Download Sequence conservation among merozoite apical complex proteins

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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