# Analysis of PfEMP1—var  Gene Sequences in Different Plasmodium falciparum Malarial Parasites

Authors: Aarti Ozarkar; D. Prakash; Dileep Deobagkar; Deepti Deobagkar
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

Plasmodium falciparum synthesizes P. falciparum erythrocyte membrane protein-1 (PfEMP-1),
a product of the multicopy var gene family, which localizes on the surface of infected erythrocytes. This protein plays
an important role in cytoadherence and immune evasion. Comparative analysis of the molecular sequences of the
DBLα domain of the var gene from different isolates of the parasite reveals variations in the number of cysteines and presence of small conserved motifs like DGEA, RGD, GAG-binding motifs. Phylogenetic analysis while highlighting the extensive diversity leads to clustered them in separate clades far apart from each other. Discriminant factor analysis of physicochemical properties of amino acid sequences revealed that the aliphatic index, isoelectric  point, and instability index have more effect in deciding the variance of different isolates sequences. The origin of diverse repertoire of the DBLα domain in the parasites highlights the complexity of host-parasite relationship in the context of parasite survival.


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## Body

## 1. Introduction

Malaria has emerged as a major health
problem especially in tropical and subtropical regions of the world [1]. P.
falciparum erythrocyte membrane protein-1 (PfEMP-1) is a product of the
multicopy var gene family [2]. The
gene has 60 copies; however, only one gene is expressed at a time. This variant
surface antigen has a major role in evasion of the host immune response and
cytoadherence [3–6]. PfEMP1 acts as a ligand for host endothelial
epithelial receptor. This results in sequestration of infected erythrocytes in
the microvasculature of the brain, placenta, and other organs thereby causing cerebral malaria and
severe malaria. PfEMP1 is a virulence factor and plays an important role in
pathophysiology of the disease (severe malaria and cerebral malaria) and
enhances survival of the parasite. Thus, it has been considered as  one of the vaccine targets [7]. PfEMP1 is composed of four domains: an N-terminal segment (NTS),
Duffy binding-like (DBL) domains, cysteine-rich interdomain region (CIDR), and
C2 domains [8]. DBLα domain is also becoming the target of immunoepidemiological
and vaccine production studies to analyze diversity in the parasite
population’s worldwide [9].We have reported distinct size
polymorphism of DBLα domain of the var gene in cultured and clinical isolates of malaria parasites [10]. There are
several reports that characterize the extent of sequence diversity of var genes in different geographical
regions [11–21]. It seems likely that the extent of
diversity reported till date indicates only the tip of the var gene diversity iceberg [22]. Malaria is prevalent and
reemerging in India.
Till date there are scanty reports regarding var gene diversity from
India [21, 23]. Thus in order to understand the divergence in the sequence and
structural motifs of var genes in
Indian and Thailand
parasite lines and field isolates from western part of India, we have analyzed
sequences from the DBLα domain of the var gene whose flanking regions are highly conserved and compared
our sequence data with the reported other Indian sequences deposited in GenBank.


## 2. Materials and Methods

### 2.1. Parasites Culture

P.
falciparum parasite lines were cultured as described earlier [10]. Briefly,
the erythrocytic stages from India (FAN5 HS,
PUNE-1, FMN-17), Thailand (SOHS,
MP-14), and Netherlands (3D7)
were cultured in RPMI 1640 medium supplemented with 0.5% Albumax II (Gibco-BRL,
Md, USA) [24]. The
field isolates from India (C, D, NS1, NS2,
and NS6) were not cultured and were used directly for isolation of genomic
DNA.


### 2.2. Isolation of Genomic DNA

Parasites were collected by
centrifugation from cultures with 20% parasitemia with mature trophozoites
stages and washed with PBS. Parasitized RBCs (p-RBC) were lysed with saponin
(0.15%), and the parasite pellet washed with PBS. DNA was isolated by treatment
with proteinase K in the presence of sodium dodecyl sulphate followed by phenol
chloroform extraction and subsequent ethanol precipitation [25].


### 2.3.var Gene Primers and PCR Amplification

DBLα primers (synthesized by Invitrogen, Md, USA)
of var gene were used for
amplification of parasite genomic DNA (forward primer: CGACACCGGCGACATTATAAGAGG
(primer1) and reverse primer:
TCGCAGGTATTGTGGCACGTAGTC (primer2)). The primers were specific for the two
highly conserved sites in DBLα domain and flank the polymorphic segment of DBLα
domain. The amino acid sequences were DTGDIIRG and DYVPQYLR (see
Figure 1 in Supplementary Material available
online at doi:10.1155/2009/824949).
PCR was carried out as described earlier [10].


### 2.4. Cloning and Sequencing

The PCR products were purified using
PCR purification system (Roche). The DNA was ligated into pGEM-T easy system
(Promega) and used to transformE. coli TOP10
cells (Invitrogen). The recombinants were sequenced using T7 and Sp6 primers
with BigDye Terminator v3.1 cycle sequencing kit  (Applied Biosystems 3730 sequencing machines).


### 2.5. Bioinformatic Analysis

The nucleotide sequences derived from
the parasite lines and field isolates were analyzed for sequence similarities
by NCBI BLAST
(http://www.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide sequences were translated
into amino acid sequences using Expasy Translation tool. Pepstat analysis was performed
to derive information about the amino acid sequences. By the SIM Alignment Tool, local similarity
program (http://ca.expasy.org/tools/sim-prot.html)
analysis was carried out. The
multiple sequences were aligned using MultAlin program (http://bioinfo.genotoul.fr/multalin/multalin.html).
Pairwise sequence alignment was generated using ISHAN-Integrated Software for
Homology Analysis (http://physics.unipune.ernet.in/~pbv/ishan.html) [26]. The
percentage of amino acid identity between sequences was calculated. An identity
matrix analysis was performed, in which each sequence from a parasite was
compared to all other sequences from the same isolate and from different
isolates. The resultant matrix contained the percent sequence identity of each
sequence relative to all other sequences. The mean and range values for the
sequence comparison data were calculated.
The percent similarity values against the sequences was plotted in
MsExcel. The sequences (38) from other Indian isolates (ICGEB-R1, R15, R35, and
MRC20) and 3D7 were derived from GenBank database and were also used for comparative
analysis of the data. Multiple sequence alignment of the sequences for
phylogenetic analysis of the parasite isolates wascarried out using ClustalW (http://www.ebi.ac.uk/clustalw/).
Phylogenetic tree was constructed by using WebPhylip. The trees were
reconstructed using protpars program, that implements maximum parsimony method.
The phylogenetic analysis was performed to determine the relationships between
the var gene sequences. The T- and B-cell epitopes prediction was carried out, as described earlier [27].  Although the prediction was carried out for
few sequences by us, we have studied extensively the T-cell epitopes for all
the sequences.


### 2.6. Statistical Analysis

The physicochemical properties of an
amino acid sequence were determined using Protparam. The data was analyzed
using multivariate statistics, as they offer the advantage of taking into
account all the variables in a single analysis, thus making it possible to
assess variation in the molecular
weight, pI, amino acid index, aliphatic index, hydropathic index, and
instability index of an amino acid sequence. The sequences were grouped among four groups,
namely, Indian lab adapted, Indian field, ICGEB sequences [23], and MRC-20 sequences
[21]. Discriminant factor analysis (DFA) was done to find out variables which
were most useful for discriminating between the sequences of different groups. We
performed Pillai’s trace statistics to find out whether the clusters were
significantly different from each other [28].


## 2.1. Parasites Culture

P.
falciparum parasite lines were cultured as described earlier [10]. Briefly,
the erythrocytic stages from India (FAN5 HS,
PUNE-1, FMN-17), Thailand (SOHS,
MP-14), and Netherlands (3D7)
were cultured in RPMI 1640 medium supplemented with 0.5% Albumax II (Gibco-BRL,
Md, USA) [24]. The
field isolates from India (C, D, NS1, NS2,
and NS6) were not cultured and were used directly for isolation of genomic
DNA.


## 2.2. Isolation of Genomic DNA

Parasites were collected by
centrifugation from cultures with 20% parasitemia with mature trophozoites
stages and washed with PBS. Parasitized RBCs (p-RBC) were lysed with saponin
(0.15%), and the parasite pellet washed with PBS. DNA was isolated by treatment
with proteinase K in the presence of sodium dodecyl sulphate followed by phenol
chloroform extraction and subsequent ethanol precipitation [25].


## 2.3.var Gene Primers and PCR Amplification

DBLα primers (synthesized by Invitrogen, Md, USA)
of var gene were used for
amplification of parasite genomic DNA (forward primer: CGACACCGGCGACATTATAAGAGG
(primer1) and reverse primer:
TCGCAGGTATTGTGGCACGTAGTC (primer2)). The primers were specific for the two
highly conserved sites in DBLα domain and flank the polymorphic segment of DBLα
domain. The amino acid sequences were DTGDIIRG and DYVPQYLR (see
Figure 1 in Supplementary Material available
online at doi:10.1155/2009/824949).
PCR was carried out as described earlier [10].


## 2.4. Cloning and Sequencing

The PCR products were purified using
PCR purification system (Roche). The DNA was ligated into pGEM-T easy system
(Promega) and used to transformE. coli TOP10
cells (Invitrogen). The recombinants were sequenced using T7 and Sp6 primers
with BigDye Terminator v3.1 cycle sequencing kit  (Applied Biosystems 3730 sequencing machines).


## 2.5. Bioinformatic Analysis

The nucleotide sequences derived from
the parasite lines and field isolates were analyzed for sequence similarities
by NCBI BLAST
(http://www.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide sequences were translated
into amino acid sequences using Expasy Translation tool. Pepstat analysis was performed
to derive information about the amino acid sequences. By the SIM Alignment Tool, local similarity
program (http://ca.expasy.org/tools/sim-prot.html)
analysis was carried out. The
multiple sequences were aligned using MultAlin program (http://bioinfo.genotoul.fr/multalin/multalin.html).
Pairwise sequence alignment was generated using ISHAN-Integrated Software for
Homology Analysis (http://physics.unipune.ernet.in/~pbv/ishan.html) [26]. The
percentage of amino acid identity between sequences was calculated. An identity
matrix analysis was performed, in which each sequence from a parasite was
compared to all other sequences from the same isolate and from different
isolates. The resultant matrix contained the percent sequence identity of each
sequence relative to all other sequences. The mean and range values for the
sequence comparison data were calculated.
The percent similarity values against the sequences was plotted in
MsExcel. The sequences (38) from other Indian isolates (ICGEB-R1, R15, R35, and
MRC20) and 3D7 were derived from GenBank database and were also used for comparative
analysis of the data. Multiple sequence alignment of the sequences for
phylogenetic analysis of the parasite isolates wascarried out using ClustalW (http://www.ebi.ac.uk/clustalw/).
Phylogenetic tree was constructed by using WebPhylip. The trees were
reconstructed using protpars program, that implements maximum parsimony method.
The phylogenetic analysis was performed to determine the relationships between
the var gene sequences. The T- and B-cell epitopes prediction was carried out, as described earlier [27].  Although the prediction was carried out for
few sequences by us, we have studied extensively the T-cell epitopes for all
the sequences.


## 2.6. Statistical Analysis

The physicochemical properties of an
amino acid sequence were determined using Protparam. The data was analyzed
using multivariate statistics, as they offer the advantage of taking into
account all the variables in a single analysis, thus making it possible to
assess variation in the molecular
weight, pI, amino acid index, aliphatic index, hydropathic index, and
instability index of an amino acid sequence. The sequences were grouped among four groups,
namely, Indian lab adapted, Indian field, ICGEB sequences [23], and MRC-20 sequences
[21]. Discriminant factor analysis (DFA) was done to find out variables which
were most useful for discriminating between the sequences of different groups. We
performed Pillai’s trace statistics to find out whether the clusters were
significantly different from each other [28].


## 3. Results

The genomic DNA from parasites amplifies
multiple bands in the size range of 350–700 base pair
(bp) when amplified with the primer1 and primer2  (see Figure 2 in Supplementary Material available online at doi:10.1155/2009/824949) [10]. The PCR products were cloned, and 328
plasmids were screened. The Indian isolate FAN5
HS showed maximum number of clones whereas in Thai isolate SOHS yielded maximum number of clones. A total of 189 nucleotide sequences from
Indian and Thai parasite lines and field isolates were obtained. The nucleotide
sequences have been deposited in GenBank (accession numbers: DQ 364441–364452, DQ 408208–408223, EF 143927–EF 143965, EU 333323–EU 333374).
SIM analysis of amino acid sequences predicted 99 unique sequences—Indian lab
adapted (FAN5 HS-12, PUNE-1-5, FMN17-4),
Thai lab adapted (SOHS-55, MP-14-7), and field isolates (C-8, D-4, NS1-20, NS2-14, NS6-6). The nucleotide sequences when submitted
for NCBI-blast showed more than 50% homology to sequences from different
parasite lines and isolates in the GenBank.The nucleotide sequences of DBLα were
translated into amino acid sequence and analyzed by PepStats analysis. It
provides information about various features of the amino acid sequence.
Majority of the sequences had small, polar, and charged residues (see Table 1).
The amino acid sequence was analyzed using Multalin (see Figures 1 and 2). The
analysis of the aligned DBLα sequences indicates presence of universally
conserved blocks in the sequences (Supplementary Figure 1). Comparison of
sequences showed that there is marked conservation in the positions of certain
residues in DBLα domain. The conserved blocks were interspersed with variable
blocks, and these sequences varied extensively in both sequence and length. The
conserved block LREDWW was conserved throughout the lab adapted parasite lines
(Indian and Thai), whereas it was semiconserved in field isolates of India. The
sequences showed presence of conserved large bulky aromatic amino acids such as
phenylalanine (F), tryptophan (W), and tyrosine(Y) and both hydrophobic as well
as hydrophilic residues. It is interesting to note that a few sequences are
shared and show similarity with other parasite lines while most sequences show
extensive variation.Table 1
Comparative summary of Pepstat analysis.
Sr. no.Property       (Residues)FAN5 HS-1MP14-7C23ICGEB25MRC20-70Number (mole %)1Tiny3232362726(A + C + G + S + T)(25%)(26%)(27%)(23%)(21%)2Small6161635453(A + B + C + D + G + N  + P  +  S + T + V)(48%)(49%)(46%)(45%)(43%)3Aliphatic1716191818(I + L + V)(13%)(13%)(14%)(15%)(15%)4Aromatic1817171716(F + H + W + Y)(14%)(14%)(12%)(14%)(13%)5Nonpolar5855595354(A + C + F + G + I + L + M+ P + V + W + Y)(45%)(45%)(43%)(44%)(44%)6Polar7168776668(D + E + H + K + N + Q + R + S + T + Z)(55%)(55%)(57%)(55%)(55%)7Charged4242443845(B + D + E + H + K + R + Z)(32%)(34%)(32%)(31%)(37%)Figure 1
MuItiple sequence alignment of sequences derived from Indian lab adapted isolates. Multiple alignments of sequences derived from the DBLα domain of the parasite lines from India (FAN5 HS = Fa, PUNE-1 = Pu, and FMN-17 = F). Dots indicate gaps necessary for alignment. The Mulltalin program was used to align amino acid sequences, with the program default parameters.  DGEA motif was observed in FMN-17 isolate (F1 and F2) and GAG binding motifs were observed in all the sequences. Positions of limited variability 2 (PoLV2 = LREDWW) and CRC are conserved in all sequences.Figure 2
MuItiple sequence Alignment of DBLα domain from field isolates from India. Multiple alignments of sequences derived from the DBLα domain of the Indian field isolates (C, D, NS1, NS2, and NS6). Dots indicate gaps necessary for alignment. The Mulltalin program was used to align amino acid sequences with the program default parameters. Positions of limited variability 2
(PoLV2 = LREDWW) and CRC are not conserved in all sequences.Although the DBLα domain has an
average of 4
cysteine residues, the number varied from 2 to 9 cysteines. The parasite line
from Thailand (S61) showed presence
of 2 cysteines and was shorter in length (120 amino acids). The cysteines at
CRC position were conserved throughout the Indian parasite lines whereasin field isolates and parasite lines
from Thailand, it was semiconserved. The cysteine position in the homology
block VWKAiTC is conserved in all sequences whereas in homology block YFraTC , it is semiconserved in parasite lines
from Thailand. In parasite line from India, FAN5
HS, a single sequence had 3 cysteines and the cysteine in YFraTC block was
absent.Two of theFAN5 HS sequences showed extra long sequences of 276 and 250 amino
acids with additional 156 and 148 amino acids at 5′ end, respectively. Similar
case was noted in a field isolate NS6. A single sequence of the NS6 field isolate
has showed presence of an extra long sequence of length of 165 amino acids with
additional 35 amino acids at the 5′ end of the sequence. There was a
duplication of 6 amino acid residues (DTGDII) at the 5′ end of the sequence.
The FAN5 HS sequences showed presence
of conserved blocks in DBLα domain namely GACAPYRRLH and CTLARSFADIGDI while
NS6 sequence showed presence of CTLARSFADIGDI motif (Supplementary Figure 1).The
presence of small motifs (GAG-glycosaminoglycan binding, DGEA motifs, RGD
motifs) was observed both in semiconserved and highly variable regions (see Table2). The number of GAG binding motifs varied in all the sequences. The DGEA
motif was observed only in the Indian parasite line, FMN-17 (F1, F2) while RGD motif was observed only in parasite line
from Thailand, namely, SOHS (S64).Table 2
Summary of motifs present.
Sr. No.Motifs presentIsolates name1DGEA
motifFMN-17-1 and FMN-17-22RGD
motifSOHS-S643LDV
motifFew
3D7 and ICGEB sequences4GAG—bindingMajority
of parasite lines of India,motifsThailand,
and field isolates sequencesPairwise
sequence comparisons were carried out to determine average sequence identity.
It can be seen that although most of the sequences varied in their degrees of
identity between 19%–62%, a few
sequences were 99% identical to each other (see Table3). There is a low
sequence identity among the var sequences of Indian lab isolate, FAN5 HS. The sequences showed variation
and were separated in different peaks (see Figure 3). The sequences analyzed in this study were
grouped close together than other reported Indian sequences, whereas all Indian
isolates showed similarity to each other when compared to 3D7. A given parasite
line appears to contain sequences which cluster in distinctly different groups
showing more similarity to sequences from other isolates and differed
extensively from each other.Table 3
Pairwise comparison fromvar genes
from Plasmodium falciparum Indian and
Thai parasite lines
and Indian field isolates.
Sr.
no.Isolates
nameRange
(%)Average
identity (%)Indian
parasite lines1FAN5 HS/FAN5 HS19–99592PUNE-1/PUNE-146–98723FMN-17/FMN-1749–99734FAN5 HS/PUNE-119–99595FAN5 HS/FMN-1747–56516PUNE-1/FMN-1748–5752Thai  parasite lines1SOHS/SOHS43–99712MP-14/MP-1453–99763MP-14/SOHS49–9974Indian
field Isolates1C/C48–97722D/D52–59553NS1/NS145–99724NS2/NS253–99765NS6/NS641–99706C/D48–99737C/NS144–99718C/NS248–62559C/NS639–615010D/NS150–987411D/NS246–605312D/NS642–605113NS1/NS245–605214NS1/NS638–996815NS2/NS640–9969Figure 3
Pairwise sequence alignment graph of Indian isolates and 3D7. Pairwise sequence alignment was performed using ISHAN software and the matrix was plotted using MsExcel. x-axis shows isolates sequences whereas y-axis shows percent identity.The phylogenetic unrooted tree was
constructed using amino acid sequences from parasite lines of India and
Thailand and field isolates  (see Figure4). These sequences were compared to
3D7 sequences and other previously reported Indian sequences available from
GenBank database. The sequences of the individual isolates were clustered
markedly in separate clades apart from each other. The parasite lines from India, FAN5 HS and PUNE-1, were closely related to each other whereas FMN-17 was far away from them. The
parasite line from Thailand
and field isolates was clustered in distinctly separate groups. A few field
isolates were close together to parasite lines from India and Thailand.
However when compared with 3D7 sequences, the 3D7 sequences fall in separate
clusters apart from parasite lines from India and Thailand
and field isolates.Figure 4
Phylogenetic tree of sequences of parasite lines from India lab adapted and field isolates and ICGEB and MRC20 sequences and 3D7. Unrooted phylogram was constructed by using WebPhylip—protpars program that implements maximum parsimony method. Yellow block represents 3D7 DBLα sequences, light blue block represents Indian lab adapted parasite lines (FAN5 HS, Pune-1, FMN-17), green block represents Indian field isolates (C, D, NS1, NS2, NS6), dark blue represents ICGEB sequences, and pink block represents MRC-20 sequences. The sequences of the isolates are clustered in separate groups apart from each other.Discriminant factor analysis of physicochemical properties of
amino acid sequences derived from different parasite lines revealed that the
four groups, that is, Indian lab adapted, Indian field, ICGEB isolates, and
MRC-20, do not form significantly different clusters  (see Figure5(a)).  The
aliphatic index, isoelectric point, and instability index have more effect in
deciding the variance of different isolates sequences, where as molecular
weight of the protein, its amino acid index and hydropathicity index are not
significant in deciding the variation (see Figure 5(b)).Discriminant factor analysis of physicochemical properties of an amino acid sequence. (a) Clusters of different groups, (b) variables which discriminate between the clusters. Group 1 = Indian lab adapted isolates (red), 2 = Indian field isolates (green), 3 = ICGEB isolates (pink), and 4 = MRC-20 (blue). Aliphatic index, isoelectric point and instability index have more effect in deciding the variance of different isolates, whereas molecular weight, amino acid index, and hydropathicity index are not significant.
(a)(b)The T-cell epitope prediction revealed
that majority of the sequences showed weak binding motifs; however few
sequences (11) showed strong binding motifs (see Table4).  The ability to bind T cells seems to show
differences in the various genes. The sequences showed presence of B-cell epitopes
in all sequences.Table 4
Summary of strong binding T-cell epitopes in Indian isolates.
Sr.
No.Isolates
nameSequence1PUNE-1-4ETDQVPTYY2C2,
D12CSADRGNAY3NS1-24CSADTGNAY4NS1-66,
NS6-9,YQDESGNYYNS6-12, NS6-155ICGEB-5SDRCGHNYN6ICGEB-16LTDAKAKSY,
CSAEENDRY7MRC20-1LTDPKAKEY


## 4. Discussion

The diversity in thevar gene
in malarial parasite has been reported. In the present study, we have
critically analyzed var gene sequences in terms of motifs, amino acid,
physicochemical properties, and constructed phylogenetic trees from var gene sequences derived from parasite
lines from India and Thailand and also field isolates from Western part of
India. In this analysis, the variation in the DBLα domain in sequences derived
from Indian and Thai
laboratory adapted parasite lines and field isolates was compared to sequences
reported in GenBank from other Indian isolates and 3D7. Extensive polymorphism
and variations were
observed in DBLα domain. The multicopy DBLα domain seems to be highly diversified while conserving
the salient features of the domain. Thus we have observed a highly dynamic and
variable picture of var gene
organization. Polymorphism within the hypervariable region leads to length
variation and sequence variability. The var gene diversity at genomic level among parasite isolates both within and
among endemic areas has been reported earlier [11–21]. The parasite line from India
(FAN5 HS) and field isolate (NS6) both show presence of extra long sequences where DTGDII motif is repeated. It
is likely that events such as duplication and DNA recombination may be
possible. Thus gene recombination and duplication have been an important mechanism for generating
diversity in var genes [12, 13, 29–31].
It has been shown that children with symptomatic infections had a greater
repertoire of variant-specific antibodies [16]. Additionally, the var genes of P. falciparum have
been reported to undergo constant changes due to frequent recombinationor rearrangements that generate a vast
repertoire of var genes in nature.NaturalPlasmodium falciparum populations are genetically diverse, to an
extent that within some geographic regions nearly all isolates contain unique
parasite genotypes with regard to polymorphic single copy genes [11]. The repertoire is so immense that it
raises questions about the possible molecular genetic mechanism instrumental in
creating such high variability while conserving the important functionally
conserved regions. The possible implications of this in immune evasion and
persistence of parasites in host are of great importance to pathogenicity and virulence. It was
reported that global var gene repertoire was immense even among
geographically close isolates. High degree of similarity was observed among
sequences from 3D7 [14]. In three different geographical parasite lines, highly
divergent var gene sequences were observed [20]. Vast amount of global var gene diversity was reported whereas limited amount of diversity was observed in
PNG isolates [21]. Limited diversity was observed in India
as the isolates were
collected from an area having low malaria transmission rate. However, we
observed considerable diversity among clones, even within a single isolate. It
was reported that varCOMMON type gene
family is found in few Kenyan
and Indian ICGEB isolate R35 [32]. However, our sequences derived from parasite
lines and field isolates data analysis do not show varCOMMON type gene.It
was reported that DBLα domain
comprises of 400 amino acids and contains 16–18 conserved
cysteine residues [8, 12, 33]. The number of cysteine residues is important
for classification of DBLα domain and severity of disease [8, 16]. Cysteine 8
was conserved in all sequences. The position of cysteine at CRC motif
was conserved in all Sudanese var genes.  We also observed the similar
conserved cysteines (CRCs)
in the Indian isolates. Variants with unusual number of cysteines may form a
subset with altered antigenic and adhesive properties. Sequences predominantly
expressed in patients with severe malaria could be subgrouped on the basis of
number of cysteine residues. These sequences were commonly found in the var gene repertoire of parasites from
patients with mild malaria [34]. This
suggests that positions of cysteines are important for DBLα structure, possibly
because they are involved in disulphide bonding or other aspects of
conformation or folding. The cysteine disulphide bonds may be important for
stabilizing the surface-located variable loops and protuberances which appear to
play a critical role in folding or structural conformation of whole PfEMP1
protein. Presence of conserved cysteines and hydrophobic amino acid residues suggests that they are
structurally important.Thevar genes in Plasmodium falciparum genome are classified into 17 different
protein architectural types based on domain compositions [35]. The genome is
divided into 3 major subgroups (A, B, C) and two intermediate groups (B/A, B/C)
on the basis of 5′ upstream (Ups) sequence and chromosomal location [36, 37].
Group A and B/A genes have DBLα 1 type domain whereas groups B, C, B/C, or B/A have DBLα domain.
Based on the number of conserved cysteine residues in the sequences ,var genes are grouped as DBLα
and DBLα1 domains. DBLα domain has 4 conserved cysteine residues whereas DBLα1
has 2 cysteine residues [38]. However, we too report that majority of the
sequences have DBLα domain, that is, 4 cysteine residues, and all the isolates
are from severe malaria cases.A number of charged potentially exposed
amino acid residues are also present in these segments. Divergent segments
contain multiple hydrophilic residues suggesting that they are likely to be
exposed and may serve as epitopes for agglutinating antibodies. Presence of
common segments even in divergent regions of DBLα domains and the hydrophilic
nature of the sequences suggest
that it is likely that they are both exposed and form epitopes for antibody
recognition.  The presence of conserved
cysteines and tryptophan (W) residues is amongst the most highly conserved residues
suggesting that DBL domains share conserved three-dimensional structures. We
have shown that there is variation in T-cell and B-cell epitopes and also in
their 3D structures. The difference in number and position of cysteine residues
suggests a rearrangement of disulfide bonds leading to different three-dimensional
structures [39].Sequences consisting of position of
limited variability (PoLVs 1–4) and the number of cysteine residues have been
suggested as signature sequences of the DBLα domain [16]. Each PoLV was four
amino acids long and situated adjacent to conserved amino acid residues at the
boundary of previously defined islands of homology. In the sequences from
parasite lines from India,
PoLV2 (LREDW) is conserved whereas in parasite lines from Thailand
and field isolates all
PoLV 1–4 are different. Thus the sequences derived from parasite lines and
field isolates differed from each other, and variation was also observed within
sequences of a single parasite line.The glycosaminoglycan (GAG) binding
motifs are clusters of positively charged amino acid residues. These small
amino acid motifs are linked to the severe states of malaria [22]. We observed
them in sequences obtained from parasite lines from India and Thailand
and field isolates. These motifs are responsible for involvement of the protein
in rosette formation of iRBC. It was observed that similar motifs could not be
linked to severity of the disease [15]. It has been suggested that intravenous
injection of glycosaminoglycan disrupts rosettes and releases already
sequestered parasites into circulation. Thus it can act as candidate for
adjunct therapy in severe malaria [40]. Heparin sulfate on the surfaces of
uninfected RBC may act as receptors for rosetting and that GAG-binding motifs
of PfEMP1 mediate this binding [39].
DGEA mediates integrin-collagen interactions in platelets. It has been
suggested that it is important in cell adhesion molecules and can therefore be
a target for therapeutic intervention [41]. The RGD motif was observed in parasite line from Thailand
SOHS and in 3D7 sequences. The LDV motif was observed in 3D7 and
other Indian isolates (ICGEB-R1, R15, R35), but it was not present in any of
our sequences. The RGD and LDV motifs are associated with protein-protein interaction and cell
attachment [42]. They are cell adhesion motifs involved in ligand-receptor
interactions involving the integrin family [43]. Thus it can act as a
potential therapeutic agent for the treatment of disease.The T- and B-cell epitopes in
different sequences of DBLα domain of parasites show extensive variation. The
epitopes varied in its location and amino acid composition. Due to such
variation, it is difficult to make an effective vaccine. Thus the conserved
epitopes are required for an effective vaccine design against malaria.The evolutionary relationships
of conserved cysteine rich motifs in adhesive molecules of malaria parasites
have been studied [44].  The report
suggests that rapid divergence originated from multiple gene duplication
events. There is a sequential pattern of repeated duplication and
diversification. The unrooted phylograms showed that the genes show diversity
even among the lab adapted and field isolates from India.  The sequences in the genes of Indian and Thai isolates are quite
diverse from that of 3D7. This
interesting pattern demonstrates the variations in the sequence organization of
the var gene domains and raises
interesting possibility about role of mutations and recombination in the generation
of this diversity. The role of host-induced immune pressure remains to be elucidated.The
DBLα domain of pfEMP1 displays extensive divergence in both sequence and length
even among the same parasite line. Despite the extent of sequence diversity in
DBLα domains, it is predicted that due to the presence of conserved cysteines
and homology blocks between invariant cysteines, the DBL domain may have a
common fold. As a result, the receptor-binding pockets may lie in the same
region of diverse DBL domains. The evolution of variation in the var gene
sequences in different geographical parasite lines and field isolates with
respect to small motifs seems
to play important role for parasite survival as well as to evade immunity.



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*Source: 824949-2009-03-25.xml*