|Dittmer, Neal - KANSAS STATE UNIV|
|Suderman, Richard - KANSAS STATE UNIV|
|Jiang, Haobo - KANSAS STATE UNIV|
|ZHU, YU CHENG|
|Gorman, Maureen - UNIV WISCONSIN|
|Kanost, Michael - KANSAS STATE UNIV|
Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 14, 2003
Publication Date: January 1, 2004
Citation: Dittmer, N.T., Suderman, R.J., Jiang, H., Zhu, Y., Gorman, M.J., Kramer, K.J., Kanost, M.R. 2004. Characterization of CDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae. Insect Biochemistry and Molecular Biology 34: 29-41. Interpretive Summary: This paper is the first report of the sequences of several DNA fragments for some very important enzymes in insect developmental biology, called laccases which catalyze protein cross-linking reactions during hardening of the cuticle (outer protective layer of insects). Laccases help to control the physical properties of the exoskeleton. In the past, cuticle-hardening enzymes have been very difficult to study because of their intractable nature and instability. Now, however, obtaining cloned cDNAs is a significant step that will allow production of these proteins and the development of reagents for use in future research on the expression, properties and regulation of laccase, as well as for the development of compounds that inhibit laccase activities. Together with scientists at Kansas State University and the University of Wisconsin, we analyzed and discussed the nucleotide and amino acid sequences, and made comparisons with similar enzymes from other organisms, including nematodes and fungi. The results obtained provide new information about structure-function relationships for insect enzymes and add to the knowledge base for insect molecular science. The long-term goal of this research is to facilitate a more effective application of Cis for the manipulation of cuticle tanning and for control of insect pests.
Technical Abstract: The laccase-type of phenoloxidases may play an important role in insect physiology by catalyzing protein cross-linking reactions during cuticle sclerotization. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the ten histidines and one cysteine that bind to copper ions in fungal laccases. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino terminal sequence, as well as a methionine in the T1 copper center. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that the AgLac1 mRNA was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malphigian tubules and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. Both of the M. sexta laccase mRNAs were constitutively expressed, and also showed enhanced expression in the pharate pupal and early pupal epidermis; a result consistent with the laccases' presumed role in cuticle sclerotization.