 19 cytosolic Cu/Zn-SODS from other species (Table I) with all of the copper/zinc binding sites and activity sites conserved. Sequence comparison between CT Cut Zn-SOD and SP CulZn-SOD of schistosomes. The two schistosome SOD sequences were compared at both the protein and DNA levels (Fig. 4). At the protein level they contain 63 identical residues which are highlighted in bold type in Fig. 4. This makes the two proteins 45% homologous within the overlapping region. Most of the identical residues are also present at the corresponding position in SODS from the 19 other species and represent areas of the protein known to be important in metal binding and catalysis. The number of species having a particular residue in common with both schistosome SODS is given in Fig. 4 above the boldface amino acid in CT SOD. Only four residues (A,,, Qh8, NiZ1, and 4~~ in CT SOD) are conserved in both schistosome SODS which are not present in any other SOD species. Of particular note is Qa, which is a K in 17 out of the 19 other species. When each schistosome SOD is compared to the SODS of the other species, homologies range from 40-45% for SP-SOD to 60-65% for CT SOD. Thus the two schistosome SOD sequences appear to be no

Schistosoma

mansoni:

CYTOSOLIC

C&n

315

SOD

TABLE I Amino Acid Comparison between Schistosome CT Cu/Zn-SOD and 20 Cu/Zn-SODs from Various Species

-TV YSYLVILFIL

BGVKGVVKFT MKAVCYHTGT PYIGA-WFTP

LDNYCSAYGY GYSYYHERHF DP-IASF-KE A T-----LK-D A-G---LNSS " "-----IN-. ”

“-----IN-.

A L-----LK-D A T-----LX-D v---~,-LNss “---A-U-A ------LK-D V---A-“R-D A T-----LK-D ------LK-T I\ T-----LK-D A ------LK-D ?, T-----LK-D *T K---*-LKv-----m-s " L-----LR-A

.DA--T-F-E

QETDNGP""" HGDY"WNGS AKG.D.T-VH-GNG.ATT--SSGT--K-

.DA--T-F-E

--GEGC--K-

TG+-p-M-

*.---------

-N----“-S-

GP-H--IH-E GP-Q-IIN-E E--S-TYL-D,---TIF-~ GP-Q-TIH-E

-QPEG---“L -KBS----X-vGv~,,-TT--G-G,-TT-~sGE--“L --SESA-TTI LKG.EKT-L-.$,D---TL -KGTG.--"-USGEm-“AKG.D.K-V--D-G,-TT-QD-GD,-T-

KGFIE--TRWCSy&--TENGNI----pT‘SI--e-PSGQIT--TETYDI--NDPN TGTIK--AEKGSIT--TPKGRIT--TESGQIT--TETGSIT--TENt.q--MEGKIE--TDGIILK--TP-

D.--------L.--------L.------&LL.------ALQ.------QYA-R---I-T-D.------Q--.----e-&-e L.------Q-E,------QyD.------P--,----Lee-N.----I-V-E.------G--

-N-Q---T--W-A----------+-T-----++T-N-*------

GP-Q-TIH-E E----TIF--

SN---T-I-E

GP-Q-TIY-E GE-T-T-L-E GP-E-TIH-E GP-Q--I”-E GP-Q-TIR-E SN-E---TLGD-----&E GETT-T-Y-E

“*--A-LK-D --S-----E 100

1

DGNAVYNATD G-V-KFDFW _____T-DK N-“-I”DI”-----II”-D --T-TpTI-. -----IE-TG -CPTK”-I------II-NG --PTP”-IC------T-DE .-K-D”DMK------T-OK --“-D”SIE-----IT”-E --T-SFTI-------T--E --““~-*-------*--K .-“-N”SIE-----IETDQ---KGT”-------T.-K --“-T”yIE------E-NG N-“-EFEIK------T--S N-“-D”,JE------A--K --“-N”SIE------K-DK N-“-I”JI”--.--I--NT --“-EATI”--.-.-T-E, G-“-*F-F---...-T.rJN-“.g(IDI---M---KTDE N-“-j(GSFJ(-

HFNPTKQEB

GAPEDSIRBV

GOLGN”“AGA

----FN-R-

-PRHGYP--A

-----IR"-R

----LSKK-

-G-K-EP---

----DGKT----yGK-----YQK-----LSKK----LSRK-y--*GK-----“GK-----HSKK----“GAT----ESKK-Y--Fp,KN----LsKK----HSKf(----LSKK----D-KT----*NKN---.*c$qf(----F-KT-

-----&NT-* ---“-EN-L ---T-EN--L -G-K-EE---G-K-EE-------p-“--I\ -----ED--A -G-*-E---DRTm”---G-K-Q---G-D-EE---G-K-EE---G-&-EE---G-K-EE-------E”--R -S-K-AD--AG-K-ED-----T-E”---

KLISLNGSHS TIKG-GPFDG p----S-E*5*-p-S-p*SK-T-F-*DcK-T-L-ANS”---S-K-S”---S-D-C -Q-p-T-p*SQ-p-*-p-R”---S-E-N-“K-I-PES”-A-S-D-RQLf+++ERs”---S-DMR”---S-E-p----S-EyNQ-P-T-PNPQ---K-ER-.---ppyS--K-I-PT-

--GNANA-GK

INEFSGLRAG V-...--PPTGSIT--TETGTV---RPSG-"C--mK-

K.ILGI‘BYBEFG -LL-T---RYD.------Q-L.------ALL.-----v---

D.XZNGCTSii GLG-M-LE--N-Q------N----W-T-N---+,-S-

-N--------

-ASESE-V- SY-**-NSpNAm---r----

IIGRSW F---AL---A ----T--V--“--A*-“+, ----TV-“-A ----TV-“-A ----T--“-----TL-“-----&“-“-A ---+“-“-A ----T--“-“---TV-“-A ----T--“-----TL-“-“---TL-“----Tee”-----T--V-W--AL-“----+-A-“---T----““---“---A

23EWUBEH -R-----MD KP-------N DP----K--D*----*--DP----K--m K*---K--N K&---K--N DP----K--DP----K--KQ---K--N GT----K--N Kp-------N

KE----K--D KE----K--N KQ----K--N KP-------N LE----K--KG----K--D j(A--.----N GQ----K-DT

ELSKVTGNAG -G-RT---S-E-TK-------LS--------+-------T----w -E-TK-----E-TK-------+--------se----E-TX-----E-m-----E-TK-----E-~----DE-TK-----E-TK-----E-TK-------PT----DE-*-----E-LX----.E-LK-----

-N-P------N----I---N-Q------N-*---T--N-Q-----------&T-N----L---N----I---

-x----y---

GRIACGWGL P----ATI-F S------I-I --“---II-A-IG---I-I A-I‘+--I-I S------I-I S------I-I --I---II---“---II-S------I-I p-p----I-I S------I-I S------I-I $,------I-I $,------I-I -------I-I -_--__---_ -------I-F S------I-T p-p----I--

153

ME R-P -K ,-&. -K” -KI -p, -Q, Qc, pG, -Q, SQ, TQ, -KD -p. -Q. -p. Tp” cp, E., TN.

Note. The top line is the amino acid sequence of schistosome CT Cu/Zn-SOD. The second line is that of schistosome SP Cu/Zn-SOD. The copper/zinc binding sites (in bold type) are formed by 6 His residues (Hd5, H4,, H,,, H,,, H,,, and H,,,) and one Asp (Da,) residue. The two cys (C,, and C,,,) residues (in bold type) are believed to form a disulfide bond. The Arg (R& residue is believed necessary to guide the superoxide to the activity site (Tamer et al. 1982). All of the Cu/Zn binding sites and activity sites are conserved. The identical residues are represented by short dashes (-). Deletions are represented by dots (.). The underlined amino acids in the schistosome CT Cu/Zn-SOD (CT SMSOD) indicate sequences which were determined by peptide sequence analysis.

more related to each other than they are to any other SOD, and the conserved residues are those which are also conserved in other species. Differences between the two schistosome proteins are summarized in Table II. The only conserved regions in the two schistosome sequences at the DNA level are those which encode the conserved protein sequences discussed above. However, of the 63 possible codons which could be identical because of amino acid homology, only 30 are. The remaining 32 codons have differences in the first or third position of

the codon. This suggests that the two genes are quite distinct and highly diverged. Expression of enzymatically active schistosome CT CulZn-SOD in E. coli. The entire coding region of CT Cu/Zn-SOD cDNA was inserted into the bacterial expression vector pBAce, as described under Materials and Methods (Fig. 2). To examine expression, a culture of E. coli (DHSol strain) containing the recombinant plasmid pBAce-CTSOD was induced by phosphate depletion of the medium (Craig er al. 1991). As a control, a culture of bacteria containing the parental vector pBAce was also in-

316

HONG ET AL. TABLE II Differences between CT Cu/Zn SOD and SP Cu/Zn SOD of S. Munsoni Properties

CT Cu/Zn-SOD

SP Cu/Zn-SOD

Length Molecular weight Predicted pI value Net charge Hydropathicity Hbmoiogy to bther SODS Hyrophobic signal peptide

153 a.a. 15,693 6.6 -5 More hydrophilic

184 a.a. 20,346 10.2 +7 More hvdroohobic

duced. Total bacterial extracts were prepared from both cultures and were analyzed for protein content and SOD enzyme activity. An initial analysis was made by using SDS-PAGE. After electrophoresis, the gel was stained with Coomassie brilliant blue (Fig. 5, lanes 1 and 2). A 16-kDa protein band was observed in pBAce-CTSOD extract (lane 2) that was not present in the extract derived from the vector control (lane 1). The total extract was then separated into a soluble supematant and an insoluble fraction by low-speed centrifugation. The 16-kDA band remained predominantly in the soluble supematant (Fig. 5, lane 3) although a small amount was also present in the insoluble cellular debris (Fig. 5, lane 4). The size of the induced protein is consistent with the observed molecular weight of parasite-derived CT Cu/Zn-SOD on SDS gels. The soluble supernatants of the bacterial extracts were also analyzed for SOD activity using the SOD nitrite assay. To do this, 1 mg of total protein from the extracts (in a volume of 200 ~1) was loaded onto a FPLC Superose 12-gel filtration column (Pharmacia). Fractions from the column were collected and aliquots of each fraction were assayed for SOD activity with or without the addition of cyanide. The results of this assay are shown in Fig. 6. A large peak of Cu/Zn-SOD activity was observed in the column fractions from the pBAce-CTSOD culture (Fig. 6A). This activity was cyanide

6025% -

4ck”ro

2&30 a.a.

sensitive in that it was reduced about 45 fold in the presence of 1 mM cyanide. On the other hand, a much lower amount of SOD activity was observed in the column fractions from the control extract (Fig. 6B). This activity was largely cyanide insensitive. These results indicate that the schistosome enzyme was efficiently expressed in the bacterial culture containing the expression plasmid. The position of the peak of cyanide sensitive activity corresponds to a molecular mass of 30-40 kDa, suggesting a dimer form of the enzyme, which is a property common to other native cytosolic Cu/Zn SODS. The SOD activity observed in the presence of cyanide and in the control culture probably corresponds to the cyanide-insensitive Mn-SOD/Fe-SOD known to exist in bacterial cells (Keele et al. 1970; Yost and Fridovich 1973). Purification of recombinant CT CulZnSOD. The bacterially produced schisto-

some SOD was purified to homogeneity. A typical purification started with 4 liters of bacterial culture which yielded 7 g of bacterial cells (wet weight), containing a total protein content of 900 mg. The bacterial cells were disrupted in 20 ml of PBS by a French pressure cell and the insoluble cellular debris was spun down by centrifugation. Soluble extract was first precipitated with 0.4% Polyethylenimine (PEI) to remove nucleic acids and then with (NH&SO+ The SOD stayed in the soluble fraction after PEI precipitation (Fig. 5, lane 5) but was precipitated with (NH&SO4 in

CT:

-22

SP:

-6

CT: SP:

-TTTTTTACAAAGTCATACGAGG . ..ATTAAAATGACAGTAl'ATTCC...TATTATCATCGTAGACAT M T V Y S . ..Y Y H RR

19 6 MKAVCVMTG TAGVKGVVKFT 1 ATGAAAGCTGTTTGTGTTATGACTGGTACAGCTGGCGTAAT I I I I I II I I I I I IIIII II 97 TTTGATCCGGCTATTGCTTCATTTACGAAGGRACCATATATATA~TGC~TGTGGTTC~ FDPAIA GAVWFT SFTKEPYI ;'E

CT: SP:

CT:

SP:

96 H

218

D2 N

G

3 60

II

II 157

13 0 19 17 VHVH. AXE F SGLKAG 61 CA?d%AACTGATAACGGACCTGTTCATGTTCAC...GCCGAATTCAGTGG?.CTCAAGGCTGGT III I III I III III II II III II 158 CAACATGGAGATTATATGTACGTTAATGGAAGCGTGGCA.........~TCCCACCTOOA QHGDYMY VNGSVA...GLPPG

121

T

18

P

2 19 19 15 19 19 11 19 KH. GFXVBEFGDT TNGCTSA AAACAT...WXTTCCkTGTTCATGAATTTGGTGATACAACAAATGGATGTACTTCAGCT IIII II IIIIllIII I III I III III AAACTGTTGGGTACACAl!GTTCATCGTTATlXAGGACTTGG~CATGTGTCTTGAAGCT KLLGTBVBRYGGLGNMSLEA 19

19

17

19

0 19 18 KQssEIIGAPEDSIR& GGAGCTCATTTCAATCCAACTRRACAAGAACATGGAACAC II IIIIIIIIIIIII II III Illll Ill CCTCCTCATTTCAATCCTTTCAACCAACGTCATGGTCCAC GPHFNPFNQRB GPRHGYPRH

17 120

IIII

217

13 180 I

I I I I 277

19

19

19

GABFNPT

CT: SP:

181 278

CT:

241

SP:

238

CT: SP:

CT: SP:

CT:

301 398

361 458

421

SP:

518

CT:

481

SP:

CT:

240 I

I

I

II

337

19 19 18 19 19 7 18 16 VGPLGNVVA GADGNAVYNAT GTTGGTGACCTGGGAAA TGTTGTGGCGGGTGCTGATCCGCAGTTTATAACGCGACT I III II I lllll II I II I III II I I I GCTGGAGATTTAGGAAACATTAGAGTTGGACGAGGTGGTGTAGCGAAATTTGATTTCTAT RVGRGGVAKFDFY AGQLGNI 16 19 19 19 D K L I SLNGSH SIIGRSMVIH GACAAATTAATATCTTTGAACGGCTCTCATTCAATCATTG JIJI II I I I I II I I llllllll I I GTTACTATAAAAGGTCTAGGACCATTTGATGATGGATTTATT~TCGAGCACTTGTTAT~T VT I KG L G P F D GFIGRALVIB

300 I 397 19

2

19 360

lllllllll

19 19 19 19 4 17 19 0 19 DDLGRGGHELS KVTGNA E N121E GAAAATGAAGATGATTTAGCGTGGTGGTCATGAGCTCAGCAAAGTAACTGGTAATGCT I IIll IIllllIII I II I II III I I III llllll GCAAATAGGGATGATCTTGGAAGAAATCGAGACGUGGCAGTAGAACAACGGGTAATTCT A NRDDLGRNRDEGS RTTGNS 19 12 17 19 19 0 19 GGBLAC G V V G LA A152E l GGTGGTCGTTTAGCTTGTGGCGTTGTTGGTTTAGCTGCTGAGTAGAGTGGTAGATTTATG III I IIIII II I IIIII III I III I GCTCCCAGATTAGCATGGTCTACAATAGGTTTTTCGT~CCATG~TG..A..TTTTTG GPBLACATI GFRAP*

457 19

19 420 II 517

480 III

II

577 540

578

TTTTTACGTTTTTCATGCCTATTCTCCCTGTGAAACAGTATGATCATTATTGTCAAARAG I I I I I I I I I I II I I Ii II II .TTGGACGTACCACATAATATCAGATAATAAA TTCATACTGAAAGTTC...

541

GGAAGTTTTGTGTTCCAAATCATGCAC

586

ABBTBBBGTACATACATAT...

617

FIG. 4. Sequence comparison between schistosomal CT Cu/Zn-SOD and SP Cu/Zn-SOD. The top strand DNA sequence represents that of the CT Cu/Zn-SOD cDNA and the bottom strand represents that of the SP Cu/Zn-SOD cDNA. The amino acid sequence above the top strand DNA sequence is derived from the CT Cu/Zn-SOD cDNA and the amino acid sequence below the bottom strand is from SP Cu/Zn-SOD cDNA. Both DNA sequences are numbered separately starting from their start codon. Ammo acid residues and nucleotides which are identical between the two are given in boldface type. The numbers above the boldface amino acids in the CT Cu/Zn-SOD protein sequence indicate the number of Cu/Zn-SODS from 19 other species which contain an identical residue at that particular position. All of the known Cu/Zn binding sites are underlined. These include 6 Hs, 1 D, 2 Cs, and 1 R. The N-linked glycosylation site in SP Cu/Zn-SOD (NGS) is also underlined, as are the poly(A) addition signals in both cDNAs. The asterisks (*) indicate the stop codons. Deletions are represented by dots (.). The 5’ untranslated region and the first 11 codons of the SP SOD cDNA sequence are deleted for convenience.

318

HONG ET AL. 12

3

4

5

67

Comparison between bacterially produced CT CulZn-SOD and schistosomederived CT CulZn-SOD. The bacterially 4-69

kd

c-44

kd

e29

kd

Cl8

kd

t-14

kd

FIG. 5. Pm&cation of recombinant CT Cu/Zn-SOD from E. coli. The protein composition at each purification step was analyzed by 15% SDS-PAGE using extracts equivalent to 0.5 ml of a bacterial culture. After electrophoresis, the gel was stained with Coomassie brilliant blue. Lane 1, Total extract of bacteria containing the control vector; lane 2, total extract of bacteria containing the expression plasmid, pBAceCTSOD; lane 3, soluble fraction of the total extract from bacteria represented in lane 2; lane 4, insoluble fraction of the total extract from bacteria represented in lane 2; lane 5, supematant after 0.4% PEI precipitation of the soluble fraction in lane 3; lane 6, 65-90% saturated ammonium sulfate precipitate of the supernatant of lane 5, resuspended in 10 mM Tris-HCl (pH 7.5); lane 7, Pooled Mono Q fractions (10 mkf TrisHCl, O-O. 1 M NaCl) from protein suspension in lane 6.

the fraction that was between 65%-90% saturated (Fig. 5, lane 6). The pellet was then solubilized in 10 mM Tris-HCl (pH 7.5) and the enzyme was purified to homogeneity on a FLPC Mono Q column (Fig. 5, lane 7). Calculations of protein concentration and SOD activity indicate that the specific activity in the starting bacterial extract was about 5200 NU/mg protein (equivalent to 610 cytochrome c units/mg protein). The purified protein had a specific activity of 35,000 NU/mg protein (equivalent to 4100 cytochrome c units/mg protein), which represented a sevenfold purification. Approximately 30-40 mg of purified SOD was obtained per 4 liters of starting culture.

synthesized Cu/Zn-SOD was compared to the native protein purified from the soluble adult worm extract. Several different criteria were used to evaluate their similarity. (1) Specific activity. The specific activities of the two proteins were compared by an indirect immunological method, since the yield of parasite-derived SOD was too low to determine protein content by conventional assays. For this procedure, we started with preparations of each enzyme that had the same activity per milliliter and carried out a series of twofold dilutions. The diluted enzymes were then analyzed by Western blot using the cross-reactive antiSP SOD polyclonal antiserum to develop the blot (Fig. 7A). The relative amount of immunoreactive protein in each lane was determined using a laser densitometer. From this analysis we can conclude that the native enzyme is about 1.6-1.8 times more active then the bacterially produced protein, assuming that these proteins have the same immunoreactivity toward the anti-SP SOD serum. The 40% loss of the activity in the bacterially produced SOD can be partially compensated by incubation with 100 pJ4 Cu’+/Zn*+ (10% increase). There is no effect of incubation with Cu*+/Zn*’ on the native SOD activity (data not shown). This suggests that at least part of the activity loss in the bacterial preparation may be due to the lack of the metal ions. (2) ZEF analysis. Purified bacterially produced CT Cu/ZnSOD was analyzed by IEF gel electrophoresis on native activity gels and compared to the native protein (Fig. 7B). Both preparations of protein gave two bands which focused at the same positions (p1 = 6.0 and 6.3). (3) N-terminal sequence. The aminoterminus of the bacterially produced SOD was determined by automated Edman degradation using an automated sequencer (Applied Biosystems 470A) (data not shown). It has the same sequence as that of

Schistosoma

mansoni:

CYTOSOLIC Cu/Zn SOD

319

S4lkd 30kd

‘““1B

2

--CN

1

400

-+CN

300

.P .5 ” ;: a

200

40 80 kd

100

20

01 1

0

FPLC Superose-12

Fractions (ml)

4

I

2

3

4

5

6

FPLC Superose-I2

7

6

9

10

11

12

Fractions (ml)

FIG. 6. Analysis of schistosome CT Cu/Zn-SOD activity in E. coli. Bacterial cells were lysed using a French pressure cell and extracted in PBS (pH 7.2). The soluble fraction was injected onto an FPLC Superose 12-gel filtration column and eluted with 30 ml PBS at a rate of 0.5 ml/mitt. The eluent was collected as l-ml fractions. Fraction 1 represents the first milliliter after the void volume (MW % 200 kDa). Fraction 12 corresponds to a MW of several hundred daltons. The MW of each fraction was calibrated using proteins of known MW as standards. An aliquot from each fraction was taken and SOD activity was determined. A shows the activity profile of an extract from bacterial cells containing the pBAce-CTSOD expression plasmid. Panel B shows the activity profile from bacteria1 cells containing the control plasmid. 0 No KCN added to the assay; n 1 n&f KCN added to the assay.

the parasite-derived SOD for at least 10 residues and it is also unblocked. Taken together, the above characterizations lead us to conclude that the bacterially produced schistosome CT Cu/Zn-SOD has essentially the same properties as the native protein isolated from schistosomes. DISCUSSION

Superoxide dismutase has been studied extensively in many species and its biological function is generally believed to protect cells from oxidant-related injuries. It forms the first line of defense against deleterious superoxide radicals (Hassan and Schellhorn 1988). In vivo studies on the biological function of SOD have been carried out mostly in E. coli possessing double mutations (sodA- sodB -) in the endogeneous SOD genes (Carlioz and Touati 1986). In these mutants, it was observed that lack of SOD activity enhanced oxygen-dependent mutagenesis of the bacterial genome (Farr et al. 1986). This provided direct evidence

that SOD prevents oxidative damage to DNA. In other studies expression of human Cu/Zn-SOD in the E. coli SOD mutant has been shown to complement the SODdeficient phenotype and to increase cell growth (Natvig et al. 1987). Overexpression of bovine Cu/Zn-SOD in Drosophila melanogaster has also been shown to augment resistance to oxidative stress and to increase the longevity of Drosophila (Phillips et al. 1989; Reveillaud et al. 1991). Schistosomes are notorious for their abilities to circumvent the immune responses of the host (Smithers and Terry 1969). One possible mechanism of immune evasion could be the expression of antioxidant enzymes that would suppress oxidative killing by the host effector cells (Callahan et al. 1988). SOD is believed to play a major role in the antioxidant system of the parasite (Mkoji et al. 1988; Hong et al. 1992). Studies which demonstrate the presence of SOD activity in many parasitic species (Paul and Barrett 1980; Fairfield et al. 1983; Rhoads

320

HONG ET AL.

(B) PI

s.mmsoni

(A) Activity

(NU)

0.25

0.5

1.0

2.0

E. coli 1

S. mansoni 2

E.coli 4.0

0.25

0.5

1.0

2.0

4.0

6.0 + 6.3 -+

I

FIG. 7. Comparison of CT Cu/Zn-SOD isolated from bacteria and schistosomes. (A) Quantitative Western blot analysis of the purified CT Cu/Zn-SOD. The indicated amount of SOD activity derived from purified E. coli and S. mansoni protein preparations was loaded onto the lanes of a 15% SDS polyacrylamide gel. The gel was electroblotted to an Immobilon-P membrane and developed using a cross-reactive anti-SP Cu/Zn-SOD antiserum and alkaline phosphatase-conjugated second antibodies. The immunoreactive protein content of each lane was determined by scanning the blot with a laser densitometer. (B) IEF activity gel analysis of purified CT Cu/Zn-SOD. Purified CT Cu/Zn-SOD from E. co/i (lane 1) and S. mansoni (lane 2) was analyzed on native IEF gels. Following isoelectrofocusing, the gels were stained for SOD activity as described under Materials and Methods.

1983; Kazura and Meshnick 1984; Leid and Suquet 1986; Sanchez et al. 1988; Callahan et al. 1991; Henkle et al. 1991; Michalski and Prowse 1991) as well as those which demonstrate acquisition of SOD from the host (Fairfield et al. 1983) further support the hypothesis that a role for SOD may be to protect the parasite against host toxic oxidants. However, direct evidence which demonstrates the function of SOD in the parasite defense system is lacking. In the present study, we have cloned, sequenced, and expressed a new species of schistosome SOD. The Cu/Zn-SOD we have studied represents the major species of SOD activity present in the parasite. Other studies, to be presented elsewhere, suggest that a great deal of this enzyme is present at or near the surface of the adult parasite (Hong ef al., 1992). As such, it is an excellent candidate for involvement in the parasite defense system. Our data demonstrate that the enzyme produced in bacteria behaves essentially the same as the enzyme derived from schis-

tosomes. We thus have available for the first time, large quantities of a highly purified, enzymatically active protein for further study. This will facilitate functional and structural studies of the protein, including its crystallization. These studies may ultimately lead to the design of antischistosomal drugs targeted specifically against the schistosome SOD. Schistosome CT Cu/Zn-SOD will also be tested in in vivo protection assays as a potential vaccine candidate, since we have shown that this enzyme is enriched in extracts of the surface tegument fraction. Two other schistosome proteins (glutathione S-transferase and paramyosin) have been shown to provide some degree of protection to host animals against schistosome infection (Capron ef al. 1987; Pearce et al. 1988). In the present case, it should also be possible to raise specific anti-schistosome SOD monoclonal antibodies and determine if they will enhance the oxidative killing of the parasites by the host effector cells. Similar experiments have been performed suc-

Schistosoma

mansoni:

cessfully in the study of Nocardia asteroides infection, in which anti-nocardial SOD monoclonal antibodies potentiate the effects of host immune attacks (Beaman and Beaman 1990). Note added in proof. Cordeiro da Silva et al. (1992, Molecular and Biochemical Parasitology 52,275-278)

recently published a short communication reporting the sequence of a cDNA clone encoding a cytosolic Cu/Zn SOD identical to the sequence reported herein except that amino acid 115 is a serine instead of a threonine and amino acid 148 is a valine instead of an isoleucine. Both of these amino acid changes are due to single base changes in the first position of their respective codons. ACKNOWLEDGMENTS This research was supported in part by National Institutes of Health Grant AI 18867 and World Health Organization Grant 890217.

AUSTIN, F. E., BARBIERI, J. T., CORIN, R. E., GRIGAS, K. E., AND Cox, C. D. 1981. Distribution of superoxide dismutase, catalase, and peroxidase activities among Treponema pallidum and other spirochetes. Infection and Immunity 33, 372-379. BADWEY, J. A., AND KARNOVSKY, M. L. 1980. Active oxygen species and the functions of phagocytic Annual

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49,695-

BEAMAN, L., AND BEAMAN, B. L. 1990. Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host. Infection and Immunity 58, 3122-3128. BEAUCHAMP, C., AND FRIDOVICH, I. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 44, 276-287.

CALLAHAN, H. L., CROUCH, R. K., AND JAMES, E. R. 1988. Helminth anti-oxidant enzymes: A protective mechanism against host oxidants? Parasitology Today 4, 218-225. CALLAHAN, H. L, CROUCH, R. K., AND JAMES, E. R. 1991. Dirofilaria immitis superoxide dismutase: Purification and characterization. Molecular and Biochemical Parasitology 49, 245-25 1. CAPRON, A., DESSAINT, J. P., CAPRON, M., OUMA, J. H., AND BUTTERWORTH, A. E. 1987. Immunity to schistosomes: Progress toward vaccine. Science 238, 1065-1072.

CARLIOZ, A., AND TOUATI, D. 1986. Isolation of superoxide dismutase mutants in Escherichia co/i: Is

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