Characterization of the multicopper oxidase gene family in Anopheles gambiae

Abstract

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron, copper or ascorbic acid. We have identified five putative MCO genes in the genome of Anopheles gambiae and have cloned cDNAs encompassing the full coding region for each gene. MCO1 mRNA was detected in all developmental stages and in all of the larval and adult tissues tested. We observed an increase in MCO1 transcript abundance in the midguts and Malphighian tubules of adult females following a blood meal and in adult abdominal carcasses in response to an immune challenge. Two alternatively spliced isoforms of MCO2 mRNA were identified. The A isoform of MCO2 was previously detected in larval and pupal cuticle where it probably catalyzes sclerotization reactions (He et al., 2007). The B isoform was transcriptionally upregulated in ovaries in response to a blood meal. MCO3 mRNA was detected in the adult midgut, Malpighian tubules, and male reproductive tissues; like MCO1, it was upregulated in response to an immune challenge or a blood meal. MCO4 and MCO5 were observed primarily in eggs and in the abdominal carcass of larvae. A phylogenetic analysis of insect MCO genes identified putative orthologs of MCO1 and MCO2 in all of the insect genomes tested, whereas MCO3, MCO4 and MCO5 were found only in the two mosquito species analyzed. MCO2 orthologs have especially high sequence similarity, suggesting that they are under strong purifying selection; the A isoforms are more conserved than the B isoforms. The mosquito specific group shares a common ancestor with MCO2. This initial study of mosquito MCOs suggests that MCO2 may be required for egg development or eggshell tanning in addition to cuticle tanning, while MCO1 and MCO3 may be involved in metal metabolism or immunity.

Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster

“…Previous studies of gene expression in D. melanogaster found that MCO1 mRNA is most abundant in late stage embryos, feeding stage larvae, and adults, and that MCO1 is expressed predominantly in the digestive system (midgut, hindgut, and crop) and Malpighian tubules (17,18). These expression profiles are similar to those of MCO1 orthologues in Anopheles gambiae and Manduca sexta (19)(20)(21). To establish whether MCO1 functions as a ferroxidase in D. melanogaster, we purified recombinant MCO1 and analyzed its enzymatic activity, performed immunohistochemistry to determine the location of MCO1 within the insect, and evaluated the loss-of-function phenotypes generated by RNAi-mediated knockdown.…”

“…Excised gel slices containing the recombinant protein were sent to Open Biosystems for the production of polyclonal antiserum in a rabbit. To remove antibodies that might react with conserved epitopes in other MCOs, the antiserum was incubated with purified A. gambiae MCO4 (19), and the unbound fraction was retained.…”

“…Alternatively, oxidation of ferrous iron followed by transferrin loading may play a protective role by limiting toxic radical formation, which is generated by ferrous but not ferric iron (1), or, it may have an immune function, given that ferric-transferrin is less likely than free iron to serve as a source of iron for pathogens (32). The possibility of an immune function is supported by up-regulation of MCO1 in D. melanogaster (33) and A. gambiae (19) in response to infection. Finally, the oxidation of ferrous iron by MCO1 may supply ferric iron to melanotransferrin, which is required for septate junction formation during epithelial maturation in D. melanogaster (34).…”

A review of the immune molecules in the sea cucumber

“…Most gene sequences of invertebrate POs have an open reading frame (ORF) approximately 2000 bp in length, and the deduced amino acid sequences contain two conserved copper centers [110,111]. In addition, PO mRNA expression is correlated with the challenge with microbial polysaccharide or pathogens [112e114].…”

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera,Apidae)

“…Its enzymatic activity has been characterized in the integument of Diptera (Barrett & Andersen 1981;Barrett 1987a,b;Binnington & Barrett 1988;Sugumaran et al 1992) and Lepidoptera (Dittmer et al 2009). In addition, the expression of genes encoding laccases has also been described in Diptera (Gorman et al 2008), Lepidoptera (Dittmer et al 2004;Yatsu & Asano 2009), Coleoptera (Arakane et al 2005;Niu et al 2008), and more recently, in Hymenoptera (Elias-Neto et al 2010) and Hemiptera (Futahashi et al 2010(Futahashi et al , 2011.…”