Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta

Department of Biochemistry, 141 Chalmers Hall, Kansas State University, Manhattan, KS 66506-0116, USA.
Insect biochemistry and molecular biology (Impact Factor: 3.45). 08/2009; 39(9):596-606. DOI: 10.1016/j.ibmb.2009.06.006
Source: PubMed


Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect's cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-beta-alanyldopamine-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis-Menten kinetics when NADA was used as a substrate, with K(m) values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent k(cat) values were 100 min(-1), 80 min(-1), and 290 min(-1). The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with K(m) values of 1.9 mM and 0.47 mM, respectively, and apparent k(cat) values of 200 min(-1) and 180 min(-1). These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.

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    • "In the melanin metabolism pathway of insects, tyrosine is converted into four types of catecholamines, specifically, dopa, dopamine, N-acetyl dopamine (NADA) and N-β-alanyldopamine (NBAD), through a series of catalytic reactions, such as hydroxylation, decarboxylation, and addition of acetyl or β-alanyl groups [3], [4], [5], [6], [7], [8]. After oxidation by phenoloxidases, such as Laccase2, these catecholamines participate in insect cuticle tanning [9], [10], [11], [12]. "
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    ABSTRACT: Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild-type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.
    Full-text · Article · Aug 2012 · PLoS ONE
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    • "Approximately 15 µg protein was loaded per lane unless otherwise mentioned, and SDS-PAGE and Western blot assay were performed as previously described [28]. Antibody against the silkworm PPO (1∶5,000), or lysozyme (a gift from Dr. K. Suzuki; 1∶5,000) [29], or Manduca sexta laccase (a gift from Dr. M. Kanost; 1∶2,000) [30] was used as the primary antibody, and the AP-conjugated goat anti-rabbit IgG (1∶5,000) was used as the second antibody. "
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    Full-text · Article · Jul 2012 · PLoS ONE
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    • "Previous studies suggest that laccase-2A from B. mori is expressed as a proenzyme and is activated by proteolytic cleavage (Yamazaki, 1989; Yatsu and Asano, 2009), but that laccase-2A from M. sexta is constitutively active, and that removal of the amino-terminal region of the protein is not associated with increased activity (Dittmer et al., 2009). Our results strongly suggest that lacase-2 from T. castaneum and A. gambiae are expressed as constitutively active enzymes, and we predict, based on studies of laccase-2 from B. mori, M. sexta and Papilio xuthus, that TcLac2A and AgMCO2A activity is regulated by the availability of substrate in newly synthesized cuticle (Dittmer et al., 2009; Futahashi et al., 2010; Yatsu and Asano, 2009). "
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