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

Abstract

In this paper, the identification and functional analysis of a fatty acyl-CoA-binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum are described. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ACBPs (i.e. approximately 90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 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 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane.

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. Pf = Plasmodium falciparum. Tg = Toxoplasma gondii. At = Arabidopsis thaliana. Hs = Homo sapiens. Numbers following the species abbreviations are the accession numbers in GenBank or the corresponding apicomplexan genome databases (http://www.PlasmoDB.org or http://www.ToxoDB.org). Solid dots indicate amino acids critical to the acyl-CoA binding activity.

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.

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) or [³H] palmitic acid as determined by Lipidex 1000 assay. Radioactivity was normalized using MBP-tag a control.

Fig. 5

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

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 5, the same molar amount of non-radioactive palmitoyl-CoA was also included to compete with the radioactive acyl-CoA, which resulted in the reduced intensity of radioactivity in these lanes. Positions of CpACBP1 monomers, dimers and polymers are indicated. Some acyl-CoA molecules were retained in the loading wells as indicated.

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 was presented as the percent displacement of radioactive palmitoyl-CoA by non-radioactive acyl-CoA. SEM values were determined from at least three individual samples.

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 conjugated with FITC. B) Direct dual-labeling of intracellular parasites grown for 24 hr using an Alexa Fluor 546-conjugated antibody against CpACBP1 and an Alexa Fluor 488-conjugated antibody against parasite total membrane protein (TMP) that mainly labels parasitophorous vacuole membrane and feeder organ. Both antibodies displayed the same labeling pattern on the surface of a meront. C) Dual-labeling of intracellular parasites grown for 48 hr using an Alexa Fluor 488-conjugated antibody against CpACBP1 and an Alexa Fluor 546-conjugated antibody against cytosolic CpSFP-PPT, showing CpACBP1 mainly on the surface of meronts, rather than in merozoites. Phase = phase contrast. DIC = differential interference contrast. DAPI = 4′,6-diamidino-2-phenylindole for counterstaining nuclei. Bar = 5 μm.