Insect allantoinase: cDNA cloning, purification, and characterization of the native protein from the cat flea, Ctenocephalides felis

• Published in: Insect biochemistry and molecular biology Vol. 34; no. 3; pp. 203 - 214
• Main Authors: Gaines, Patrick J, Tang, Liang, Wisnewski, Nancy
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.03.2004
• Subjects: 2,4-dinitrophenylhydrazine; Allantoinase; Amidohydrolase; Amidohydrolases - genetics; Amidohydrolases - isolation & purification; Amidohydrolases - metabolism; Amino Acid Sequence; amino acid sequences; Animals; cDNA; Cloning, Molecular; complementary DNA; Ctenocephalides felis; DNA, Complementary - genetics; enzyme activity; Flea; gene expression; glycosylation; hindgut; hydrolases; Immunohistochemistry; Malpighian tubule; Malpighian tubules; messenger RNA; Molecular Sequence Data; nucleotide sequences; Protein Sorting Signals - genetics; Pulicidae; purification; Purine degradation pathway; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; sequence alignment; sequence homology; Sequence Homology, Amino Acid; Siphonaptera - enzymology; Siphonaptera - genetics; tissue distribution; Ctenocephalides felis; Flea; Malpighian tubule; 2,4-dinitrophenylhydrazine; Amidohydrolase; Purine degradation pathway; cDNA; Allantoinase
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2003.10.002

Allantoinase catalyses the hydrolysis of allantoin to allantoic acid. This reaction is a step in the purine degradation pathway, which produces nitrogenous waste for excretion. A cDNA encoding full-length allantoinase was cloned from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. The cDNA encoded a 483 amino acid protein that had 43% identity with the bullfrog Rana catesbeiana allantoinase and contained the conserved histidine and aspartic acid residues required for zinc-binding and catalytic activity. Unlike the bullfrog allantoinase, the C. felis allantoinase sequence was predicted to contain a 22 amino acid signal sequence, which targets the protein to the secretory pathway. Expression of the mRNA was detected by Northern blot in the first, third, and wandering larval stages as well as in fed and unfed adults, but was not seen in eggs or pupae. In adults, mRNA encoding allantoinase was detected only in the HMT tissues. Immunohistochemistry performed using affinity-purified rabbit immune serum generated against purified recombinant flea allantoinase showed that the native protein localized to the HMT tissues in adult fleas. The anti-allantoinase serum recognized two proteins in an adult flea soluble protein extract, one migrating at 56 kDa and the other at 53 kDa. The two proteins were separated by gel filtration chromatography and were both associated with allantoinase activity. The difference in size appeared to be due to a difference in glycosylation of the proteins. The 53 kDa protein was further purified to near homogeneity by affinity chromatography and retained allantoinase activity. A comparison of the sizes of the native and recombinant C. felis proteins indicated that the 53 kDa native protein may be the product of a post-translational cleavage event, possibly at the putative 22 amino acid signal sequence at the N-terminus of the protein.