Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans.
A chitinase cDNA, GChit1 was isolated from Glossina morsitans morsitans and shown to be specifically expressed in fat body tissue. GChit1 is encoded by a 1.6 kb mRNA with a putative open reading frame (ORF) of 460 amino acids (predicted pI=7.5, m.w.=51kDa) that contains a signal peptide domain and two potential N-linked glycosylation sites. The ORF exhibits homology to various chitinases characterized from insects. It has the conserved catalytic site residues and the cysteine-rich 3'-end domain associated with chitin binding although the serine/threonine rich domain is apparently missing. Southern blot data indicate that GChit1 is present as a single-copy locus in the Glossina genome. Northern analysis indicates that transcripts for GChit1 can be detected only from the fat body of adult flies. Similarly, chitinase activity could be detected in fat body but not in the gut or salivary gland tissues. The full-length cDNA was expressed in vitro in Drosophila S2 cells and the molecule was produced in a soluble form. Polyclonal antibodies raised against recGChit1 could recognize a protein of about 50 kDa in adult fat body extracts. In addition to fat body, chitinase protein was detected by Western analysis from the milk gland tissue of pregnant females as well as from the intrauterine larval and pupal developmental stages. No chitinase specific mRNA transcripts could be observed, however from larvae and pupae. The intrauterine larva of tsetse may receive the protein from its mother via the milk gland route. The molecular characteristics of GChit and its product and the potential role of this chitinase in tsetse biology are discussed.
Yan
J
J
Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
Cheng
Q
Q
Narashimhan
S
S
Li
C-B
CB
Aksoy
S
S
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005216:Fat Body / Q000201:enzymology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods; D008969:Molecular Sequence Data; D012333:RNA, Messenger; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014312:Trisaccharides / Q000378:metabolism; D014370:Tsetse Flies / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D014312:Trisaccharides; C054851:allosamidin; D002688:Chitinases; D000117:Acetylglucosamine