Cloning, Expression, and Characterization of the acyl-CoA-binding Protein in African Trypanosomes

Abstract

African trypanosomes are shielded from their hosts' defenses by a coat of variant surface glycoprotein molecules, each of which is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. During the later stages of glycosylphosphatidylinositol biosynthesis, myristic acid is incorporated into the anchor from the donor myristoyl-CoA by a series of unique fatty acid remodeling and exchange reactions. We have cloned and expressed a recombinant trypanosome acylCoA-binding protein that has a preference for binding relatively short chain acyl-CoAs and that has a high affinity for binding myristoyl-CoA (K d ‫؍‬ 3.5 ؋ 10This protein enhances fatty acid remodeling of glycosylphosphatidylinositol precursors in the trypanosome cell-free system. We speculate that the trypanosome acyl-CoA-binding protein plays an active role in supplying myristoyl-CoA to the fatty acid remodeling machinery in the parasite.

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

“…In African trypanosomes, ACBP has been found to be responsible for supplying myristoyl-CoA to the fatty acid remodelling machinery during GPI synthesis (Milne & Ferguson, 2000;Milne et al, 2001). Although it is as 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 C. parvum sporozoites (Priest et al, 2003).…”

“…Rather, it reflects the competitive binding between ACBP and Lipidex 1000 (Rasmussen et al, 1994). The K d values determined by fluorescence or dialyser-based methods are typically lower, in the range of 1-10 nM (Chao et al, 2002;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 of ACBPs, this method can be used as a qualitative assessment, such as the ligand competition assay.…”

“…However, CpACBP1 is incapable of binding acyl-CoA esters with an acyl chain of 20 carbons or longer. This feature makes CpACBP1 different to many other ACBPs, including those from bovines and trypanosomes that can bind to acyl-CoA esters with an acyl chain of 24 or more carbons (Milne & Ferguson, 2000; van Aalten et al, 2001). 2) and short ACBP-domain only (lanes 3 and 4) fusion proteins after native PAGE fractionation.…”

“…T-ACBP in testis, L-ACBP in liver and I-ACBP in intestine) . In Trypanosoma brucei, an ACBP was found to be involved in the synthesis of the glycosylphosphatidylinositol (GPI) anchor in variant surface glycoproteins (Milne & Ferguson, 2000;Milne et al, 2001). Although ACBPs are capable of binding mediumto long-chain fatty acyl-CoA esters, they may vary in their substrate preference and binding affinities.…”

“…Although ACBPs are capable of binding mediumto long-chain fatty acyl-CoA esters, they may vary in their substrate preference and binding affinities. For example, the highest affinities of ACBPs from bovines (liver) and trypanosomes (or P. falciparum) are C18 stearoyl-and C14 lauroyl-CoA, respectively (Milne & Ferguson, 2000;van Aalten et al, 2001).…”