FUNCTIONAL CHARACTERIZATION OF ACYL-CoA BINDING PROTEIN (ACBP) AND OXYSTEROL BINDING PROTEIN-RELATED PROTEINS (ORPS) FROM CRYPTOSPORIDIUM PARVUM

Functional Characterization of Acyl-CoA Binding Protein (ACBP) and Oxysterol Binding Protein-Related Proteins (ORPs) from Cryptosporidium parvum. (December 2006) Bin Zeng, B.S., Jiangxi Agricultural University; M.S., China Agricultural University Chair of Advisory Committee: Dr. Guan Zhu From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs 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 open reading fragment (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.