Sequence conservation among merozoite apical complex proteins of Babesia bovis, Babesia bigemina and other apicomplexa

Molecular and Biochemical Parasitology, 49 (1991) 329-332 © 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 ADONIS 0166685191004925

329

MOLBIO 01644

Short Communication

Sequence conservation among merozoite apical complex proteins of Babesia boris, Babesia bigemina and other apicomplexa Carlos E. Suarez 1, Terry F. M c E l w a i n l, E d w a r d B. S t e p h e n s 2, V i s h n u S. M i s h r a 2 a n d G u y H. P a l m e r 1 tDepartment of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, U.S.A. and 2Department qf lnfectious Diseases, University of Florida, Gainesville, FL, U.S.A. (Received 19 August 1991; accepted 21 August 1991)

Key words: Babesia rhoptry protein; Apicomplexa; Plasmodium; Rhoptry protein; Theileria

Obligate intracellular parasites of the phylum Apicomplexa cause important hemoparasitic diseases of humans and animals including malaria, babesiosis, coccidiosis and toxoplasmosis. Apicomplexan parasites contain several secretory organelles, including rhoptries and micronemes that are necessary for important parasite functions, including host cell invasion. However, analysis of apicomplexan organellar function on a molecular level has been limited. We recently reported the sequences for the genes encoding p58, a neutralization-sensitive Babesia bigemina merozoite surface protein [1] and Bv60, a Babesia boris merozoite surface protein [2]. These two polypeptides share an immunofluorescence pattern typical of apical complex polypeptides [3,4], and the Bv60 polypeptide has been localized to the rhoptry by immunoelectron microscopy (manuscript in preparation). In contrast to other babesial merozoite surface proteins which are antigenically polymorphic among strains, surface exposed epitopes on p58 and Bv60 are conserved among all examined strains of B. Correspondence address." Carlos E. Suarez, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, 99164-7040, USA.

bigemina [3] and B. boris, respectively [4]. Based on these similarities, we hypothesize that p58 and Bv60 have similar functions in merozoite invasion of erythrocytes, the target cell for both B. bigemina and B. bovis in cattle. Examination of p58 and Bv60 amino acid sequences was used to identify conserved regions that may direct common functions. Computer aided comparison between both nucleotide sequences using the software from the GCG package of the University of Wisconsin [5], revealed 47% identity. The amino acid sequences have 35% identity and 57% similarity as determined using the BESTFIT program [5]. Homology was greatest in the 300-amino acid block located at the amino terminus (45% identity and 65% similarity); this sequence is shown in Fig. 1A. The 14-amino acid sequence PLSLPNPYQLDAAF is strictly conserved in both polypeptides. The identical amino acids in the 14residue stretch occur despite nucleotide changes that result in different codon usage in 7 out of 14 codons, with the codons for the first leucine and serine residues differing in 2 nucleotides each (Fig. 1B). In addition, there are several shorter oligopeptides within the amino terminal 300 residues that are identically conserved between p58 and Bv60 (Fig.

330 A p58 By60 p58 By60 p58 Bv60

..FLGVCFGILLLV.ARSOSAIRYTHRSOVM.SAEVVODVSKTLLEANEV

46

:==..-[.1:11 -: , l l : l - - : l I : .llllll:.-tl .1:.: MRIISGVVG~LFLVFSHHVSAFRHNQRVGSLAPAEWGDLTSTLETADTL

50

VNAEMEATQVNKDMQSQLSNVKETIVGEVCEKVAGNSTCGESVIAYVNRC :. • .:.111. lli.:l ll.:li...:::l,l I i ,I.:II

MTLRD~MHNITKDM~HVLSNGREQIVNDV~SNAPEDSN~REVVNNYADR[

96

i00

OEGDCLTLDSNK. . . . . . YKPLSLPNPYQ{DAAFMLFRESDSNPAKNEVK 140 : :I:I:Io:I I.IIIlllllllllll-II:tl.ll[lll-II EMYGCFTIDNVKYPLYQEYQPLSLPNPYQLDAAFRLFKESASNPAKNSVK 150

BV60

RFWMRSRSS[.HGDYHHFVVSLLKKNVVRDPESNDVENFASQYFYMTTLY I l:i I-: !illi-ll-:ll..ill::.:.-ill :-.. :II.I: REWLRFRNGANHGDYHYFVTGLLNNNVVHEEGTTDVEYLVNKVLYMATMN

200

p58

YKTYLTVDFTAAKFFNKLAFTTRLFGFGIQKALKRLVRSNLPVDLGTHPE

238

p58

BY60 p58 Bv60

IIIIIll:

.lllil::.lll::l:

I,.-I.

::l.l:l

I::-:.

YKTYLTVNSMNAKFFNRFSFTTKIFSRRIRQTLSDIIRWNVPEDFEERSI

188

250

ATIREIASGYGEYMMTQVPAMTSFAERFSKMATKTLLVTVSDYVHLPAYK 288 • I ::.]:[::l[:il:i.:--ll I:..I..I.I1.-:.-II. I II ERITQLTSSYEDYMLTQIPTLSKFARRYADMVKKVLLGSLYSYVEAPWYK 300

p58

RWYRKFKEFIVNFFTDPAK

By60

RWIKKFRDFFSKNVTQPTK

B P

L

S

L

P

N

P

Y

Q

L

D

A

A

F

p58

n

525

CCG TTG

AGT

CTG

CCA l~,T

CCT

TAC

~G

TTG

GAC

GCT

GCC

TTC

By60

n

482

CCT

TCT

CTT

CCA

CCT

TAC

CAG

TTG

GAT

GCT

GCG

TTC

CTA

AAC

Fig. 1. (A) Comparison of the blocks containing amino acids 1 307 of p58 from B. bigemina and 1-319 of Bv60 from B. bovis using the program BESTF1T [5]. Cysteine residues are underlined. The program display the symbols '.' between residues whose comparison value [5] is greater than or equal to 0.1 or ':' when greater than or equal to 0.5. A vertical bar denotes identity between residues. The symbol '.,...' denotes a gap used to achieve optimal alignment between the two polypeptide sequences. (B) Comparison of codon usage for the region encoding the peptide (PLSLPNPYQLDAAF) common to p58 and By60. ~n', nucleotide sequence; *, denotes nucleotide changes. 1A). Between amino acids 76-101 in p58, and 80-105 in Bv60, there is a region containing 4 cysteine residues located in identical positions, suggesting that b o t h polypeptides have a similar tertiary structure. Published r h o p t r y and m i c r o n e m e protein sequences o f other a p i c o m p l e x a n organisms were searched for similarity to the p58 and Bv60 polypeptide sequences (sequences o f the Theileria parva 104-kDa m i c r o n e m e - r h o p t r y protein [6], Plasmodium falciparum r h o p t r y associated protein-1 [7], Plasmodium yoelii r h o p t r y protein (3' region) [8], and the Plasmodium knowlesi D u f f y b l o o d g r o u p receptor m i c r o n e m e polypeptide [9]). T h e identical or a closely related 14-amino acid sequence was not present in the published

sequences, indicating that the 14-amino acid oligopeptide does not mediate a c o m m o n function in all r h o p t r y polypeptides. H o w ever, evaluation o f overall sequence similarity a m o n g these apical complex polypeptides, using the p r o g r a m R D F from the Protein Resource Package [10], indicated significant sequence similarity a m o n g all published apic o m p l e x a n r h o p t r y and m i c r o n e m e sequences. The R D F p r o g r a m c o m p a r e s a test sequence with r a n d o m l y p e r m u t e d versions o f each potentially related sequence identified by the F A S T P p r o g r a m [10]. Highly significant scores ( > 3 s t a n d a r d deviations above the mean for p e r m u t e d versions) were o b t a i n e d a m o n g all the apical complex polypeptides tested (Table I), except between Bv60 and the P. knowlesi

331 TABLE I Evaluation of the statistical significance of sequence similarities among merozoite apical complex polypeptides using the program RDF

Bv60 p58 Tp Pf Py Dr

Bv60

p58

Tp

71 15 7.8 3.7 1.9

71 13 8.4 4.3 6.5

15 13 9.0 5.0 2.9

Pf 7.8 8.4 9.0 14.7 11.4

Py

Dr

Bv42

3.7 4.3 5.0 14.7

1.9 6.5 2.9 11.4 11.5

- 0.4 1.6 2.8 1.7 0.1 1.4

11.5

The RDF value indicated is the number of standard deviations above the mean optimized FASTP score of 20 shuffled sequences, p58: B. bigemina 58-kDa apical complex protein (GenBank accession No. M60878); Bv60: B. boris 60-kDa apical complex protein (GenBank accession No. M38218); Tp: Theileria parva 104-kDa microneme-rhoptry protein (GenBank accession No. M29954); Pf: Plasmodium falciparum rhoptry associated protein-1 (GenBank accession No. M32853); Py, Plasmodium yoelii rhoptry protein (3' region) (GenBank accession No. M34281); Dr, Duffy receptor family of Plasmodium knowlesi (located in micronemes) (GenBank accession No. M37513); Bv42: B. bovis merozoite outer membrane surface exposed protein.

polypeptide. In contrast, when apical complex proteins were compared to a B. bovis merozoite surface protein (Bv42) unrelated to the apical complex (submitted for publication), the R D F scores were < 3 standard deviations above the means. Conservation of the 14 residue oligopeptide in p58 and Bv60 from different Babesia species, despite changes in other regions of the polypeptide, suggests that this region mediates critical functions. Computer searches of data bases (EMBL release 26.0, Pir-Protein release 27.0, SwissProt release 17.0) did not identify any significant similarity of the 14-amino acid region to oligopeptides of known function such as enzymes or receptors. We hypothesize that the conserved oligopeptide is required for invasion of bovine erythrocytes. Invasion of different target cells by the related apicomplexan parasites may involve a similar mechanism mediated by different oligopeptides.

Acknowledgements The authors wish to thank Steven M. Thompson for consultation on sequence analysis using the VADAMS Laboratory at Washington State University. The work was supported by grants from the Agency for International Development DAN-4178-A-007056-00 and the INTA Argentina Fellowship

Program sponsored by the Inter-American Development Bank and coordinated by Winrock International.

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332 Mol. Biochem. Parasitol, 41, 125 134. 8 Keen, J., Holder, A., Playfair, J., Lockyer, M. and Lewis, A. (1990) Identification of the gene for a Plasmodium yoelii rhoptry protein. Multiple copies in the parasite genome. Mol. Biochem. Parasitol. 42, 241 246. 9 Adams, J.H., Hudson, D.E., Torii, M., Ward, G.E.,

Wellems, T.H., Aikawa, H. and Miller, L. (1990) The duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 63, 141 153. 10 Lipman, D.J. and Pearson, W.R. (1985) Rapid and sensitive protein similarity searches. Science 227, 1435 1441.