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. ∼90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankyrin 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 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.

Cryptosporidium parvum Long-Chain Fatty Acid Elongase

“…In addition to membrane localization in sporozoites, immunostaining has localized CpLCE1 primarily to the PVM, similar to the case for both CpACBP1 and the C. parvum oxysterol binding protein-related protein 1 (CpORP1) (35,36). Cryptosporidium parvum is an intracellular parasite, but it is considered extracytoplasmic because of being covered by a PVM on the host intestinal epithelial cells (6).…”

“…5B). It has previously been shown that CpACBP1 also colocalizes with the PVM proteins (35). An additional dual-labeling experiment using rabbit polyclonal antibodies against CpACBP1 colocalized CpLCE1 and CpACBP1 proteins (Fig.…”

“…Cryptosporidium parvum is an intracellular parasite, but it is considered extracytoplasmic because of being covered by a PVM on the host intestinal epithelial cells (6). Although association with the feeder organelle is still undetermined, it is interesting that CpLCE1 localizes to the PVM along with CpACBP1 and CpORP1, which could possibly be involved in lipid uptake across the PVM (35,36). Whether CpLCE1 acts in conjunction with these two in either lipid uptake or formation of the PVM is not understood at this time.…”

“…The anti-TMP antibodies and FITC-conjugated secondary antibodies were incubated simultaneously with anti-CpLCE1 and corresponding secondary antibodies, respectively; whereas the CpACBP1 antibodies were directly labeled with Alexa Fluor 488 using the appropriate fluorophore labeling kit (Invitrogen). Colocalization of CpLCE1 with TMP or CpACBP1 was selected because the TMP antibodies have been shown to label the parasitophorous vacuolar membrane (PVM) and feeder organelle (7) and CpACBP1 localizes to the surface of merozoites as well as colocalizes with TMPs (35). All samples were mounted with a SlowFade Gold antifade reagent containing 4Ј,6Ј-diamidino-2-phenylindole (DAPI) for DNA counterstaining (Invitrogen) and examined with an Olympus BX51 epifluorescence microscope equipped with differential interference contrast and TRITC/ FITC/DAPI filters.…”

Arabidopsis ACBP6 is an acyl-CoA-binding protein associated with phospholipid metabolism

“…2 Other than the 10-kD ACBP, much larger forms of ACBPs have been reported in A. thaliana, [3][4][5] Caenorhabditis elegans 6 and Cryptosporidium parvum. 7 In Arabidopsis, six genes have been identified to encode ACBPs and they have been designated ACBP1 to ACBP6. 5 ACBP1 and ACBP2 are membrane-associated proteins, ACBP3 is extracellularly-targeted, and ACBP4 and ACBP5 contain kelch-motifs.…”