(Received for publication, April 7, 1995)
From the
Leishmania parasites are exposed to pronounced changes
in their environment during their life cycle as they migrate from the
sandfly midgut to the insect proboscis and then into the phagolysosomes
of the vertebrate macrophages. The developmental transformations that
produce each life cycle stage of the parasite may be signaled in part
by binding of environmental ligands to receptors which mediate
transduction of extracellular signals. We have identified a family of
five clustered genes in Leishmania donovani which may encode
signal transduction receptors. The coding regions of two of these
genes, designated rac-A and rac-B, have been
sequenced and shown to code for proteins with an
NH
Parasitic protozoa of the genus Leishmania experience
markedly different environments as they transit through their life
cycle. Promastigotes live in the midgut of the sandfly vector,
infectious metacyclic parasites reside in the mouthparts of the insect,
and amastigotes live within the acidic phagolysosomal vesicles of the
vertebrate host macrophages(1) . As the parasite migrates from
the midgut to the mouthparts of the sandfly and subsequently into the
phagolysosomes of the macrophage, it must be able to sense its changing
extracellular milieu and respond by initiating a developmental
transformation that culminates with the establishment of each
successive life cycle stage. Although the factors that induce these
developmental transformations are not fully understood, it seems likely
that some environmental signals will act via binding of ligands to
specific receptors in the parasite membrane which transduce the
extracellular signal to the interior of the cell. However, to date no
such ligands or their receptors have been identified in Leishmania parasites.
Nonetheless, a body of evidence suggests that the
second messenger cAMP is probably involved in signal transduction
events and in life cycle transformations in Leishmania and in
other related kinetoplastid protozoa. Thus, various life cycle stages
contain significantly different intracellular concentrations of cAMP in
the related parasites Trypanosoma brucei(2) and in Trypanosoma cruzi(3) . Furthermore, cAMP analogs and
inhibitors of cAMP phosphodiesterase promote the in vitro differentiation of non-infectious insect stage epimastigotes of T. cruzi into infectious metacyclic
trypomastigotes(4) , whereas such reagents inhibit the
transformation of amastigotes of Leishmania donovani into
promastigotes (5, 6) or of long slender bloodstream
forms of T. brucei into short stumpy forms that are infectious
to the insect(7) . Perhaps the strongest case for the
involvement of cAMP in the differentiation of kinetoplastid parasites
is the observation that peptides which are digestion products of
vertebrate
Although the cognate
ligands and receptors involved in such signaling processes have not
been identified for any of these kinetoplastid protozoa, a family of
genes encoding adenylate cyclases have been cloned in T. brucei(9) and in the related parasite T.
equiperdum(10) . The proteins encoded by these genes
possess a large extracellular hydrophilic NH
The probable role
of cAMP in life cycle transformations in kinetoplastid protozoa has led
us to investigate adenylate cyclases in L.donovani.
In this paper, we demonstrate that L. donovani parasites
contain a family of genes that encode putative receptor-adenylate
cyclases (RACs)
Figure 1:
Comparison of deduced amino acid
sequences for RAC-A and RAC-B and alignment with T. brucei GRESAG4.3. Numbers at the right specify the last amino
acid in each line. Vertical lines indicate identical amino
acids. Predicted (42) transmembrane segments I and II are
indicated by underlining. The alignments were generated using
the program PILEUP(43) .
To determine the
detailed structure of the Leishmania genes, we radiolabeled
the 175-bp PCR fragment and used this reagent as a probe to screen a
cosmid library of L. donovani genomic DNA. Two hybridizing
cosmid clones, designated AC6 and AC9, were chosen for detailed
structural analysis. The results of this analysis (see below and Fig. 3B) reveal that these two overlapping clones
contain five clustered adenylate cyclase genes. Two of these genes,
designated rac-A and rac-B, were then chosen for
complete sequence analysis. The DNA sequence for each of these genes
has been entered into the GenBank data base (rac-A and rac-B), and deduced protein sequences of rac-A and rac-B and their alignments with the T. brucei receptor-adenylate cyclase GRESAG4.3 (9) are shown in Fig. 1. The sequences of the RAC-A (1380 amino acids) and RAC-B
(1331 amino acids) proteins clearly reveal that they are members of the
receptor-adenylate cyclase family and are closely related in sequence
and structure to the trypanosome proteins that are members of this
family. RAC-A and RAC-B both contain: (i) a putative transmembrane
segment near the NH
Figure 3:
Genomic arrangement of rac genes
in L. donovani. A, Southern blot showing PstI digested DNA from L. donovani strain DI700
genomic DNA (genomic) or from cosmid clones AC9 and AC6. The blot was
probed with the 1.9-kb PstI fragment of rac-B encoding part of the conserved adenylate cyclase catalytic domain
(AC-B probe, ``Experimental Procedures''). Numbers (kilobase pairs) at the left indicate the mobility of DNA
molecular weight markers. Letters A-E indicate the five
hybridizing PstI fragments present in both genomic DNA and in
the cosmid inserts. B, restriction map of the rac gene cluster indicating the exact (rac-A and rac-B) or approximate (rac-C, rac-D and rac-E) locations of the rac gene coding regions. The
restriction map was generated from single and multiple digestions with
different restriction enzymes (A, AseI; B, BglII; D, DraI; E, EcoRI; N, NotI; P, PstI; S, SpeI). Sites with asterisks denote a partial
restriction map for this enzyme, indicating that other sites exist
within the insert which have not been mapped. Hatched bars at
the top of the figure indicate the positions of the inserts for the AC6
and AC9 cosmid clones. The open letters A-E underneath the
restriction map define the location of the rac-A through rac-E genes. The positions of the coding regions of rac-A and rac-B (open boxes) are known precisely from
the restriction maps and the DNA sequence of these coding regions. The arrows underneath these boxes indicate the direction of
transcription for these genes. The position of the 2.7-kb PstI
fragment containing the adenylate cyclase conserved catalytic domain of rac-E (black box) is known because a PstI
restriction fragment of this size that hybridizes to an adenylate
cyclase catalytic domain probe (AC-CD probe, ``Experimental
Procedures'') is present at this location in the restriction map.
Hence, this box defines the approximate position of the adenylate
cyclase domain of rac-E. The 1.6-kb PstI fragment
from rac-C is located between the EcoRI site and the
right hand terminus of AC6; this region is shown as a cross-hatched
box. The 1.2-kb PstI restriction fragment from rac-D that hybridizes to the adenylate cyclase probe is contained within
the EcoRI/BglII restriction fragment of AC9, but is
not present in AC6; hence, its approximate location is also shown as a cross-hatched box.
Figure 2:
Adenylate cyclase activity of rac-A expressed in Xenopus oocytes. A, the
cAMP-mediated activation of the CFTR Cl
To further investigate the arrangement
of the multiple rac genes, we have constructed a restriction
map of the AC6 and AC9 inserts, and we have mapped the locations of the
adenylate cyclase genes by hybridization of restriction fragments to a
second probe (AC-CD probe) derived exclusively from the conserved
catalytic domain (Fig. 3B). This restriction map
confirms that AC6 and AC9 do overlap and defines the precise location
of rac-A and rac-B as well as the approximate
locations of three other rac genes, designated rac-C, rac-D, and rac-E. Thus the open boxes in Fig. 3B represent the protein coding regions of rac-A and rac-B, as defined by the sequences of the
subclones and the locations of specific restriction sites. These
results also define the direction of transcription for these two genes (arrows under each gene) and the probable direction for
transcription of the gene cluster. Together, these results suggest that
there are five related rac genes located within this single
cluster.
To confirm the existence of three additional rac genes, we subcloned the PstI fragments containing rac-C, rac-D, and rac-E and obtained partial
DNA sequence from the termini of these subclones. The deduced amino
acid sequences derived from one terminus of each subclone show
pronounced similarity to the extracellular domains of RAC-A and RAC-B (Fig. 4A), whereas the deduced amino acid sequence
derived from the other terminus of each clone shows identity to the
adenylate cyclase catalytic domain of RAC-B (Fig. 4B).
The similarities between each of the RAC proteins is shown
schematically in Fig. 4C. These results confirm that
each of these genomic fragments represents another rac gene
and that there are five tandemly arrayed genes in this cluster. The
limited sequence from RAC-E is identical to the corresponding regions
of RAC-B, suggesting that these two proteins may be very closely
related to each other; however, the presence of an additional PstI site in rac-B reveals that there is at least one
polymorphism at the DNA level that generates the two differently sized rac-B and rac-E PstI fragments observed on the
genomic Southern blot (Fig. 3A).
Figure 4:
Partial deduced amino acid sequences
derived from cloned PstI fragments encoding RAC-C, RAC-D, and
RAC-E. Each subcloned PstI insert was partially sequenced from
each terminus, and the deduced amino acid sequences were aligned with
either RAC-A or RAC-B. In addition, an internal segment of the RAC-E
insert was sequenced using an internal oligonucleotide primer. Amino
acid identities are indicated by vertical lines. These
sequences aligned with either the extracellular domain (RAC-C aligned
maximally with RAC-A, whereas RAC-D and RAC-E aligned maximally with
RAC-B) (A) or the catalytic adenylate cyclase domain of RAC-B (B). A schematic diagram (C) shows the regions of
RAC-A and RAC-B that are homologous to the sequenced segments of RAC-C,
RAC-D, and RAC-E. ECD refers to the extracellular domain, TMD to the transmembrane domain, and ACD to the
adenylate cyclase catalytic domain.
Figure 5:
Northern blots showing expression of rac mRNAs in promastigotes and amastigotes of L.
donovani. Total RNA (10 µg) from promastigotes (P)
and amastigotes (A) of the LV9 strain of L. donovani was separated on a 1.2% agarose-formaldehyde gel, blotted onto a
nylon filter, and hybridized with probes representing the adenylate
cyclase conserved catalytic domain (AC, hybridized with the AC-CD
probe), or the 3`-untranslated regions of rac-A (rac-A) or rac-B (rac-B). To
demonstrate that lanes contained similar amounts of total RNA, one of
these blots was rehybridized with a rRNA (44) probe (rRNA). Numbers at the left indicate the mobility of
RNA molecular weight markers indicated in
kilobases.
The results presented here demonstrate that Leishmania parasites possess a family of putative receptor-adenylate cyclases
that are related in structure to those described in the trypanosomes
and that bear structural similarities to receptors with known ligands
and functions from higher eukaryotes. Consequently, this family of
probable receptors exists in kinetoplastid parasites that exhibit an
intracellular mode of existence within their mammalian hosts and is not
restricted to trypanosomes that live free in the bloodstream or to
Kinetoplastida such as T. brucei and T. equiperdum that undergo antigenic variation as a mode of immune evasion. The
two most significant questions that now require examination are: (i)
what specific role do these putative receptors play in signal
transduction, and (ii) what ligands do they recognize?
One possible
role for the RAC proteins in signal transduction could be to bind
ligands present in the insect vector or inside the host macrophages and
induce a life cycle switch by elevating intracellular cAMP. The
potential analogy to T. cruzi, where peptide ligands present
inside the insect gut activate parasite adenylate cyclase and promote
the developmental transformation between non-infectious epimastigotes
and infectious metacyclics(8) , is obvious. It may be possible
to assess the role of the RACs in development or in other physiological
processes by genetic approaches, either by performing targeted gene
disruptions (36) of individual rac genes, or by
generating dominant negative mutants of these genes and overexpressing
these altered polypeptides within the parasites. Finally, extensive
studies on various RAC family members will be required to determine
whether they all function in closely related signal transduction
pathways and recognize different but possibly related ligands, or
whether each RAC transduces a different signal with a distinct effect
on parasite biochemistry and physiology.
The observation that
co-expression of RAC-B markedly inhibits the adenylate cyclase activity
of RAC-A (Fig. 2B) suggests an intriguing mechanism for
receptor function. These two proteins may oligomerize in the membrane
to maintain the innate adenylate cyclase activity of RAC-A in a
repressed state. This repression might then be relieved by binding of
ligand to the hetero-oligomer. If this interpretation is correct, it
should be possible to detect complexes between RAC-A and RAC-B using
specific antibodies against each protein or by epitope tagging
individual receptors(32) . Furthermore, the observation that
the truncated RAC-A
Ligands for orphan receptor-guanylate
cyclases have been identified in several higher
eukaryotes(37, 38, 39, 40) , usually
by assaying crude extracts for ability to stimulate guanylate cyclase
activity in a target tissue followed by biochemical purification of the
active peptide. By analogy, it may be possible to search for potential
ligands for the RAC proteins by applying extracts of macrophages or of
sandflies to intact parasites or to a heterologous system expressing
individual rac genes and assaying for an increase in adenylate
cyclase activity. The assay that we have used for detection of RAC-A
adenylate cyclase activity involving activation of the co-expressed
CFTR channel, or the more conventional radioimmunoassay, may be useful
for identifying ligands for each putative receptor. Thus application of
a mixture containing a ligand to oocytes expressing RAC-A or RAC-B or
co-expressing both proteins may increase the cAMP level above that
observed in the absence of ligand.
The observation that rac-A and rac-B mRNAs are detectable in promastigotes but not
amastigotes implies that these putative receptors function either in
the insect or during the transformation between the insect and the
macrophage stage of the parasite life cycle. If these proteins are
involved in developmental transitions, they could function in the
switch between non-infectious procyclic and infectious metacyclic
promastigotes(41) , or they could promote the differentiation
of promastigotes to amastigotes by interacting with a ligand present in
macrophages.
Clearly many questions remain to be answered concerning
the functions of the rac genes and their protein products. At
present, the RACs represent orphan receptors identified by molecular
genetics but with unknown biological functions. However, as the first
putative signal transduction receptors identified in Leishmania or related kinetoplastids(9, 10) , these proteins
provide the opportunity to study signal transduction and its potential
role in the life cycle of these medically important and biologically
interesting parasites.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s) U17042 [GenBank® Link]and U17043[GenBank® Link].
We thank Weibin Zhang for isolation and injection of Xenopus oocytes, Mark Drew for performing control experiments
involving inositol transport assays on oocytes, and Richard Burchmore
for culturing amastigotes of L. donovani. We also thank Buddy
Ullman for thoughtful comments on this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal hydrophilic domain, an intervening putative
transmembrane segment, and a COOH-terminal domain that has high
sequence identity to the catalytic domain from adenylate cyclases in
other eukaryotes. We have expressed the receptor-adenylate cyclase
protein (RAC)-A protein in Xenopus oocytes and demonstrated
that it functions as an adenylate cyclase. Although RAC-B exhibits no
catalytic activity when expressed in oocytes, co-expression of RAC-A
and RAC-B negatively regulates the adenylate cyclase activity of RAC-A,
suggesting that these two proteins interact in the membrane.
Furthermore, a truncated version of RAC-A functions as a dominant
negative mutant that inhibits the catalytic activity of the wild type
receptor. The rac-A and rac-B genes encode
developmentally regulated mRNAs which are expressed in the insect stage
but not in the mammalian host stage of the parasite life cycle.
-globin, and which are found in the gut of the
triatomid insect vector following a blood meal, will activate a
membrane-bound adenylate cyclase in non-infectious epimastigotes of T. cruzi and promote the differentiation of these
epimastigotes into infectious metacyclic trypomastigotes in
vitro(8) . These results underscore the importance for
parasite development of environmental ligands which probably bind to
receptors and activate adenylate cyclase.
-terminal
domain which is separated by a putative transmembrane segment from a
COOH-terminal domain that bears high sequence homology to the catalytic
domains of adenylate cyclases from other eukaryotes. These trypanosome
genes have been expressed in Saccharomyces cerevisiae where
they complement a temperature-sensitive defect in the yeast adenylate
cyclase(10, 11) . The structural organization of these
trypanosome proteins suggests that they may be receptors whose
NH
-terminal domains bind specific extracellular ligands
that activate the intracellular adenylate cyclase domain. This
suggestion is reinforced by the structural similarity between these
trypanosome proteins and several well-characterized receptors from
higher eukaryotes(12) . Thus, mammalian atrial natriuretic
peptide receptors bind their ligands via their NH
-terminal
extracellular domains and activate the cytoplasmic guanylate cyclase
domain(13, 14) . However, ligands for these putative
trypanosome receptors have not yet been identified, and their specific
role in signal transduction is currently unknown.
(
)that are structurally very
similar to the predicted proteins described in T. brucei and T. equiperdum. Five of these genes are arranged in a cluster
in the L. donovani genome and are expressed as mRNAs of
different sizes. One of these genes have been expressed in Xenopus oocytes and shown to encode a protein that functions as an
adenylate cyclase. These results open the way to investigating the role
of the Leishmania RAC proteins in signal transduction and
their possible involvement in the life cycle transformations of this
parasite. Studies on the RAC proteins of the intracellular Leishmania parasites may provide interesting parallels and
distinctions compared to the RAC proteins identified in the
extracellular parasites T. brucei and T. equiperdum.
Growth of Parasites and Isolation of Nucleic
Acids
Promastigotes of either the DI700 (15) or the LV9
strain of L. donovani were cultured in Dulbecco's
modified Eagle's-L medium (15) containing 5%
heat-inactivated fetal calf serum (Life Technologies, Inc.) and 5%
bovine embryonic fluid (Sigma) at 27 °C. For preparation of LV9
amastigotes, J774-G8 macrophage-like cells were cultured in plastic
tissue culture flasks containing RPMI medium supplemented with 20
mM HEPES (Sigma), pH 7.3, and 20% heat-inactivated fetal calf
serum at 37 °C in the presence of 5% CO and were
infected with promastigotes at a multiplicity of
10
parasites/cell. After 24 h the medium and remaining promastigotes were
aspirated, the adherent cells were washed twice with sterile
phosphate-buffered saline, fresh medium was added to the culture, and
the infection was continued for a further 24 h. Cells were removed from
the tissue culture flask by scraping with a rubber policeman and were
lysed by passage through a 30-gauge needle 20 times to achieve
95%
breakage of cells, as monitored by staining with Trypan Blue. The
liberated amastigotes were purified on a Percoll gradient as
described(16) , and RNA was prepared immediately. Total
parasite RNA was prepared by the guanidinium isothiocyanate-phenol
method(17) .
Polymerase Chain Reaction
Degenerate
oligonucleotides were purchased from Oligos Etc. and utilized in the
PCR to amplify regions of the adenylate cyclase conserved catalytic
domain from genomic DNA of L. donovani. The conserved peptide
sequences and the oligonucleotides (bases in parentheses represent
degenerate positions) designed from them were: peptide 1, TLIFTDI;
oligonucleotide 1, AC(T/G/C)CT(T/C/G)AT(T/C)TT(T/C)AC(T/G/C)GA(T/C)AT;
peptide 2, EVKTVGD; oligonucleotide 2,
GA(G/A)GT(T/G/C)AAGAC(T/G/C)GT(T/G/C)GG(T/G/C)GA; peptide 3, GDSFMIA;
oligonucleotide 3 (reverse complement),
GC(A/G)ATCAT(A/G)AA(G/A/C)(G/C)(T/A)(A/G)TC(G/C/A)CC; peptide 4,
RVGIHTG; oligonucleotide 4 (reverse complement):
CC(G/A/C)GT(A/G)TG(A/G)AT(G/A/C)CC(G/A/C)AC(G/A/C)C(G/T). The
degeneracies in these oligonucleotides were selected on the basis of
the codon biases of previously sequenced Leishmania genes (18) . The PCRs were performed in a total volume of 50 µl
and contained 10 mM Tris, pH 8.3, 50 mM KCl, 3
mM MgCl, 200 µM each dNTP, 2
µM each oligonucleotide, 2 µg of genomic DNA from the
DI700 strain of L. donovani, and 1 unit of Taq Polymerase (Promega). Each PCR was performed on a Perkin Elmer DNA
Thermal Cycler utilizing 30 cycles with a 95 °C melting temperature
for 1 min, a 30 °C annealing temperature for 90 s, a 72 °C
extension temperature for 1 min, with a final incubation at 72 °C
for 5 min.
Isolation of Adenylate Cyclase Cosmid Clones
A
library generated from a Sau 3A partial digest of genomic DNA
from L. donovani strain DI700 and cloned into the cosmid
vector SuperCos1 (Stratagene) was obtained from Dr. Buddy Ullman
(Oregon Health Sciences University, Portland, OR) and screened with the
175-bp PCR product obtained using oligonucleotides 1 and 3. The product
of this PCR was purified by phenol-chloroform extraction, and 150
ng was radiolabeled with [
-
[P]dATP
(New England Nuclear) using random oligonucleotide
priming(19) . Four nylon filters (Schleicher and Schuell)
containing approximately 2,000 colonies each were screened by filter
hybridization (20) uitlizing the radiolabeled PCR fragment at a
concentration of 2
10
counts/min
ml
, and eight positive clones were colony purified
for further analysis. Two of these positive cosmid clones, AC6 and AC9,
which contained between them the entire rac gene family, were
selected for detailed analysis.
DNA Sequencing
Segments of the AC6 and AC9 cosmids
containing the coding regions of the rac-A and rac-B genes were subcloned into the plasmid vector Bluescript SK+
(Stratagene), and nested deletions were prepared by digestion with
exonuclease III as described previously(21) . Single-stranded
DNA was prepared from these deletions (22) and sequenced by the
dideoxy chain terminating method(23) , as described (21) . In addition, some regions were sequenced utilizing
synthetic oligonucleotides complementary to known regions of sequence.
All sequences were obtained from both strands of the DNA. The partial
sequences of RAC-C and RAC-D were obtained from double-stranded plasmid
DNA using an A.L.F. automated DNA Sequencer (Pharmacia), and RAC-E
partial sequence was obtained from double-stranded DNA by manual
sequencing.
Adenylate Cyclase Probes
The AC-B probe was a
1.9-kb PstI restriction fragment from rac-B encoding
amino acids 545-1174 (Fig. 1). This probe contains part of the
conserved adenylate cyclase catalytic domain and part of the
extracellular domain of RAC-B. The 1.4-kb AC-CD probe representing the
conserved adenylate cyclase catalytic domain of RAC-A was prepared
using the PCR and a forward primer (20-mer) initiating immediately
downstream from the second putative transmembrane segment (II in Fig. 1) and a reverse primer initiating at the RAC-A
stop codon and proceeding upstream for 20 nucleotides. The probes for
the 3`-untranslated regions of rac-A and rac-B were a
1.0-kb EcoRI/SalI fragment and a 410-bp NheI/XhoI fragment, respectively, initiating 100
bp downstream from each stop codon. Hybridizations of Southern and
Northern blots were performed in 50% formamide, 5
x SSC, 10
Denhart's solution, 0.1% SDS, 5 mM EDTA at 42
°C, and washes were performed in 0.1
SSC, 0.1% SDS, 5
mM EDTA at 55 °C.
Expression of rac-A and rac-B mRNAs in Xenopus
oocytes
For expression of rac mRNAs, the protein coding
regions of rac-A and rac-B were subcloned into the
oocyte expression vector pL2-5(24) . Capped transcripts
were synthesized from these vectors utilizing T7 RNA polymerase as
described previously(25) . Approximately 0.5 ng of each
transcript, as well as 2.5 ng of transcript from a similar plasmid
containing the coding region of the CFTR chloride channel, were
injected into defolliculated stage V-VI oocytes from X. laevis as detailed previously(25) . After 4-5 days of
incubation at 17 °C in ND-96 buffer containing 96 mM NaCl,
2 mM KCl, 1.8 mM CaCl, 1 mM
MgCl
, 5 mM HEPES, pH 7.4, membrane currents were
measured from each oocyte using the two-microelectrode voltage clamp
technique(26) .
Construction of a Dominant Negative Deletion Mutant of
rac-A
For expression of a dominant negative version of the RAC-A
protein, RAC-A, the plasmid containing the rac-A insert
within the pL2-5 plasmid was digested with XhoI,
religated, and transformed into bacteria. Plasmids prepared from
individual colonies were tested by restriction digestion to identify
those which were missing the terminal 0.7-kb XhoI fragment and
hence were missing the sequence encoding the 218 COOH-terminal amino
acids of RAC-A. This plasmid was transcribed with T7 RNA polymerase,
and the RNA was microinjected into oocytes as described above.
cAMP Assays
Quantitation of cAMP was performed
using the radioimmunoassay kit from Biomedical Technologies Inc. and
the manufacturer's instructions. Oocytes were injected with 5 ng
of either RAC-A or RAC-B cRNA and incubated in ND-96 for 3 days.
Lysates were prepared from 15 oocytes containing each RNA and from 15
uninjected oocytes as follows. Each batch of oocytes was homogenized in
270 µl 10% trichloroacetic acid using a pestle and a
microcentrifuge tube. This homogenate was clarified by centrifugation
for 5 min in an Eppendorf microcentrifuge (Brinkmann Instruments, Inc.)
for 5 min at 14,000 revolutions/min. The supernatant was extracted
three times with 5 volumes of water-saturated ether, residual ether was
removed by heating to 50 °C for 30 min, and the samples were stored
at -20 °C. Aliquots of each lysate were diluted and assayed
in duplicate by radioimmunoassay.
Cloning and Sequencing of Adenylate Cyclase Genes from
Leishmania donovani
A PCR-based strategy was used to clone
receptor-adenylate cyclase genes from L. donovani based on the
assumption that they would contain sequences related to those found in
the highly conserved catalytic domains of the trypanosome proteins (9, 10) . We chose four highly conserved peptides (see
``Experimental Procedures'') present in the trypanosome
cyclases in order to design degenerate oligonucleotide primers
(oligonucleotides 1-4). When these oligonucleotides were used in
the PCR (27) in conjunction with genomic DNA from L.
donovani, L. mexicana, or L. enriettii, they
amplified DNA fragments of sizes 175 bp (oligonucleotides 1 and 3), 275
bp (oligonucleotides 2 and 4), and 400 bp (oligonucleotides 1 and 4)
(data not shown). These are the sizes of fragments that would be
predicted for PCR amplification from DNA sequences closely related to
the catalytic adenylate cyclase domains of the trypanosome genes, and
these results suggested that L. donovani does contain genes
related to the trypanosome adenylate cyclases.
terminus (underlined sequences
marked I in Fig. 1), (ii) a large (approximately 830
amino acid) hydrophilic domain that follows this first transmembrane
segment, (iii) a second putative transmembrane segment (underlined sequence marked II in Fig. 1), and (iv) a
COOH-terminal hydrophilic domain with a high degree of identity to the
conserved catalytic domain of eukaryotic adenylate cyclases. In
addition to these structural similarities, there is a high degree of
sequence identity between the trypanosome proteins and the RAC-A and
RAC-B proteins. Direct comparison between each of the three proteins
indicates that RAC-A is 30.6% identical to the T. brucei GRESAG4.3 receptor-adenylate cyclase, RAC-B is 31.2% identical to
this trypanosome protein, and RAC-A is 55.5% identical to RAC-B. The
highest degree of identity occurs over the conserved catalytic domains
which are 52.7% identical between RAC-A and GRESAG4.3, 52.4% identical
between RAC-B and GRESAG4.3, and 88.0% identical between RAC-A and
RAC-B. Within the putative extracellular domains, the identities
between the RAC-A, RAC-B, and GRESAG4.3 are clustered in islands of
high identity separated by stretches of low identity.
There are
several other structural features that are worthy of comment. First, it
is not clear whether the first putative transmembrane segment is
retained in the mature proteins and tethers the NH terminus
to the membrane or whether it is cleaved during maturation of the
polypeptide, as is the case in the structurally related atrial
natriuretic peptide receptor(14) . However, analysis of these
sequences using von Heijne's algorithm (28) reveals a
high similarity to known signal sequence cleavage sites at residue 53
of RAC-A and 59 of RAC-B, suggesting that these NH
-terminal
hydrophobic sequences may be removed during processing. Second, RAC-A
and RAC-B both contain multiple consensus sequences for
asparagine-linked glycosylation (29) (not shown), both in the
putative extracellular domains and within the RAC-A conserved catalytic
domain. However, the extent to which the RAC-A and RAC-B proteins are
modified by glycosylation is currently unknown. Finally while the
adenylate cyclase catalytic domains are highly conserved, the extreme
COOH terminus of RAC-A (amino acids 1307-1380) and RAC-B (amino
acids 1315-1331) bears no similarity to each other, suggesting
that antibodies directed against these sequences should be able to
specifically recognize each distinct isoform and may be useful for
studies on subcellular localization of each protein.
Functional Expression of RAC-A and RAC-B Proteins in
Xenopus oocytes
Although the rac-A and rac-B genes encode proteins with pronounced sequence similarity to known
adenylate cyclases, it is nonetheless important to demonstrate
experimentally that these Leishmania proteins possess
adenylate cyclase activity. A strategy that was employed successfully
with the adenylate cyclase genes from T. brucei and T.
equiperdum was to complement a temperature sensitive adenylate
cyclase mutant of S. cerevisiae by transformation with the
trypanosome gene and demonstrate the ability of the recombinant yeast
to grow at the non-permissive temperature. However, in similar
experiments, we were unable to complement either temperature-sensitive
or missense mutants of the yeast cyr1 adenylate cyclase gene;
hence, we attempted expression of rac-A and rac-B in Xenopus oocytes. This novel approach involves
co-expression of the rac gene with the human CFTR gene, which
encodes a chloride channel that is stimulated by phosphorylation of its
regulatory domain via the cAMP-activated protein kinase A(30) .
Hence, if the rac genes encode functional adenylate cyclases,
their co-expression in oocytes with CFTR will increase the
intracellular cAMP, activate protein kinase A, stimulate the chloride
channel activity of CFTR, and induce an increased current of
Cl ions that can be measured by voltage clamping the
oocytes. The results of one such experiment are shown in Fig. 2A. Oocytes that were injected with both rac-A and CFTR mRNAs (filled circles) exhibited increased
transmembrane conductance compared to oocytes that were uninjected (crosses), injected with CFTR mRNA only (squares), or
injected with rac-A mRNA only (open circles). This
conductance increase exhibited a reversal potential of -25 to
-30 mV, corresponding to the Cl
equilibrium
potential in Xenopus oocytes. This result strongly suggests
that the RAC-A protein does possess adenylate cyclase activity which
results in a CFTR-mediated Cl
conductance increase.
It is noteworthy that this adenylate cyclase activity is present in the
absence of any exogenous ligands which might bind to the RAC-A
extracellular domain and stimulate catalytic activity. A similar
observation of enzymatic activity in the absence of added ligands was
also reported for the trypanosome proteins expressed in
yeast(10, 11) . Co-injection of several batches of
oocytes with mRNAs encoding the rac-B protein and the CFTR
protein produced no statistically significant increase in
Cl
conductances. These results suggest the
possibility that there is no basal enzymatic activity of RAC-B in the
absence of ligand or that it is significantly lower than that of RAC-A.
channel was
measured by a two-electrode voltage clamp experiment. Oocytes were
injected with RNA encoding CFTR alone (squares), RAC-A alone (open circles), RAC-A plus CFTR (filled circles), or
uninjected controls (crosses). After incubation for 4-5
days, each oocyte was impaled with two electrodes and continually
superfused with ND96 buffer. Each data point represents the steady
state current (mean ± S.E., n = 3-4) at
the indicated potential. Currents were recorded during a 250 ms pulse
from a holding potential of -60 mV to the indicated test
potential. B, the level of cAMP present in uninjected (nude) oocytes or in oocytes injected with 5 ng of cRNA
encoding RAC-A (RAC-A), RAC-B (RAC-B), or RAC-A
(RAC-A
), or with cRNAs encoding both RAC-A and RAC-B (RAC-A + RAC-B), or RAC-A and RAC-A
(RAC-A
+ RAC-A
) was measured by radioimminoassay. The
measurements were performed on lysates from 15 pooled oocytes of each
type and represent the averages of two cAMP determinations. The largest
difference between duplicates was in the RAC-A expressing oocytes,
where the standard deviation was 135 pmol
oocyte
.
To confirm the results obtained by electrophysiology, we have
assayed oocytes expressing RAC-A and RAC-B by a more conventional
radioimmunoassay (Fig. 2B). This quantitation reveals
that rac-A-injected oocytes accumulate significantly more cAMP
than control uninjected oocytes but that cAMP levels are not elevated
in rac-B injected oocytes. Furthermore, co-expression of RAC-A
and RAC-B in the same oocytes led to a marked attenuation of the
adenylate cyclase activity compared to the level observed in RAC-A
expressing oocytes. To confirm that attenuation of RAC-A activity was
not simply due to some contaminant in rac-B RNA which either
degraded rac-A cRNA or inhibited its translation, we
co-expressed rac-B RNA with cRNA encoding a Leishmania
myo-inositol transporter. The co-expressing oocytes exhibited myo-inositol transport activity that was equivalent to that of
oocytes injected with the transporter RNA only (data not shown); hence,
the rac-B RNA does not contain an inhibitor that prevents
expression of other co-injected RNAs. The inhibition or the RAC-A
adenylate cyclase by RAC-B has been confirmed in three independent
experiments and suggests that the RAC-B protein may interact with the
RAC-A protein to negatively regulate its adenylate cyclase activity.
Expression of a Dominant Negative Form of
RAC-A
Dominant negative forms of many signal transduction
receptors can be prepared by expressing truncated proteins containing
deletions in the cytoplasmic domain involved in transducing the
signal(31) . Thus, deletion of part of the guanylate cyclase
domain of the ANP receptor generates a truncated receptor that
oligomerizes with the wild type protein and inhibits the guanylate
cyclase activity of the complex(32) . We have prepared a
truncated version of the rac-A gene that encodes a receptor,
the RAC-A protein, that terminates at amino acid 1162 of the RAC-A
protein and which is missing the last 218 amino acids of the adenylate
cyclase catalytic domain. Co-expression of this truncated receptor with
the wild type RAC-A protein (Fig. 2B, RAC-A + RAC-A
) markedly inhibits the adenylate cyclase activity of
RAC-A. In control experiments similar to those described above, the
co-expression of rac-A
RNA with inositol transporter RNA
failed to inhibit functional expression of the transporter protein.
Hence RAC-A
functions as a dominant negative receptor, strongly
suggesting that the wild type and deleted proteins physically interact
in the oocyte membrane.
Genomic Arrangement of rac Genes
The existence of
related but distinct rac-A and rac-B genes
raises the question of how many members there are within this gene
family. Since the catalytic domains of rac-A and rac-B are highly conserved, as is the case for adenylate cyclase genes
in other eukaryotes(33) , a probe containing this domain should
be able to detect other family members. Consequently, we utilized a
1.9-kb PstI fragment from rac-B (AC-B probe)
containing part of this conserved domain to probe a Southern blot
containing PstI digests of L. donovani genomic DNA
and of AC6 and AC9 cosmid DNA. This blot (Fig. 3A)
reveals five distinct bands of hybridization in genomic DNA marked A
through E; bands A-D are also present in AC9 DNA, and bands B, C, and E
are present in AC6 DNA. (In this nomenclature, the bands designated A
and B are those derived from the rac-A and rac-B genes, respectively.) These results imply that the AC6 and AC9
inserts overlap, and that these cosmids contain between them all five
hybridization bands present in the genomic DNA. This pattern suggests
that the L. donovani genome contains at least five adenylate
cyclase genes which are clustered together and hence present on two
overlapping cosmid clones. Clustered arrays of functionally related
genes are common among the kinetoplastid protozoa such as Leishmania and Trypanosoma and appear to comprise
single transcription units which are synthesized as polycistronic
precursor RNAs(34) .
Furthermore, the
concordance between the five hybridizing PstI fragments
present in genomic DNA (Fig. 3A) and the five
hybridizing PstI fragments present in the AC6 and AC9 cosmid
clones suggests that either: (i) this cluster contains all the
adenylate cyclase genes present in the L. donovani genome, or
that (ii) any other adenylate cyclase genes that might exist do not
hybridize to the probe containing the conserved catalytic domain under
the hybridization conditions used here and hence are relatively
divergent in sequence. Another result which is consistent with a single
clustered arrangement of adenylate cyclase genes is the observation
(not shown) that Southern blots from pulsed-field gels (35) of L. donovani chromosomes reveal a single band of hybridization
to the adenylate cyclase probe representing a chromosome of 850
kilobase pairs.
Expression of rac mRNAs during the Leishmania Life
Cycle
To define the expression of the rac-A and rac-B mRNAs during the parasite life cycle,
we have hybridized Northern blots containing equal amounts of total
promastigote and amastigote RNA with probes representing the conserved
adenylate cyclase catalytic domain and the unique 3`-untranslated
regions of each mRNA (Fig. 5). These blots reveal that the rac-A (9 kb) and rac-B (
5
kb) mRNAs are expressed in promastigotes but are not detectable in
amastigotes; hence, these are developmentally regulated mRNAs.
Rehybridization of these blots with a rRNA probe confirms that similar
amounts of promastigote and amastigote RNA were present in each lane.
The conserved adenylate cyclase catalytic probe hybridizes to bands of
sizes
5,
7, and
9 kb, revealing that there are at least
three different adenylate cyclase mRNAs. This probe also hybridizes to
a
5 kb band present at a low level in the amastigote RNA,
suggesting that one of the rac-C, -D, or -E mRNAs may be expressed in amastigotes and may comigrate with the rac-B mRNA.
protein functions as a dominant negative
receptor (Fig. 2B) strongly suggests that these
proteins oligomerize within the membrane and can affect the catalytic
function of other members of the complex. The development of a dominant
negative mutant of the rac-A gene also provides a genetic tool
which may be used to illuminate the biological function of these
receptors in the parasite.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.