An immune-induced Reeler protein is involved in the Bombyx mori melanization cascade
Graphical abstract
Highlights
► Two reeler domain-containing cDNA clones, reeler1 and reeler2 were isolated from Bombyx mori. ► Reeler1 expression is inducible by bacterial challenge in B. mori larvae, while reeler2 expression is not inducible. ► Reeler1 is involved in the nodulation responses. ► Reeler1 participates in the prophenoloxidase activation cascade.
Introduction
Insects rely on an innate immunity response to defend themselves against microbial infection. The melanization cascade is an important component of the insect immune defense system, and it is involved in numerous defense responses, including nodulation, phagocytosis and melanotic encapsulation (Cerenius and Soderhall, 2004, Kanost et al., 2004, Mavrouli et al., 2005, Wilson et al., 1999).
Nodule formation is the earliest and predominant cellular defense response to bacterial infections in insects (Bedick et al., 2000). Nodulation is thought to begin with the formation of hemocyte aggregates and to end with melanization, forming darkened nodules. The nodulation process contributes to clearance of the bacteria from the circulating hemolymph (Miller and Stanley, 2000). Noduler is a novel immune-induced protein that was purified from bacteria-challenged hemolymph in wild silkworm, Antheraea mylitta, and it was first reported to mediate the nodulation response (Gandhe et al., 2007). Several genes that share sequence similarity with A. mylitta noduler have been identified from immunized larval fat bodies of the fall webworm, Hyphantria cunea (Shin et al., 1998), and from Manduca sexta (Zhu et al., 2003), Samia cynthia ricini (Bao et al., 2003), Lonomia obliqua (Veiga et al., 2005) and Bombyx mori (GenBank accession no. DQ898221). The deduced amino acid sequences of these proteins include a characteristic reeler domain (Zhu et al., 2003). The reeler domain was initially identified in the mouse reelin protein (D’Arcangelo et al., 1995), a secreted glycoprotein that plays a pivotal role in the development of the central nervous system in mammals (Quattrocchi et al., 2002). The reeler domain-containing genes identified in lepidopteran insects may have immune-related functions, as shown in studies reporting that bacterial challenge induced their expression in S. c. ricini, A. mylitta, H. cunea and M. sexta larvae (Bao et al., 2003, Gandhe et al., 2007, Shin et al., 1998, Zhu et al., 2003). However, the detailed functions of these reeler domain-containing molecules in the insect defense system remain largely unknown. The silkworm, B. mori, is a major model for insect biochemical research and the first lepidopteran insect for which draft genome sequences became available (Xia et al., 2004). However, the functions of the B. mori reeler proteins have not yet been investigated. Two reeler domain-containing genes are predicted in the silkworm genome (http://silkworm.genomics.org.cn/). One contains the complete open reading frame (ORF) sequence (BGIBMGA014360-TA), and the other lacks some coding sequence at the 3′ terminus of the ORF (BGIBMGA005981-TA). It is interesting to know if both of the reeler genes were all expressed in B. mori and if they had the expression differences in response to bacterial challenge.
Gandhe et al. (2007) reported that Noduler in A. mylitta functions in nodule formation in the cellular immune response to bacterial infection. Further study is needed to understand how this reeler domain-containing protein participates in the nodulation response. Melanization is of central importance in the nodulation and capsulation responses in some lepidopteran and dipteran insects, such as Pseudoplusia and Drosophila, and results in melanin formation (Lavine and Strand, 2002, Lavine and Strand, 2003, Sideri et al., 2008). Prophenoloxidase (proPO) activation is an important step in the insect melanization cascade; proPO is activated by microbial infection and leads to the proteolytic conversion of proPO into phenoloxidase (PO) in the hemolymph (Ji et al., 2004). PO catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine (dopa) and the oxidation of dopa to dopaquinone (Ashida and Brey, 1998, Kan et al., 2008), which allows for wound healing and the melanization of foreign microorganisms that are trapped in hemocyte nodules (Kanost et al., 2004). Dopamine present in hemolymph is also a significant substrate for PO. PO-based oxidation of dopamine produces dopaminequinone and consequently the cross-linking and melanization of proteins (Sideri et al., 2008). A relationship between Reeler protein and the proPO activation system has not yet been reported in insects; therefore, a functional analysis of Reeler is of considerable interest. In this report, we investigated the possible relationship between reeler gene expression and PO activity. Our findings reveal that the Reeler protein is a necessary component of the insect melanization cascade.
Section snippets
Insects, bacteria and cell lines
Silkworms, B. mori (Qiufeng × Baiyu strain), were provided by the College of Animal Sciences, Zhejiang University. Fifth instar larvae were used in these experiments. Escherichia coli K12 and Bacillus subtilis strains were provided by the College of Life Sciences, Zhejiang University. The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were maintained at 27 °C in TNM-FH insect medium supplemented with 10% (v/v) fetal bovine serum (Gibco-BRL). A Bac-to-Bac baculovirus expression system
Isolation and sequence analysis of B. mori reeler cDNA clones
We searched the silkworm genomic sequence (http://silkworm.genomics.org.cn/) using a nucleotide sequence encoding an immune-related protein (GenBank accession no. DQ898221). The coding sequence of a B. mori immune-related gene (Gene ID: BGIBMGA014360-TA) was identified in the B. mori genome. Additionally, 432 bp of nucleotide sequence (Gene ID: BGIBMGA005981-TA) that lacked the 3′ end of the coding sequence showed significant identity with the B. mori immune-related gene. In order to obtain the
Discussion
In this study, we analyzed the functions of an immune-induced reeler domain-containing protein in B. mori larvae. The reeler domain was first identified in Reelin, a large glycoprotein consisting of 3461 amino acid residues in mouse (D’Arcangelo et al., 1995). Reelin plays central roles in mammalian brain development, such as neuronal cell migration and dendritic growth (Kohno and Hattori, 2010). Although reeler domain-containing genes have been identified in insect species, their molecular
Acknowledgments
We thank Professor Xing-Meng Lu of the College of Animal Sciences of Zhejiang University and Professor Cheng-Liang Gong of the College of Life Sciences of Soochow University for kindly providing us the Silkworm strain. This work was supported by the National Natural Science Foundation of China (Grant No. 31071692), the National Basic Research Program of China (2010CB126205) and the Qianjiang project of Zhejiang Province, China (Grant No. 2009R10007).
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