Molecular and Biochemical Parasitology 90 (1997) 155 – 168

Organization, sequence and stage-specific expression of the

phosphoglycerate kinase genes of Leishmania mexicana
mexicana 1

C.A. Adjé, F.R. Opperdoes, P.A.M. Michels *

Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology (ICP),
and Laboratory of Biochemistry, Catholic Uni6ersity of Lou6ain, ICP-TROP/74.39, a6enue Hippocrate 74,
B-1200 Brussels, Belgium

Received 24 May 1997; received in revised form 25 August 1997; accepted 26 August 1997

Abstract

In Leishmania mexicana two genes were detected coding for different isoforms of the glycolytic enzyme phospho-
glycerate kinase. This situation contrasts with that observed in other Trypanosomatidae (Trypanosoma brucei,
Trypanosoma congolense, Crithidia fasciculata) analyzed previously, which all contain three different genes coding for
isoenzymes A, B and C, respectively. All attempts to detect in L. mexicana a type A PGK, or a gene encoding it,
proved unsuccesful. We have cloned and characterized the genes PGKB and PGKC. They code for polypeptides of
416 and 478 amino acids with a molecular mass of 45146 and 51318 Da, respectively. The two polypeptides are 99%
identical. PGKC is characterized by a 62 residue C-terminal extension with alternating stretches of hydrophobic and
charged, mainly positive amino acids. As in other Trypanosomatidae, PGKB is located in the cytosol, PGKC in the
glycosomes. However, Leishmania mexicana distinguishes itself from other trypanosomatids by the simultaneous
expression of these isoenzymes: :80% of PGK activity is found in the cytosol and 20% in the glycosomes, both in
promastigotes and in the amastigote-like form of the parasite. © 1997 Elsevier Science B.V.

Keywords: Phosphoglycerate kinase; Gene analysis; Isoenzymes; Glycolysis; Leishmania mexicana

Abbre6iations: G6PDH, glucose-6-phosphate dehydrogenase; HK, hexokinase; MDH, malate dehydrogenase; PCR, polymerase
chain reaction; PEP, phosphoenolpyruvate; PEPCK, PEP-carboxykinase; PGK, phosphoglycerate kinase; PTS, peroxisome-targeting
signal; PYK, pyruvate kinase.
* Corresponding author: Tel.: +32 2 7647473; fax: + 32 2 7626853; e-mail: michels@trop.ucl.ac.be
1
Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB data bases under the
accession numbers X98486 (PGKB) and X98487 (PGKC)

0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.

PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 1 5 2 - 7
156 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
1. Introduction
In Kinetoplastida, the glycolytic pathway is
organized in a peculiar manner. The majority of
the glycolytic enzymes reside in organelles called
glycosomes [1]. Only the last part of the pathway
is present in the cytosol. Activity of some gly-
colytic enzymes has been detected in both the
glycosome and the cytosol. That is, for instance,
the case for phosphoglycerate kinase (PGK; EC
2.7.2.3.).
The structure and kinetics of Trypanosoma bru-
cei PGK, and the organization and expression of
its genes has been studied in detail [2 – 4]. Three
tandemly arranged genes (PGKA, PGKB and
PGKC) have been detected, encoding different
isoenzymes of PGK. One gene (PGKB) codes for
a cytosolic isoenzyme of 45 kDa, another (PGKC)
for an enzyme of 47 kDa located in the glyco-
some. PGKC is post-translationally targeted to
the glycosome by a hexapeptide -NRWSSL at the
end of a 20 amino-acid C-terminal extension [5].
Furthermore, the PGK gene complex contains a
third gene (PGKA) encoding a 56 kDa PGK-like
glycosomal protein of unknown function. In com-
parison with PGKB and PGKC, PGKA has an 8
amino-acid extension at the N-terminus and an 80
amino-acid insertion in the N-terminal half of the
protein [4,6]. PGKB and PGKC are very similar:
95% amino-acid identity. PGKA is more different
(80% identical to B and C). It has been estab-
lished that PGKB and PGKC are monomeric
proteins [3,7], similar to PGK in most other or-
ganisms [8].
Levels of the major PGK isoenzymes are stage
regulated, with PGKC abundant in the mam-
malian stage and absent in the procyclic stage,
whereas the reverse was observed for PGKB
[9,10]. PGKA is constitutively expressed at low
level within the two main stages of T. brucei.
The subcellular localization of PGK and the
gene organization have also been studied in
Crithidia fasciculata [11] and Trypanosoma congo-
lense [12]. The situation in these organisms ap-
peared to be similar to that in T. brucei: three
tandemly-linked genes A, B and C, with similar
expression patterns and coding for proteins with
similar subcellular distribution as in T. brucei.
Striking differences are, however, that C. fascicu-
lata has a much longer C-terminal extension of 38
amino acids ending in -MVLASP and that in T.
congolense genes B and C are identical as result of
a gene conversion. The encoded protein (PGKC1/
C2) is cytosolic, because of the lack of the C-ter-
minal extension.
Here we report our analysis of PGK in Leish-
mania mexicana mexicana, where the situation
appears somewhat different. Only two genes
rather than three have been observed in the PGK
locus.
2. Materials and methods
2.1. Growth of parasites
Promastigotes of L. m. mexicana (NHOM/B2/
84/BEL46) were grown at 27°C in the semi-
defined medium SDM-79, supplemented with 10%
heat-inactivated fetal calf serum (Gibco, UK) [13].
L. mexicana was adapted to grow as amastigote-
like forms in axenic culture at 36°C, with an
atmosphere containing 5% CO2 in a medium as
described in [14], but modified as follows: growth
medium RPMI 1640 was supplemented with 1/40
vol. Eagles Basal vitamin mix (Gibco)/25 mM
Mes, pH 5.5/100 mM adenosine/28 mM folic acid.
Fetal calf serum and hemin were added at a final
concentration of 20% and 5 mg l − 1, respectively.
2.2. Cloning and sequencing of PGK genes
Purification of high molecular weight DNA
from L. mexicana promastigotes and the construc-
tion of a genomic library in Escherichia coli
MB406, using the phage vector lGEM11
(Promega) have been described [15]. The library
was screened for recombinant clones containing
the Leishmania PGK genes by hybridization with
the complete PGKB gene of T. brucei [2]. Screen-
ing was performed at moderately stringent condi-
tions: 3×SSC/0.1% SDS, in the presence of 10%
dextran sulfate, at 65°C (1× SSC= 150 mM
NaCl/15 mM sodium citrate, pH 7.0). Post-hy-
bridization washes were carried out at 65°C for 1
h with 3× SSC/0.1% SDS, followed for 40 min
 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
with 1×SSC/0.1% SDS and finally for 40 min
with 0.3×SSC/0.1% SDS. Positive recombinant
lGEM11 phages were purified and rescreened.
High-titre phage lysates were prepared and DNA
was purified from phages as described [16].
Exonuclease III (Erase-a-base, Promega) was
used for progressive shortening of the subcloned
fragments for a serial sequencing approach on one
strand. The other strand was sequenced using
specific oligonucleotides. DNA sequence analysis
was performed using the T7 DNA polymerase kit
of Pharmacia (Sweden). Other procedures used
for DNA analysis have been described previously
[17].
2.3. Subcellular fractionation by digitonin
treatment of intact cells
Cells in the late exponential phase of growth
( : 8 ×107 cells ml − 1) were harvested by centrifu-
gation at 2000× g for 10 min at 4°C. The pellet
was washed twice with an iso-osmotic buffer con-
taining 25 mM Tris – HCl, pH 7.4/1 mM EDTA/
250 mM sucrose and finally resuspended in this
same solution. Shortly before performing the en-
zyme assays, aliquots of the cell suspension were
diluted in Hanks’ balanced salt solution [18] to a
density of 1 mg protein ml − 1 and a variable
amount of digitonin was added. The mixture was
incubated at 25°C for 5 min and then centrifuged
for 2 min at 13 000×g. Enzymes were assayed, as
described [7], immediately after centrifugation, in
the supernatant. Total cellular enzyme activities
were determined after adding 0.1% (v/v) Triton
X-100 to the cells. Protein concentration were
measured by the fluorescamine method [19] with
bovine serum albumin as standard.
2.4. Electrophoretic analysis of proteins
Polyacrylamide gel electrophoresis in the pres-
ence of 0.1% SDS [20] was done following stan-
dard techniques. Isoelectric focusing was
performed using an Ampholine PAGplate, pH
3.5 – 9.3, following the instructions of the manu-
facturer (Pharmacia).
157
2.5. Immunoblotting
Immunoblotting of proteins after size-fractiona-
tion by electrophoresis, was carried out as de-
scribed [21]. After blotting, the Immobilon PVDF
membranes (Millipore) were blocked by incuba-
tion in Tris-buffered saline (10 mM Tris–HCl, pH
8/150 mM NaCl) containing 4% bovine serum
albumin. The blots were immunostained by incu-
bation first with a rabbit polyclonal antiserum
raised against PGK purified from T. brucei glyco-
somes. The antiserum was used in a dilution of
1/250. Subsequently, the blot was incubated with
the second antibody coupled to alkaline phos-
phatase at a dilution of 1/7500 and a combination
of Nitroblue tetrazolium (0.34 mg ml − 1, Sigma)
and 5-bromo-4-chloro-3-indolyl phosphate (0.17
mg ml − 1, Sigma).
2.6. DNA amplification by PCR
The existence of a PGKA gene in L. mexicana
was tested by PCR, using various sets of specific
primers. Two sets of oligonucleotides were de-
signed based on conserved (60–80%) regions at
the borders of all available PGKA sequences
Fig. 1. Schematic representation of aligned PGK genes of
Trypanosomatidae and the position of oligonucleotides used
for PCR. The black box in PGKA represents the 240 bp
specific insertion. The grey and hatched boxes indicate con-
served segments of trypanosomatid PGK genes. The arrows
represent the oligonucleotides used: 1, PGKsA1; 5%CTGCAT-
CCTCATCAGCCACCTC3%; 2, PGKsA2; 5%GCTTCTCTTC-
CTCTGGCAG3%; 3, PGKsA3; 5%TTGA(CT)CAGGGTGG-
(CT)T(CT)TG3%; 4, PGKsA4; 5%CTC(AT)GG(AC)AGGCGC-
GCAAA3%; 6, PGKs6; 5%CT(GC)(AC)TAGCCACCT(GTC)-
GG3%; 7, PGKs7; 5%(GAC)(AT)T(GAC)TCCT(GT)CTC(AC)-
(GC)(AT)CA3%; 12, PGKL12; 5%GAAGGTGCTCATCCGC-
GTG3%; 13, PGKL13; 5%CAGTGCCAAAGGCATCGC3%.
158 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168

Fig. 2. Physical map of the genomic clones of L. mexicana PGK in lGEM11, called lLmPGK1-3. Parts were subcloned in plasmid
pBSSK+ , and called pCAO1-02. Boxes represent the protein-coding regions of the PGK genes; the hatched area corresponds to the
part of the PGKC gene encoding the C-terminal extension. The black bars correspond to the phage vector sequences. The arrows
below the map indicate the DNA segments subcloned in plasmids. The thick lines underneath represent the areas sequenced. Every
part indicated has been sequenced several times; sequencing of coding regions was performed in both directions. C, ClaI; E, EcoRI;
H, HincII; Hd, HindIII; N, NdeI; P, P6uII; S, SacI; X, XhoI.

(primers 1 + 2 and 3 +4, Fig. 1). Two other sets
of oligonucleotides (primers 6+7 and 12+ 13)
were selected on the basis of an alignment of all
available trypanosomatid PGK sequences. These
latter oligonucleotides correspond to partially
conserved areas of the PGK genes, located on
both sides of the PGKA specific insert (Fig. 1).
Amplification was performed using 1 mg of total
genomic DNA from L. mexicana as template, and
in some cases also DNA from L. dono6ani. As
positive control, 1 mg of total DNA from other
trypanosomatid species (C. fasciculata, T. brucei )
was used. The reaction was done in a total vol-
ume of 100 ml containing 10 mM Tris – HCl, pH
8.8/0.1% Triton X-100/50 mM KCl/1.5 mM
MgCl2/200 mM each of the deoxynucleotides and
0.1 mM of each primer. Amplifications were car-
ried out in a hybaid thermal reactor (Hybaid,
UK) and started by the addition of five units of
Primezyme DNA polymerase (Biometra, Ger-
many). The program for amplification was as
follows: first cycle: 5 min 95°C, 30 cycles: denatu-
ration 30 s, 93°C; annealing 30 s− 1 min, 48°–
60°C, dependent on the primer–DNA
combination; extension 1 min, 72°C and a final
cycle of 10 min, 72°C. The PCR products thus
obtained were run on agarose gels and either
checked by Southern blot analysis or cloned into
pBSIIKS having T-overhanging 3% ends (T-vec-
tor), prepared as described [22], and sequenced.
2.7. Northern blot analysis
Total RNA was isolated from L. mexicana
amastigote-like form cells (3× 109 cells) and pro-
mastigotes (5× 108 cells) according to standard
procedures (Qiagen, Germany). Poly(A) + RNA
was then purified using the polyAT tract mRNA
isolation system of Promega. The procedure used for
RNA blot analysis has been described before [17].
 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
Fig. 3. The primary structure of the C-terminal extension of L. major and L. mexicana PGKC. The areas rich in charged residues
are in bold characters, the stretches containing many hydrophobic amino acids are underlined. The arrow indicates a segment of
amino acids with a high probability to form an a-helix.
3. Results
3.1. Cloning and organization of PGK genes of L.
mexicana
The PGKC gene of T. brucei was used as probe
to screen a L. mexicana genomic library in
lGEM11, at moderately stringent conditions
(0.3 × SSC, 65°C). Ten positive clones were ob-
tained. Three of them were taken for further
analysis. Hybridizing restriction fragments were
subcloned in plasmid pBSIISK for subsequent
sequence identification (Fig. 2).
Clone lLmPGK1 (subclone pCA01) contains a
single PGK gene coding for an amino-acid se-
quence with a high level of similarity with PGK of
T. brucei (see below). This sequence has a very
long C-terminal extension, suggesting that it codes
for a glycosomal PGK protein. By analogy to the
situation in other trypanosomatids, this gene was
named PGKC.
A second clone (lLmPGK2, subclone pCA02)
also contained a single PGK gene with an open-
reading frame highly similar to the first one, but
lacking the C-terminal extension. This second
gene was named PGKB.
Restriction site mapping of a third subclone
pCA03, derived from lLmPGK3 containing a
fragment of 12 kb of genomic DNA of L. mexi-
cana, confirmed the presence of the two genes
(PGKB and PGKC), which are tandemly arranged
as indicated in Fig. 2. The two genes are sepa-
rated by an intergenic region of about 1 kb.
3.2. Sequence analysis of L. mexicana PGK genes
Sequence analysis of the clones showed that the
first gene (PGKB) has an open-reading frame of
1248 bp encoding a polypeptide of 416 amino
acids, with a molecular mass of 45146 Da (not
159
counting the initiator methionine). The second
one is 1434 bp long and encodes an amino-acid
sequence of 478 residues with a molecular mass of
51318 Da. The last 62 amino acids form a C-ter-
minal extension, because they are absent from
most other PGKs. A C-terminal extension is a
characteristic of the glycosomal PGKs of try-
panosomatids.
Sequence comparison of the two polypeptides
shows 99.0% positional identity (Table 1). They
differ by only 65 residues, of which three are
internal substitutions, while the other 62 corre-
spond to the C-terminal extension. Two substitu-
tions are located in the N-domain of the protein.
Arg95 in polypeptide B is replaced by an Ala in
polypeptide C. Glu102 in the first polypeptide is
substituted by a Lys in the second one. The third
substitution concerns the last amino acid of
polypeptide B which is a Gln. In the second
polypeptide a Glu is found at the equivalent
position.
A highly similar situation has been described
for C. fasciculata PGK, were the B and the C
polypeptides differ by only two amino acids in the
N-domain and the presence of a C-terminal exten-
sion in the PGKC protein [11].
The calculated net charge of the polypeptide
encoded by gene B is 0, while the value for the
second polypeptide is + 8. Strikingly, the excess
of basic residues in the C-terminal extension is
responsible for this difference between the two
polypeptides.
The situation in Leishmania major appears very
similar [23]. Its PGKB and PGKC are nearly
(99.5%) identical, except for a highly-charged (+
8) C-terminal extension of also 62 residues in the
latter. Here the enzymes have a calculated net
charge of +3 and + 11, respectively. The L.
mexicana and L. major PGKB and PGKC are 89
and 92% identical. However, their C-terminal ex-
 160

Table 1
Percentage identity between the amino-acid sequences of PGK in trypanosomatids

Tbru-A Tcon-A Cfas-A Cfas-B Cfas-C Lmaj-B Lmaj-C Lmex-B Lmex-C Tcon-B Tbru-B Tbru-C
Tbru-A —
Tcon-A 82.4 —
Cfas-A 56.9 56.0 —
Cfas-B 69.5 73.1 55.3 —
Cfas-C 69.5 72.7 55.3 99.3 —
Lmaj-B 70.2 74.3 54.8 89.0 89.2 —
Lmaj-C 70.2 74.3 54.8 89.0 83.1 99.5 —
Lmex-B 70.0 73.9 54.1 86.8 87.3 92.1 92.3 —
Lmex-C 70.2 74.1 54.6 87.1 80.4 92.3 88.7 99.0 —
Tcon-B 73.1 84.3 53.2 76.7 77.0 77.7 77.9 77.9 78.2 —
Tbru-B 78.1 72.4 54.4 74.1 74.1 74.1 74.1 74.6 75.3 81.4 —
Tbru-C 78.4 72.4 54.2 73.9 74.1 74.1 71.6 74.3 71.6 82.4 92.6 —

Pairwise comparison of the trypanosomatid PGK complete amino-acid sequences, not counting insertions and deletions.
C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
tension differs more and shows only 65% posi-
tional identity. Strikingly, the extension of PGKC
of both Leishmania species displays a similar pat-
tern of alternating clusters of charged and hydro-
phobic residues (Fig. 3). Moreover, both
sequences contain a stretch of amino acids with a
very high probability to form an a-helix (PHD
program, EMBL, Heidelberg).
In order to analyze further the relationship
between the L. mexicana PGKs and other try-
panosomatid PGKs, a multiple alignment was
made with the PGK sequences available in Gen-
Bank™ and using the programme ClustalW(1.5)
(Fig. 4).
As shown in Table 1, the Leishmania PGKs are
most related to the corresponding enzymes of C.
fasciculata. The same result was obtained when the
separate domains were compared: the N-domain of
all trypanosomatid PGKs in front of the 80 amino-
acids insertion present in the PGKA polypeptide,
and the C-domain (not including the extension) of
the proteins. A striking observation was that be-
tween the L. mexicana and C. fasciculata PGKs a
higher degree of similarity was observed in the
N-domain than their C-domain, whereas in a
comparison with the enzymes of other trypanoso-
matids the C-domain appeared always to be better
conserved than the N-domain (not shown).
3.3. Search for a PGKA related protein and its
gene
In all Trypanosomatidae analysed so far (T.
brucei, T. congolense and C. fasciculata), PGK
genes are organized in a locus containing three
tandemly-linked genes A, B and C, of which gene
A contains a 240 bp insertion. However, Southern
blot analysis of the genomic clones of L. mexicana
did not show the presence of an additional gene a
short distance upstream of gene PGKB.
The possibility of the presence of a PGKA
enzyme in the cell, or a gene coding for it further
upstream of PGKB, or somewhere else in the
genome, was then further explored.
3.3.1. Sequence and hybridization analysis
Subclone pCA02 contains the B gene, a 5%
flanking region of about 3.9 kb and a 3% region of
161
about 0.8 kb. Three HincII fragments of 1.5, 0.8
and 2.5 kb, obtained by digestion of pCA02 were
analysed (Fig. 2).
When subjected to Southern blot analysis using
the L. mexicana, PGKC gene as hybridization
probe, only the 2.5 kb fragment containing 800 bp
of the PGKB gene could be detected (not shown).
Furthermore, the three HincII fragments were
subcloned and large segments of each of them
were sequenced, as indicated underneath the re-
striction map of pCAO2 in Fig. 2. No typical
PGK sequence was found in the area upstream of
the PGKB gene.
3.3.2. Western blot analysis shows only two PGK
isoenzymes in L. mexicana
Different subcellular fractions of L. mexicana
promastigotes, prepared by differential centrifuga-
tion as described [24], were subjected to SDS/
PAGE, blotted and probed with a polyclonal
antiserum raised against purified glycosomal
PGKC of T. brucei. The results are shown in Fig.
5A (lanes 1–3), together with those obtained with
total homogenates of both procyclic and blood-
stream form T. brucei as control.
In a promastigote homogenate (lane 1) two
bands of 46 and 49.5 kDa were observed. The
49.5 kDa band was predominantly found in the
small-granular fraction (lane 2), whereas the
polypeptide of 46 kDa was mainly present in the
soluble fraction (lane 3). This result confirms that
the genes PGKB (predicted Mr polypeptide 45
146) and PGKC (predicted Mr 51 318) code for a
cytosolic (soluble) and glycosomal (small granu-
lar) isoenzyme, respectively. The weak signals, at
46 kDa in the small granular fraction and at 49.5
kDa in the soluble fraction, are presumably due
to incomplete separation of both cell compart-
ments. In procyclic and bloodstream form T. bru-
cei, the 56 kDa PGKA is recognized in addition
to the 45 kDa PGKB (procyclic) and 47 kDa
PGKC (bloodstream form), although the PGKA
signal is only faintly observed in the bloodstream
form. No polypeptide of similar size could be
detected on the Western blot of L. mexicana. This
negative result may be interpreted as confirmation
for the absence of a PGKA in this latter species.
However, it is also possible that the enzyme is too
162 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168

Fig. 4. Comparison of the L. mexicana phosphoglycerate kinase amino-acid sequences with other trypanosomatid PGK sequences.
Amino acids have been aligned to obtain maximal positional identity. * indicate identity, and ’ gaps, the boundaries between the N-
and C-domains are indicated by \ B. L – mm; L. mexicana; T – bb, T. brucei; Cfas, C. fasciculata.
 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168 163

Fig. 5. Western blot analysis of PGK isoenzymes in L. mexicana. Panel A: Proteins of different subcellular fractions were size
fractionated by SDS/PAGE. Lanes 1 and 3, 200 mg of protein from a total homogenate and the soluble fraction, respectively; lane
2, 50 mg of the small granular fraction (glycosomes); lanes 4 and 5, 200 mg of protein of a total homogenate from T. brucei
bloodstream form and procyclic cells, respectively. Panel B: Proteins were fractionated by isoelectric focusing. About 300 mg of
protein from a total cell lysate of L. mexicana promastigotes (lanes 2, 4, 6) and amastigote-like forms (lanes 1, 3, 5) were loaded
in each lane of an Ampholine PAGplate, pH 3.5–9.3. Blots of both gels (panel A and B) were probed with anti-glycosomal PGK
of T. brucei. The positions of the molecular weight markers are indicated at the left (M). PI, pre-immune serum; R, antiserum raised
against recombinant PGKC; N, antiserum raised against natural PGKC.

different from the trypanosome PGK to be recog-
nized by the antiserum.
3.3.3. Application of DNA amplification by PCR in
a search for the presence of a PGKA gene in L.
mexicana
Various attempts were made to detect a PGKA
gene in L. mexicana by PCR, using either oligonu-
cleotides specific for the border of the insertion in
the PGKA gene of C. fasciculata, T. brucei and T.
congolense (primers 1+ 2 and 3+4, Figs. 1 and
6), or oligonucleotides representative of partially
conserved sequence motifs present in all available
trypanosomatid PGK sequences, including PGKA,
where they were located at both sides of the
insertion (primers 6+7 and 12 + 13, Figs. 1 and
6). With the PGKA specific primers, no fragments
of the expected length (260 bp for primers 1+2,
261 bp for primers 3+ 4) were amplified in Leish-
mania DNA. However, the proper fragments
could be detected when DNA from other Try-
panosomatidae was used. The fragments of differ-
ent length amplified in L. mexicana and L.
dono6ani DNA appeared unrelated to PGK se-
quences upon Southern analysis or cloning plus
sequencing. When using the oligonucleotides spe-
cific for conserved areas in all trypanosomatid
PGK sequences (primers 6+ 7 and 12+ 13), sev-
eral fragments were amplified, but only a single
fragment was recognized by the PGKC gene of L.
mexicana as probe. This was the fragment ex-
pected for PGKB and PGKC (420 bp, primer
6+ 7; 450 bp primer 12 + 13). A 240 bp longer
fragment, indicative for the existence of a PGKA
gene was not obtained. In contrast, the presence
of a PGKA specific band could be ascertained
after PCR with T. brucei and C. fasciculata DNA
under similar conditions. All these amplification
experiments lend further support to the notion
that there is no PGKA gene in Leishmania.
164 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168

Fig. 6. PCR amplification of segments in trypanosomatid DNA using various sets of PGK-specific primers. Lm, L. mexicana; Ld,
L. dono6ani; Cf, C. fasciculata; Tb, T. brucei; TbA, T. brucei genomic PGKA clone. For a detailed description of the experiments,
see Section 2 and Section 3.

3.4. The isoelectric point of the L. mexicana PGK 3.5. Expression and subcellular distribution of
isoenzymes PGK in L. mexicana

Isoelectric focusing of total cell lysates (pro-
mastigotes and amastigote-like forms), followed
by Western blotting, was performed to determine
the isoelectric point (pI) of the L. mexicana
PGKs. As shown in Fig. 5B, three bands were
revealed: one strong band and a doublet corre-
sponding to a pI 7.58 and a pI 9.14 – 9.20, respec-
tively. The pI 7.58 corresponds to the value
predicted for the cytosolic enzyme. The doublet
should presumably be attributed to two variants
of the glycosomal enzyme, probably products of
two slightly different alleles.
In trypanosomes, the expression of PGKB and
PGKC in the cytosol and glycosomes, respec-
tively, is subject to tight developmental control
[3,9]. Only the glycosomal PGKA is expressed at
low levels in both bloodstream and insect stages
[2,6,9]. In order to determine if the absence of
PGKA in Leishmania could be correlated with a
different expression regime of the other isoen-
zymes, the subcellular distribution of PGKs was
measured in both promastigotes and amastigote-
like forms of L. mexicana. Therefore, aliquots of
cultured parasites in the exponential phase of
 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
Fig. 7. Release of various enzymes from intact L. mexicana promastigote (A) and amastigote-like form (B) cells by treatment with
increasing concentrations of digitonin.
, Phosphoglycerate kinase; , Pyruvate kinase; 2, Hexokinase; , Glucose-6-phosphate
dehydrogenase.
growth were treated with various concentrations
of the detergent digitonin, to cause differential
release of soluble enzymes from cytosol and or-
ganelles. In the promastigote stage (Fig. 7A),
hexokinase (HK) and pyruvate kinase (PYK)
were used as glycosomal and cytosolic marker,
respectively. PYK activity is released at low deter-
gent concentration (0.02 mg digitonin (mg
protein) − 1) and 100% of activity is obtained at a
concentration of 0.06 mg mg − 1. HK activity was
detected at higher digitonin concentrations (0.15 –
0.30 mg mg − 1). 80% of PGK activity was released
together with PYK and thus, must be cytosolic,
whereas only 20% of the activity followed the
release of HK.
In the amastigote-like form, the activity of the
various glycolytic enzymes tested (HK, PYK, al-
dolase and PGK) was considerably lower than in
the promastigote stage. Moreover, PYK could not
be used as marker, because its activity did not
remain stable during the experiment. Therefore,
glucose-6-phosphate dehydrogenase (G6PDH),
which is mainly present in the cytosol [15,25], was
used as marker together with HK. The PGK
activity shows the same distribution profile as in
165
promastigotes: 80% cytosolic and 20% glycosomal
(Fig. 7B), but the distribution varies somewhat
between different batches of cells, with 80–90% in
the cytosol. This latter value is in agreement with
the result obtained in the isoelectric focusing ex-
periment (Fig. 3B).
The expression of PGKB and PGKC in pro-
mastigotes and amastigote-like forms was also
studied at the RNA level, by Northern blot analy-
sis. The result of the hybridization of the blot with
a PGK probe was in agreement with that of the
digitonin titration: two bands of 2600 and 1500
nucleotides were observed in both development
stages (not shown). These bands are attributed to
transcripts of PGKB and PGKC and indicate,
therefore, that both genes are expressed in differ-
ent parts of the life cycle.
4. Discussion
We have cloned and analyzed the sequence of
two genes coding for different isoenzymes of PGK
in L. mexicana and studied their expression. Our
results suggest that PGK in this organism is some-
166 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
what different from its counterpart in other Try-
panosomatidae analyzed (T. brucei, C. fasciculata
and T. congolense) in several respects.
First, in the other organisms three PGK genes
(A, B and C) are present; only two were found in
L. mexicana. Our extensive analysis strongly sug-
gests that L. mexicana does not contain a gene
related to PGKA of trypanosomes and Crithidia.
Presumably, this holds true for the entire genus
Leishmania, because in a less detailed study we
could not detect the gene either in L. dono6ani,
and similar data have independently been ob-
tained for L. major [23]. From a phylogenetic
analysis published elsewhere [26], we infer that the
common ancestor of Leishmania, Crithidia and
Trypanosoma must have possessed the PGKA
gene, but that it was lost in the lineage leading to
Leishmania.
A second aspect in which the Leishmania and
Trypanosoma PGK differ is the developmental
expression of the two isoenzymes. In T. brucei, the
cytosolic PGKB is only expressed in the procyclic
stage, whereas the glycosomal PGKC is only de-
tected in bloodstream form parasites. By reverse
genetics, it was shown that even low levels of
coexpression of cytosolic PGK in bloodstream
form T. brucei resulted in death of the cells (Blat-
tner J, Clayton C, personal communication, and
quoted in [27]). We have shown that, in contrast,
in L. mexicana the isoenzymes are simultaneously
present in cytosol and glycosomes, in a ratio of
:80/20, in both promastigotes and in amastig-
ote-like form cells.
The mutually exclusive expression of cytosolic
PGKB and glycosomal PGKC in bloodstream
form T. brucei was attributed to the different
metabolic activities of these cells and the impor-
tance to maintain the net balance of ATP produc-
tion as zero within the glycosomes [28]. In
bloodstream form trypanosomes, glycolysis is vir-
tually the only metabolic activity in the glyco-
somes. The glycosomal ATP consumed in the
early part of the pathway is then rephosphory-
lated again in the reaction catalyzed by PGK. In
contrast, glycolysis in procyclics occurs at a re-
duced rate and does not lead to the formation of
pyruvate. 1.3-Bisphosphoglycerate leaves the gly-
cosomes and is converted into 3-phosphoglycerate
by the cytosolic PGK. Phosphoenolpyruvate
(PEP) is redirected to the glycosomes which now
contain highly elevated levels of PEP-carboxyki-
nase (PEPCK) and malate dehydrogenase (MDH)
[29]. The malate, thus formed, is further metabo-
lized in cytosol and/or mitochondrion. The glyco-
somal ATP/ADP balance is now maintained by
PEPCK. In L. mexicana, the ratio 20/80 of glyco-
somal and cytosolic PGK reflects possibly the
specific activities of different routes for sugar
breakdown: glycolysis leading to pyruvate and the
formation of malate via PEPCK and MDH, re-
spectively. In addition, cytosolic PGK may also
be involved in other activities such as gluconeoge-
nesis that has been claimed to be a cytosolic
process [30].
Thirdly, L. mexicana PGK has some distinct
structural features. Like in C. fasciculata, the
primary structure of PKGB and PGKC is nearly
identical. They differ only in three internal
residues and in the presence of a 62 residues long
extension of PGKC. Therefore, it is very likely
that this extension is responsible for the targeting
of the protein to the glycosome. In T. brucei, it
was proven that the C-terminal hexapeptide con-
tains a targeting signal [5,31] which appeared to
be reminiscent to the type-1 peroxisome-targeting
signal (PTS1) [32]. An essential characteristic of
PTS1 is a hydrophobic C-terminal residue. This
was not found in L. mexicana and L. major
PGKC, which both end in -LIR. Therefore, the
topogenic signal of Leishmania PGKC is probably
not a PTS1, and may be present elsewhere in the
C-terminal extension.
Another peculiarity of the C-terminal extension
of the Leishmania enzyme is its length and the
nature of its sequence. It is 2–3 times longer than
the extension of C. fasciculata and T. brucei
PGKC, and it contains alternating stretches of
hydrophobic and charged, mainly positive,
residues. The function of this structure remains to
be determined. It may be involved in an associa-
tion of PGKC with other soluble or membrane-
associated glycosomal proteins, or it may bind
ligands, for instance to modulate the activity of
the enzyme.
An intriguing question remains why in L. mexi-
cana only two PGK isoenzymes are present,
 C.A. Adjé et al. / Molecular and Biochemical Parasitology 90 (1997) 155–168
whereas T. brucei and C. fasciculata contain three
isoenzymes. A definite answer will only be avail-
able when the physiological role of PGKA in the
other organisms becomes known.
Acknowledgements
We are grateful to Ms Dominique Cottem for
technical assistance in various aspects of the work
described; to Mr Joris Van Roy for providing
subcellular fractions of L. mexicana; to Ms
Françoise Van de Calseyde-Mylle for secretarial
help in the preparation of this manuscript; to Dr
David Hart (King’s College, London, UK) and
Drs Judith Blattner and Christine Clayton
(ZMBH, Heidelberg, Germany) for sharing un-
published data with us. This research was finan-
cially supported by the Belgium State — Prime
Minister’s Office— Science Policy Programming
grant No 93/98-163.
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