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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Microbiology. Author manuscript; available in PMC Aug 1, 2007.
Published in final edited form as:
PMCID: PMC1513434
NIHMSID: NIHMS9920

Functional characterization of a fatty acyl-CoA binding protein (ACBP) from the apicomplexan Cryptosporidium parvum

SUMMARY

We have identified and conducted functional analysis of a fatty acyl-CoA binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum. The CpACBP1 gene encodes a protein of 268 aa that is 3X larger than the typical ACBP proteins (i.e., ~90 aa) of humans and animals. Sequence analysis indicated that CpACBP1 consists of an N-terminal ACBP domain (~90 aa) and a C-terminal ankrin repeat sequence (~170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 was palmitoyl-CoA. RT-PCR, Western blotting and immuno-labeling analyses clearly showed that the CpACBP1 gene was mainly expressed in the intracellular developmental stages and the level increases during the parasite development. Immunofluorescence microscopy shows that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in the lipid remodeling in the PVM or the transport of fatty acids across the membrane.

Keywords: Cryptosporidium parvum, acyl-CoA binding protein, ankyrin repeats

INTRODUCTION

Cryptosporidium parvum is a globally important parasitic protist that infects both humans and animals (Chappell & Okhuysen, 2002; Thompson et al., 2005; Tzipori & Widmer, 2000). Cryptosporidium parvum belongs to the Phylum Apicomplexa that contains many important human and animal parasites (e.g. Plasmodium, Babesia, Toxoplasma and Eimeria) (Zhu et al., 2000a). This group of parasites shares some common biological features characteristic to the phylum. For example, all apicomplexans possess similar complex life cycle stages including oocyst formation, sporulation, merogony and gametogenesis. However, recent advancements in biochemistry and genome-sequencing have revealed that a number of metabolic pathways such as fatty acid biosynthesis are highly diverse within the Apicomplexa (Abrahamsen et al., 2004; Zhu et al., 2000a). Apicomplexans may possess either apicoplast-specific Type II fatty acid synthases (FASs) (e.g., P. falciparum), or Type I FAS (e.g., C. parvum), or both (e.g., T. gondii) (Roos et al., 2002; Zhu, 2004; Zhu et al., 2000b; Zhu et al., 2002; Zhu et al., 2004). Although fatty acids are one of the major components in all cells, free fatty acids cannot enter any metabolic pathways unless they are activated by thioesterification with coenzyme A (CoA) to form an acyl-CoA or with acyl carrier protein (ACP) to form an acyl-ACP. Fatty acyl-CoA can immediately enter subsequent metabolic pathways, or may be stored/transported by a family of acyl-CoA binding proteins (ACBPs).

ACBPs are a group of highly conserved proteins and have been found in animals, plants, protists, and a number of pathogenic bacteria (Burton et al., 2005). They are typically small, cytosolic molecules of ~10 kDa. However, a number of larger ACBP proteins (e.g. Mr >55-kDa) have also been identified in both animals and plants. Mammals possess multiple ACBP proteins that are differentially expressed in various tissues (e.g. T-ACBP in testis, L-ACBP in livers and I-ACBP in intestines) (Schroeder et al., 1998). In Trypanosoma brucei, an ACBP protein was found to be involved in the synthesis of the glycosyl-phosphatidylinositol (GPI) anchor in variant surface glycoproteins (VSG) (Milne & Ferguson, 2000; Milne et al., 2001). Although ACBPs are capable of binding medium to long chain fatty acyl-CoA esters, they may vary in their substrate preference and binding affinities. For example, the highest affinities of ACBP proteins from bovine (liver) and trypanosome (or P. falciparum) are C18 stearoyl- and C14 lauroyl-CoA, respectively (Milne & Ferguson, 2000; van Aalten et al., 2001).

By data-mining the recently completed C. parvum genome sequences, we have identified a unique long-type ACBP (CpACBP1) that contains an N-terminal ACBP domain and a C-terminal ankyrin-repeat sequence, which is structurally similar to the membrane-associated ACBP1 and ACBP2 in Arabidopsis thaliana. In the present study, we have expressed CpACBP1 protein as a fusion protein and characterized its primary biochemical features. We have also found that the CpACBP1 gene is differentially expressed during the parasite life cycle, and its encoded protein is chiefly located on the parasitophorous vacuole membranes (PVM), suggesting that this protein may be involved in the formation of PVM and/or uptake of fatty acids by the parasite.

METHODS

Identification of ACBP homologues from C. parvum and other apicomplexans

CpACBP1 was identified from the C. parvum genome as an intronless gene by a homology-search using characterized animal and plant ACBP proteins as queries. Similar searches were also performed to identify ACBP homologues from the Plasmodium falciparum and Toxoplasma gondii genome databases (http://www.PlasmoDB.org and http://www.ToxoDB.org, respectively) for comparison. Domains in the apicomplexan ACBP homologues were identified by searching the Conserved Domain Database at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) and by comparing with other characterized proteins in the GenBank database.

Molecular cloning and engineering of CpACBP1

To facilitate biochemical analysis, we cloned CpACBP1 and expressed CpACBP1 as a maltose-binding protein (MBP)-fusion as described below. Briefly, the entire open reading frame (ORF) of CpACBP1 was amplified from C. parvum (Iowa strain) genomic DNA (gDNA) with the following pair of primers: 5′-ATG ACT GAT ATC TTA TCC ACG AAC-3′ and 5′-atg gat ccT TAA CTG CTT TCG AGA ATT CTT-3′ (Note: lower cases represent added BamHI restriction site). A high-fidelity Pfu DNA polymerase (Stratagene) was used to minimize potential errors introduced by amplification. The PCR product was digested with BamHI to produce a cohesive 3′ end, but retained the blunt 5′ end to facilitate unidirectional cloning. The 5′ ends were phosphorylated by treating the amplicons with T4 polynucleotide kinase. After agarose gel electrophoresis, DNA fragments of the expected size were purified using a MinElute gel extraction kit (Qiagen), and ligated into an XmnI and BamHI double-digested pMAL-c2x vector (New England Biolabs) with a T4 DNA ligase. The ligated plasmids were transformed into the TOPO-10 strain of Escherichia coli cells (Invitrogen). The resulting colonies were first screened using a sense-stranded primer located upstream to the insert in the vector and the CpACBP1 antisense-stranded primer. Plasmids were isolated from PCR-positive colonies for sequencing to confirm their identity and the sequence of the inserts. The resulting construct and encoded fusion protein were named pMAL-c2x-CpACBP1 and MBP-CpACBP1, respectively.

Similarly, we also constructed an MBP-fusion containing only the ACBP domain. Because of the presence of two EcoRI restriction sites franking the ankyrin domain (i.e., nucleotides from 298 to 788), we removed the entire C-terminal ankyrin repeats by digesting pMAL-c2x-CpACBP1 plasmid with EcoRI and agarose gel separation. The remaining plasmid fragment was re-ligated back, resulting a construct (pMAL-c2x-CpACBP1-S) that encodes only the N-terminal 100 amino acids (aa) plus 7 extra aa derived from the remaining nucleotides at the 3′ end of insert and the vector's multiple cloning site (MBP-CpACBP1-S). We predict that the extra 7 aa (RKQLRIL) will not alter the function of the ACBP domain as they do not resemble any known functional sequences in the databases.

For each construct, a plasmid containing the correct insert was transformed into E. coli Rosetta cells (Novagen) for protein expression. Briefly, a single clone of the E. coli transformants was inoculated in 10 ml LB broth containing 100 μg ml−1 ampicillin and 34 μg ml−1 chloramphenicol for growing overnight at 37 °C. On the second day, the bacterial suspension was transferred into 1 L fresh medium containing the two antibiotics and grown at 30 °C until the OD495 reached to 0.3-0.5. After adding IPTG to the broth (100 μg ml−1 final concentration), bacteria were further incubated for 4 hours at 30 °C and collected by centrifugation for 10 min at 8,000 g. Bacterial pellets were suspended in 50 ml of column buffer (20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 2 mM EDTA) containing a protease inhibitor cocktail for bacteria (Sigma-Aldrich), disrupted by sonication, and centrifuged (8,000 g, 10 min) to remove cell debris.

Supernatants were applied to an amylose-resin column (New England Biolabs), washed with column buffer (>10 X bed vol.), and the MBP-CpACBP1 fusion proteins were eluted from the column with elution buffer (10 mM maltose, 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 2 mM EDTA) according to the manufacturer's protocol. The size and purity of the recombinant proteins were analyzed by SDS-PAGE. Protein concentrations were determined by the Bradford method using a commercial kit and bovine serum albumin (BSA) as standard. The aliquots of each protein sample were either used immediately or stored at −80 °C.

Semi-quantitative RT-PCR

The entire semi-quantitative RT-PCR (semi-qRT-PCR) procedure, including the isolation of parasite total RNA from various parasite life cycle stages, the normalization of parasite RNA contents in intracellular samples, reverse transcription, amplification and analysis, has been previously described in detail (Millership et al., 2004a; Millership et al., 2004b). In this study, the following pair of primers was used to amplify CpACBP1 transcripts: CpACBP1-F334 (5′-CCT TTA TTA GAA TCA AAC CTG G-3′) and CpACBP1-R334 (5′-TTG GAT AGG AGT CAA ACC ATC-3′). Another pair of previously reported primers (995F, 5′-TAG AGA TTG GAG GTT GTT CCT-3′ and 1206R, 5′-CTC CAC CAA CTA AGA ACG GCC-3′) was used to amplify 18S rRNA as a control for normalization (Abrahamsen & Schroeder, 1999). Each semi-qRT-PCR reaction contained a comparable amount of parasite RNA, and was subjected to 45-min reverse transcription and 23 (for CpACBP1) or 20 (for 18S rRNA) thermal cycles of PCR amplification. After agarose gel electrophoresis, the intensity of each product was measured using GENETOOLS program v.3 (Hitachi Software Engineering) and the relative level of CpACBP1 transcripts was determined as the signal ratio between the CpACBP1 and rRNA amplicons.

Production of polyclonal antibodies to CpACBP

Polyclonal antibodies to recombinant CpACBP1 protein were raised in a pathogen-free rabbit. Initial immunization used 0.2 mg of affinity-purified MBP-CpACBP1 protein emulsified in an equal volume of complete Freund's adjuvant. Two subsequent booster immunizations (0.1 mg) were injected on 30 and 60 days, respectively, after the primary immunization. Rabbit sera were collected prior to and after the immunization protocol. The anti-MBP portion of the polyclonal antibodies was removed by absorbing antiserum with equal volume of amyloseresin conjugated with MBP. The antibody titer and specificity were evaluated by western blot analysis.

Western blot analysis

Western blot analysis was performed to test whether CpACBP1 protein is present in various parasite life cycle stages. Oocysts (5×106/lane), free sporozoites (2×107/lane), and HCT-8 cells (1×106/lane) infected with C. parvum oocysts (oocysts : host cell ratio = 1:1) for 24, 48 and 72 hr were lysed in loading buffer containing a protease inhibitor cocktail for mammalian cells (Sigma-Aldrich) at 95 °C for 8 min. After centrifugation for 5 min, soluble materials were fractionated in a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was first blocked with 5% BSA in TBS (20 mM Tris pH 7.5, 50 mM NaCl) for 1 h, and then incubated with rabbit anti-CpACBP antibodies and a monoclonal anti-rabbit IgG antibody conjugated to alkaline phosphatase in 1% BSA in TTBS (TBS with 0.05% Tween-20). The blot was washed 3 times with TTBS after each incubation step, and all procedures were performed at room temperature. Finally, the labeled proteins were developed using 5′bromo-4-chloro-3-indolyl phosphate (BCIP).

Immunofluorescence microscopy

Intracellular parasites were prepared by infecting HCT-8 cells grown on poly-L-lysine-treated glass coverslips for 24, 48 or 72 hr. Cells were fixed with 10% formalin, rinsed with PBS, extracted with cold methanol (−20 °C for 5 min), blocked in 0.5 % BSA-PBS (10 min), labeled with primary antibodies (1 hr in 0.5 % BSA-PBS), and incubated with secondary antibodies conjugated with FITC or TRITC (60 min in 0.5 % BSA-PBS). Samples were washed after each incubation step (3 times, 5 min each) using PBS?. Free sporozoites were fixed in suspension, directly applied onto poly-L-lysine-treated coverslips, extracted, and air-dried prior to the incubations with antibodies. Co-localization of CpACBP1 with total membrane proteins (TMP). An SFP-type phosphopantetheinyl transferase (CpSFP-PPT) immunolocalization was similarly performed, except that the respective rabbit antibodies were directly labeled with Alexa Fluor 488 or Alexa Fluor 546 using the appropriate fluorophore-labeling kits (Invitrogen) prior to the immuno-labeling experiments.

No secondary antibodies were used in co-localization experiments. The TMP antibody has previously been shown to mainly label the PVM and feeder organ in intracellular parasites (Chen et al., 2003), while cytosolic localization of CpSFP-PPT has also been previously reported (Cai et al., 2005). All samples were mounted using a SlowFade Light Antifade medium containing 4′, 6′-diamidino-2-phenylindole (DAPI) for DNA counter-staining (Invitrogen) and examined with an Olympus BX51 Epi-Fluorescence microscope equipped with Differential Interference Contrast (DIC) and FITC/TRITC/DAPI filters.

Acyl-CoA binding Assay

The binding affinity of CpACBP1 with fatty acyl-CoA was measured by Lipidex 1000 assay as previously described (Rasmussen et al., 1990; Rosendal et al., 1993). Briefly, 40 pmol of recombinant CpACBP1 was mixed with [14C] palmitoyl-CoA (0 – 8 μM) in 100 μl of binding buffer (10 mM potassium phosphate, pH 7.4) and incubated at 37 °C for 30 min. The mixture was then chilled on ice for 10 min, mixed with 0.6 ml of cold Lipidex 1000 (hydroxyalkoxypropyl dextran, Type VI from Sigma-Aldrich) (50% slurry, v/v in binding buffer) with gentle rotation at 4 °C for 1 hr, and centrifuged for 5 min at 12,000 g at 4 °C to remove free acyl-CoA esters. An aliquot of 200 μl supernatant was taken from each sample for counting radioactivity in a Beckman LS6500 scintillation counter. Each experiment included negative controls using MBP-tag only for background subtraction. At least three replicates were performed for each experimental condition. The dissociation constant (Kd) was determined by plotting the amount of bound substrate against the total concentration of substrate using nonlinear regression and Prism v4.0 (GraphPad Software).

The substrate preference for CpACBP1 was determined by a competition-binding assay. It was performed in 100 μl of binding buffer containing 40 pmol of recombinant CpACBP1 (or MBP in control groups) and 80 pmol of [14C] palmitoyl-CoA in the absence or presence of 80 pmol of non-radioactive fatty acyl-CoA esters of various chain lengths (i.e., ranging from 4 to 20 carbons). All samples were incubated, extracted with Lipidex 1000, and counted for radioactivity as described above.

In addition, we also tested whether CpACBP1 could specifically bind to long chain fatty acid. In this assay, 80 pmol recombinant CpACBP1 protein was incubated with 80 pmol [3H] palmitic acid or [14C] palmitoyl-CoA in 100 μl buffer. After extraction with Lipidex 1000, the supernatants were counted for radioactivity as described above.

Autoradiography

To visualize the binding of CpACBP1 with acyl-CoA, 80 pmol of fusion protein was incubated with 80 pmol of [14C] palmitoyl-CoA in the presence or absence of 80 pmol of non-radioactive palmitoyl-CoA in 100 μl of binding buffer for 30 min at 37 °C. Subsequently, 20 μl of the reaction was mixed with 5 μl of 5X native loading buffer, and fractionated in a 10% native PAGE gel. The gel was dried on a heated gel-drier and radioactive protein bands were visualized with X-ray film in a BioMax TranScreen LE intensifying system (Kodak).

RESULTS

CpACBP1 is a “long-type” ACBP containing ankyrin repeats

CpACBP1 is the only ABCP homologue that can be identified from the C. parvum genome by repeated BLAST searches using several animal, plant and protist ABCP proteins as queries. This intronless encodes 268 aa that constitute an N-terminal ACBP domain (~ 90 aa) and a C-terminal sequence (~178 aa) containing two ankyrin repeats (Fig. 1). Other apicomplexans appear to possess more ACBP homologues in their genomes. For example, P. falciparum has 4 ACBP homologues (PfACBPs, 3 short and one long), and T. gondii has two (TgACBPs, one short and one long). The small ACBP domains in all apicomplexan proteins share many conserved residues with their homologues found in animals and plants that are characteristic to this group of proteins (Fig. 2). Amino acids that are critical to the ligand binding activity are all present in CpACBP1 and other apicomplexan ACBPs (Fig. 2, indicated by solid dots) (Burton et al., 2005). Similar to CpACBP1, the long-type ACBP in T. gondii contains two ankyrin repeats (Fig. 1). However, it also possesses an N-terminal signal peptide, possibly for secretion. On the other hand, the P. falciparum long-type ACBP contains no ankyrin repeats (Fig. 1). These observations indicate that apicomplexans may differ from each other by possessing different numbers and types of ACBP proteins.

Fig 1
Domain organization of CpACBP1 in comparison with those from other representative eukaryotic acyl-CoA binding proteins (ACBPs).
Fig. 2
Multiple alignment of conserved region in the ACBP domain between CpACBP1 and other representative eukaryotic ACBP proteins. Amino acids shared between CpACBP1 and other sequences are shaded, while residues conserved among all listed sequences are boxed. ...

CpACBP1 has a highest binding affinity to C16:0 palmitoyl-CoA

In order to investigate the binding features of CpACBP1, we have expressed the full-length protein as well as the ACBP domain as MBP-fusion proteins (i.e., MBP-CpACBP1 and MBP-CpACBP1-S). Both fusion proteins were purified to homogeneity using amylose resin-based affinity chromatography (Fig. 3). Although the fusion proteins expressed using pMAL-c2x vector contain a factor Xα cleavage site, attempts to effectively remove the MBP-tag using factor Xα were not successful. It is possible that the MBP-CpACBP1 fusion protein folded in a way that interfered with the access of factor Xα to the cleavage site. The difficult to remove the MBP-tag was also observed for the recombinant C. parvum malate and lactate dehydrogenases, in which their activities were assayed with the presence of MBP-tag (Madern et al., 2004).

Fig. 3
SDS-PAGE analysis of purified MBP-fused CpACBP1 proteins. Short = MBP-fusion protein containing ACBP domain only. Long = MBP-fusion protein containing the entire CpACBP1 sequence. M = protein molecular marker.

Using Lipidex 1000 assay, we first confirmed that the intact MBP-CpACBP1 was able to specifically bind to palmitoyl-CoA, while MBP had no or little affinity to the same ligand (Fig. 4A). With both ligand and protein concentrations at 80 μM (near the highest concentration of 100 μM in the kinetics assay), the nonspecific binding by MBP-tag was ~ 5% of the specific binding observed for MBP-CpACBP1. In another assay using both palmitic acid and palmitoyl-CoA as substrates, both MBP-tag and MBP-CpACBP1 displayed almost the same radioactivity in binding to the palmitic acid (Fig. 4B), indicating that fatty acid is not a favorite ligand for CpACBP1. Based on these observations, we hence decided to use the uncleaved fusion proteins in all subsequent analyses.

Fig. 4
A. Specific and nonspecific binding of [14C] palmitoyl-CoA (80 μM) by MBP-fused CpACP1 (40 μM) and MBP-tag (40 μM) by the Lipidex 1000 extraction assay. B. Relative binding between CpACBP1 and [14C] palmitoyl-CoA (80 μM) ...

We first studied the specific binding between CpACBP1 and palmitoyl-CoA and determined that the Kd for CpACBP1 to bind to palmitoyl-CoA was at 407 nM (Fig. 5). This value is significantly higher than those reported for many other ACBPs that are typically at 1 – 10 nM ranges (Burton et al., 2005). This observation suggests that, although Lipidex assay is a reliable assay for determining the acyl-CoA binding profile for an ACBP, it is probably not a sensitive method for determining the binding kinetics due to the binding competition between ACBP and Lipidex 1000 during the extraction step (Rasmussen et al., 1994). It is also possible that a significant portion of the CpACBP1 fusion protein was inactive and incapable of binding to acyl-CoA, and/or the N-terminal 42-kDa MBP-tag might physically interfere the binding kinetics of the recombinant proteins.

Fig. 5
Binding kinetics of recombinant CpACBP1 with palmitoyl-CoA as determined by Lipidex 1000 assay as described in detail in the Methods section.

We also tested the specific binding of recombinant CpACBP1-S to palmitoyl-CoA in comparison with that of full-length CpACBP1 using the same Lipidex assay. Under the condition of using 0.4 μM of protein and 0.8 μM of palmitoyl-CoA, both fusion proteins displayed similar specific binding activities (i.e., 0.180 and 0.206 pmol pmol−1 protein for CpACBP1 and CpACBP1-S, respectively), thus confirming that the ACBP domain was responsible for the acyl-CoA binding.

The specific binding of CpACBP1 to fatty acyl-CoAs is further confirmed by autoradiography. When [14C] palmitoyl-CoA was incubated with various fusion proteins, only recombinant CpACPB1 (full-length) or CpACBP1-S (ACBP domain only), but not MBP-tag, displays radioactivity (Fig. 6). The radioactive intensity associated with the fractionated CpACBP1 is reduced when an equal molar amount of non-radioactive fatty acyl-CoA was added into the reaction. It is also noticeable that multiple radioactive bands are observed in lanes containing CpACBP1. Since protein fractionation was performed in native PAGE gels, this observation suggests that CpACBP1 may also function as dimers or tetramers. However, it is also possible that protein aggregation might occur under the experimental conditions used.

Fig. 6
Autoradiography showing the binding of [14C]-palmitoyl-CoA by full-length (lanes 1 and 2) and short ACBP-domain only (lanes 3 and 4) fusion proteins after native PAGE fractionation. MBP-tag only (lanes 5 and 6) was used as a control. In lanes 1, 3 and ...

Based on the specific binding data, we decided to test the substrate preference for CpACBP1 using 0.8 μM of various fatty acyl-CoAs to compete with the same molar concentration of palmitoyl-CoA. This concentration is ~ 2X of the Kd value for palmitoyl-CoA, so that the protein occupancy by the ligands was neither too low or too high. The results show that CpACBP1 can bind to the medium and long chain acyl-CoAs (Fig. 7). This protein has highest affinity for palmitoyl-CoA (C16:0) with a gradually decreased binding affinity for other acyl-CoA esters. However, CpACBP1 is incapable of binding CoA esters with an acyl chain of 20 carbons or longer. This feature makes CpACBP1 different from many other ACBP proteins, including those from bovines and trypanosomes that can bind to CoA esters with an acyl chain of 24 or more carbons (Milne & Ferguson, 2000; van Aalten et al., 2001).

Fig. 7
Acyl-CoA binding specificity of CpACBP1 determined by Lipidex 1000 competition binding assay. In each reaction, [14C]-palmitoyl-CoA was mixed with the same molar amount of non-radioactive acyl-CoA with specified carbon chain length. The binding affinity ...

CpACBP1 gene is differentially expressed and its encoded protein is likely localized to the parasitophorous vacuole membranes (PVM)

Semi-quantitative RT-PCR has shown that CpACBP1 gene is expressed differentially in the complex parasite life cycle (Fig. 8). CpACBP1 transcripts were barely detectable in free sporozoites, but started to appear after parasites' invasion into host cells. The level of CpACBP1 transcripts was relatively low during early intracellular development (i.e., from 3 to 24 hr post-infection, P.I.), but gradually increased with the time of infection. This expression pattern differs from that of many other C. parvum genes, such as the replication protein A (RPA) subunits, CpSFP-PPT and β-tubulin, but is similar to those of oocyst wall proteins in this parasite (Abrahamsen & Schroeder, 1999; Cai et al., 2005; Millership et al., 2004a; Rider et al., 2005). Such a differential expression pattern is also supported by western blot and immunofluorescence microscopic analyses that only detected CpACBP1 protein in the intracellular parasites, but not in oocysts or free sporozoites (Figs. (Figs.99 and and1010).

Fig. 8
Relative levels of CpACBP1 gene transcripts in various Cryptosporidium parvum life cycle stages as determined by semi-quantitative RT-PCR. The level of transcripts is normalized using that of the parasite 18S rRNA as a control. Spz = excysted free sporozoites. ...
Fig. 9
Western blot detection of CpACBP1 protein in Cryptosporidium parvum oocysts, excysted free sporozoites, and intracellular parasites grown for 24 and 48 hr. CpACBP1 was only detected in the intracellular parasites, but not in oocysts and free sporozoites. ...
Fig. 10
Immunofluorescence microscopy of the CpACBP1 protein in intracellular Cryptosporidium parvum. A) Indirect Immuno-labeling of intracellular parasites grown for 24, 48 and 72 hr using a rabbit polyclonal antibody against CpACBP1 and a secondary antibody ...

More surprisingly, immunofluorescence microscopy indicates that CpACBP1 is probably located on the PVM (Fig. 10). Rabbit polyclonal antibodies clearly labeled the surface of meronts with a homogeneous pattern of distribution, but failed to label the merozoites within the meronts (Fig. 10A). In a dual-labeling experiment using a rabbit polyclonal antibody mainly against PVM and the electron dense connection between host cell and parasite, we co-localized CpACBP1 and PVM proteins (Fig. 10B). On the other hand, a polyclonal antibody against CpSFP-PPT clearly labeled the merozoites, but not on the surface of meronts in another dual-labeling experiment (Fig. 10C).

DISCUSSION

ACBP was originally identified as a mammalian diazepam binding inhibitor – a neuropeptide that could inhibit diazepam binding to the γ-aminobutyric acid (GABA) receptor (Guidotti et al., 1983). Typical ACBPs are small, ~10 kDa cytosolic proteins (Burton et al., 2005). However, there are a number of long-type ACBPs that are fused with ankyrin repeats, such as the ACBP1 and ACBP2 in Arabidopsis thaliana (Chye et al., 1999; Li & Chye, 2003), or with other functional domains, such as the human peroxisomal D3, D2-enoyl-CoA isomerase (Geisbrecht et al., 1999). ACBP mainly functions as an intracellular acyl-CoA transporter and pool former, and is critical to lipid metabolism in cells (Gossett et al., 1996; Knudsen et al., 2000; Schroeder et al., 1998). However, ACBP has only been found in eukaryotes, but not in prokaryotes except for a few pathogenic eubacteria that might have acquired ACBP from eukaryotic hosts via lateral gene transfer (Burton et al., 2005).

Among apicomplexans, C. parvum only possesses a single, long-type ACBP that is fused with an ankyrin repeats domain. However, other apicomplexans may have multiple ACBP proteins of various types (i.e., short-type, long-types fused with ankyrin repeats or with uncharacterized sequences) (Fig. 1), indicating that the ACBP-mediated metabolic pathways may be highly divergent in the Apicomplexa. Arabidopsis ACBP1 and ACBP2 are membrane proteins that differ from typical cytosolic ACBPs (Chye et al., 1999; Leung et al., 2005). The ankyrin repeats in these proteins are responsible for docking these proteins to the membrane by interacting with an ethylene-responsive element-binding protein. Our immuno-labeling data indicate that CpACBP1 is also a membrane protein (i.e., mainly associated with PVM) (Fig. 10). Such a membrane association is likely mediated by the interaction of ankyrin repeats with a yet unknown protein(s) in the PVM.

Although C. parvum is an intracellular parasite, it does not reside within the host cytoplasm. Instead, this parasite is extracytoplasmic, covered by PVM on the surface of intestinal epithelial cells (Chen et al., 2002). Therefore, the PVM is the only barrier separating parasites from the intestinal lumen. The localization of CpACBP1 to the PVM is thus intriguing, although it is currently uncertain whether CpACBP1 is also associated with the feeder organ at the host cell-parasite interface. It implies that CpACBP1 may be involved in the formation of the PVM or uptake of fatty acids across the PVM. However, since CpACBP1 mRNA and protein are undetectable (or barely detectable) in sporozoites and in the invasion stages (i.e., first 3 hr of infection), it seems less likely that CpACBP1 is associated with the early stage of PVM formation. On the other hand, it is known that C. parvum may have to import fatty acids from host cells or the intestinal lumen since it is likely to be incapable of synthesizing fatty acids de novo, although it is capable of elongating long chain fatty acids (Zhu, 2004). Therefore, it is possible that CpACBP1 may function as a fatty acyl-CoA scavenger in conjunction with an acyl-CoA synthetase on or around the PVM (or the feeder organ) to facilitate the fatty acid uptake by the parasite.

Another possibility is that CpACBP1 may be involved in the synthesis of glycosyl-phosphatidylinositol (GPI) anchor. In African trypanosomes, ACBP has been found to be responsible for supplying myristoyl-CoA to the fatty acid remodeling machinery during GPI synthesis (Milne & Ferguson, 2000; Milne et al., 2001). Although it is yet unclear whether GPI anchored molecules are present in the PVM, a recent comprehensive chemical analysis has clearly revealed the presence of complex glycosylinositol phospholipids in the C. parvum sporozoites (Priest et al., 2003). In addition, a number of enzymes involved in the biosynthesis of GPI anchors are also present in the C. parvum genome, which includes phosphatidylinositol N-acetylglucosaminyltransferases (e.g., Genbank accession numbers XP_628152, XP_627129 and XP_626317). Nonetheless, further investigations are necessary to test these hypotheses.

Our biochemical data show that, although CpACBP1 can bind to medium to long chain fatty acids with chain lengths up to 18-carbons, it displays highest binding affinity towards to the C16:0 palmitoyl-CoA. The Kd value at 407 nM was obtained by Lipid 1000 assay, which is comparable to those of other ACBPs determined by the same assay (Rasmussen et al., 1994). However, this value does not represent the true acyl-CoA binding affinity. Rather, it reflects the competitive binding between ACBP and Lipidex 1000 (Rasmussen et al., 1994). The Kd values determined by fluorescence or dialyzer-based methods are typically at lower nM range (i.e., 1 – 10 nM) (Chao et al., 2002; Frolov & Schroeder, 1998; Milne & Ferguson, 2000; van Aalten et al., 2001; Wadum et al., 2002). On the other hand, although Lipidex 1000 cannot be used to assess the true binding affinity for ACBPs, this method can be used as a qualitative assessment, such as the ligand competition assay. Autoradiography indicates that both the full-length CpACBP1 protein and its ACBP domain may form dimmers or even polymers (Fig. 5). However, it is unclear whether CpACBP1 is truly present as dimer or polymers in the parasites, or the observed multiple bands in autoradiography are only an artifact induced by the present experimental conditions.

Fatty acids are essential to all organisms. Recently, fatty acid metabolism has been considered as a promising target for the drug development against cryptosporidiosis and other important apicomplexans (Gornicki, 2003; Kuo et al., 2003; Ralph et al., 2001; Roberts et al., 2003; Waller et al., 2003; Zhu, 2004). Because ACBP plays a critical role in fatty acid metabolism, it is reasonable to speculate that CpACBP1 and other apicomplexan ACBPs may be explored as new drug targets for the control of cryptosporidiosis or other apicomlexan-based diseases.

ACKNOWLEDGEMEMNTS

We thank Jason Millership and Palvi Waghela for technical assistance. This research was supported by a grant from National Institutes of Health (R01 AI44594).

Abbreviations

ACBP
acyl-CoA binding protein
ACP
acyl carrier protein
FAS
fatty acid synthase
GPI
glycosyl-phosphatidylinositol
MBP
maltose-binding protein
PVM
parasitophorous vacuole membrane
TMP
total membrane proteins

Footnotes

Note: Nucleotide sequence data reported in this paper are available in the GenBank database under the accession number DQ406676

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