Cryptosporidium parvum Long-Chain Fatty Acid Elongase

ABSTRACT We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C14:0) and palmitoyl-CoA (C16:0) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C16:0 and C14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1.

[1]  Aaron T. Smith,et al.  The Protozoan Parasite Cryptosporidium parvum Possesses Two Functionally and Evolutionarily Divergent Replication Protein A Large Subunits* , 2005, Journal of Biological Chemistry.

[2]  D. Jump,et al.  Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases Published, JLR Papers in Press, January 16, 2005. DOI 10.1194/jlr.M400335-JLR200 , 2005, Journal of Lipid Research.

[3]  P. T. Englund,et al.  A fatty-acid synthesis mechanism specialized for parasitism , 2007, Nature Reviews Microbiology.

[4]  N. LaRusso,et al.  Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src. , 2003, Gastroenterology.

[5]  L. Robertson,et al.  In vitro excystation of Cryptosporidium parvum , 1993, Parasitology.

[6]  R. Garcés,et al.  Very long chain fatty acid synthesis in sunflower kernels. , 2005, Journal of agricultural and food chemistry.

[7]  M. J. LaGier,et al.  Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase. , 2002, Gene.

[8]  J. Warrington,et al.  Identification of a Mammalian Long Chain Fatty Acyl Elongase Regulated by Sterol Regulatory Element-binding Proteins* , 2001, The Journal of Biological Chemistry.

[9]  J. Jaworski,et al.  Active-site residues of a plant membrane-bound fatty acid elongase beta-ketoacyl-CoA synthase, FAE1 KCS. , 2001, Biochimica et biophysica acta.

[10]  H. Sone,et al.  Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs. , 2002, Journal of lipid research.

[11]  B. Zeng,et al.  Two distinct oxysterol binding protein-related proteins in the parasitic protist Cryptosporidium parvum (Apicomplexa). , 2006, Biochemical and biophysical research communications.

[12]  R. Welti,et al.  A Comparative Study of Lipid Compositions of Cryptosporidium parvum (Apicomplexa) and Madin‐Darby Bovine Kidney Cells , 1994, The Journal of eukaryotic microbiology.

[13]  J. Keithly,et al.  Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum. , 2004, Molecular and biochemical parasitology.

[14]  G. Zhu,et al.  Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan Cryptosporidium parvum , 2005, Eukaryotic Cell.

[15]  C. Cassagne,et al.  Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua. , 1992, Biochimica et biophysica acta.

[16]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[17]  G. Zhu,et al.  Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase. , 2007, International journal for parasitology.

[18]  D. L. Cinti,et al.  The fatty acid chain elongation system of mammalian endoplasmic reticulum. , 1992, Progress in lipid research.

[19]  G. Zhu,et al.  Functional characterization of a fatty acyl-CoA-binding protein (ACBP) from the apicomplexan Cryptosporidium parvum. , 2006, Microbiology.

[20]  R. Reski,et al.  Cloning and functional characterisation of an enzyme involved in the elongation of Delta6-polyunsaturated fatty acids from the moss Physcomitrella patens. , 2002, The Plant journal : for cell and molecular biology.

[21]  A. Uttaro,et al.  Elongation of polyunsaturated fatty acids in trypanosomatids , 2007, The FEBS journal.

[22]  D. Nugteren.  The enzymic chain elongation of fatty acids by rat-liver microsomes. , 1965, Biochimica et biophysica acta.

[23]  H. Sprecher,et al.  An analysis of partial reactions in the overall chain elongation of saturated and unsaturated fatty acids by rat liver microsomes. , 1977, The Journal of biological chemistry.

[24]  P Argos,et al.  Prediction of transmembrane segments in proteins utilising multiple sequence alignments. , 1994, Journal of molecular biology.

[25]  Stuart Smith.  The animal fatty acid synthase: one gene, one polypeptide, seven enzymes , 1994, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  E. Fukusaki,et al.  Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha. , 2007, Gene.

[27]  H. Sprecher,et al.  Elongation of long-chain fatty acids. , 2004, Progress in lipid research.

[28]  J. Horton,et al.  Identification of Two Mammalian Reductases Involved in the Two-carbon Fatty Acyl Elongation Cascade* , 2003, The Journal of Biological Chemistry.

[29]  Jie J. Zheng,et al.  The structural biology of type II fatty acid biosynthesis. , 2005, Annual review of biochemistry.

[30]  R. Reski,et al.  Cloning and functional characterisation of an enzyme involved in the elongation of Delta6-polyunsaturated fatty acids from the moss Physcomitrella patens. , 2002, The Plant journal : for cell and molecular biology.

[31]  K. Gable,et al.  Members of the Arabidopsis FAE1-like 3-Ketoacyl-CoA Synthase Gene Family Substitute for the Elop Proteins of Saccharomyces cerevisiae* , 2006, Journal of Biological Chemistry.

[32]  R. Westerberg,et al.  Fatty acid elongases in mammals: their regulation and roles in metabolism. , 2006, Progress in lipid research.

[33]  William R. Taylor,et al.  The rapid generation of mutation data matrices from protein sequences , 1992, Comput. Appl. Biosci..

[34]  P. T. Englund,et al.  Specialized fatty acid synthesis in African trypanosomes: myristate for GPI anchors. , 2000, Science.

[35]  C. Sterling,et al.  Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients. , 1987, The Journal of parasitology.

[36]  Hervé Moreau,et al.  Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesiss⃞s⃞ The online version of this article (available at http://www.jlr.org) contains an additional figure. Published, JLR Papers in Press, August 1, 2004. DOI 10.1194/jlr.M400181-JLR200 , 2004, Journal of Lipid Research.

[37]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[38]  G. Zhu,et al.  Current Progress in the Fatty Acid Metabolism in Cryptosporidium parvum1 , 2004, The Journal of eukaryotic microbiology.

[39]  S. J. Upton,et al.  Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum. , 2000, Molecular and biochemical parasitology.