Cloning and characterization of five cDNAs encoding peritrophin-A domains from the cat flea, Ctenocephalides felis.
Five cDNAs encoding peritrophin-A domains were identified as expressed sequence tags (ESTs) from flea hindgut and Malpighian tubule (HMT) cDNA libraries. The full-length cDNAs for each were subsequently isolated and sequenced. Three of the encoded proteins were similar to published peritrophin sequences, and thus were called "peritrophin-like", or PL1, PL2, and PL3. The other two sequences had similarity to both mucin and peritrophin proteins, and were called "mucin/peritrophin-like", or MPL1 and MPL2. The predicted protein sequences encoded by these cDNAs all contained a signal sequence and one or more peritrophin-A domains, which have been shown in other proteins to bind chitin. Aside from the peritrophin-A domains, the sequences shared little or no similarity to each other or to other proteins in the GenBank non-redundant database. The predicted protein sequences were variable in size, ranging in length from 81 to 453 amino acids. The two MPL proteins contained putative N-linked and O-linked glycosylation sites, including a region of seven nearly perfect tandem repeats in the MPL1 protein sequence. Northern blot analysis of different flea lifestages and fed adult timepoints showed distinct mRNA expression patterns for each gene, although all five transcripts were primarily or exclusively detected in the HMT tissues in adults. The PL1 protein was detected by immuno-blot in soluble and insoluble protein extracts from unfed and fed adult fleas. The PL1 protein from the insoluble fractions appeared to be approximately 1 kDa larger than the PL1 protein from the soluble protein fractions. Immunohistochemistry performed on flea thin sections revealed that the PL1 protein was detected in the Malpighian tubules, hindgut, rectum, and trachea. Unpurified native PL1 protein from both soluble and insoluble protein fractions was tested for chitin-binding activity but did not bind to chitin under the conditions tested. These results show that the flea peritrophin-like proteins may have biological functions that are distinct from the peritrophic matrix and from the binding of chitin.
Gaines
Patrick J
PJ
Heska Corporation, 1613 Prospect Parkway, Fort Collins, CO, USA. gainesp@heska.com
Walmsley
Scott J
SJ
Wisnewski
Nancy
N
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002686:Chitin / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D020224:Expressed Sequence Tags; D015723:Gene Library; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009077:Mucins / Q000302:isolation & purification; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D005423:Siphonaptera / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D019476:Insect Proteins; D008562:Membrane Glycoproteins; D009077:Mucins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002686:Chitin