Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a β-1,3-glucanase-related protein in Manduca sexta larval plasma☆
Graphical abstract
M. sexta microbe-binding protein (MBP), a member of the “GNBP” clade in the family of β-1,3-glucanase-related proteins (βGRPs), specifically associates with DAP-PGs, LTA and LPS, but less so with Lys-PG or laminarin. The low binding to laminarin suggests that “GNBP” may represent a evolutionary transition state from “β-glucanase” to “β-1,3-glucan recognition protein (βGRP)”. These bindings, which are affected by other plasma factors, stimulate the prophenoloxidase activation system in most cases.
Highlights
► Identification of M. sexta MBP and its relationships with other β-1,3-glucanase-related proteins in invertebrates. ► Immune inducibility and recombinant expression of M. sexta MBP. ► A complex binding pattern of exogenous and endogenous MBP to microbes and their wall components tested by various assays. ► Certain types of MBP-mediated pathogen recognition linked to the proPO activation system. ► Binding properties of the known β-1,3-glucanase-related proteins summarized based on their domain organization and evolutionary dynamics.
Introduction
Innate immunity is the first line of defense against pathogen infection in all animals including insects (Gillespie et al., 1997, Strand, 2008). It is triggered by host proteins that recognize conserved surface determinants of microbes, such as lipopolysaccharide (LPS) or peptidoglycans (PGs) from bacteria and β-1,3-glucan from fungi. These pattern recognition receptors (PRRs) bind invading cells and initiate rapid cellular and humoral responses. Complex signaling pathways become active and induce the production of antimicrobial peptides and other defense molecules (Ragan et al., 2009, Lemaitre and Hoffmann, 2007). Extracellular serine proteinase cascades coordinate multiple immune responses: one leading to spätzle processing and Toll pathway activation; another causing proteolytic activation of prophenoloxidase (proPO). Active phenoloxidase (PO) then generates quinones that polymerize to form melanin (Nappi and Christensen, 2005). Reactive compounds such as 5,6-dihydroxyindole directly kill pathogenic invaders (Zhao et al., 2007, Kohler et al., 2007).
Biochemical and genetic studies have revealed more than 10 groups of PRRs in invertebrates, including hemolin, peptidoglycan recognition proteins (PGRPs), β-1,3-glucan recognition proteins (βGRPs), C-type lectins, galectins, fibrinogen-related proteins, thioester-containing proteins, Nimrods, scavenger receptors, and Down syndrome cell adhesion molecules (Yu et al., 2002, Lemaitre and Hoffmann, 2007). These proteins bind to surface moieties of pathogens or parasites and initiate a coordinated defense reaction. One of these groups is composed of proteins with a region similar in sequence to β-glucanases from bacteria, fungi, sea urchins, and insects. Some of its members, due to differences in binding properties, are designated βGRPs, LPS- and β-1,3-glucan-binding protein, or Gram-negative bacteria-binding proteins (GNBPs) (Lee et al., 1996, Ochiai and Ashida, 2000, Ma and Kanost, 2000, Lee et al., 2000, Kim et al., 2000, Jiang et al., 2004). Other members of the family have similar names but their binding specificities have not yet been experimentally established.
To better understand the molecular basis of innate immunity in Manduca sexta, we did a pyrosequence analysis of expressed sequence tags for hemolymph proteins (Zou et al., 2008). Of 218 newly discovered cDNA contigs, six encoded polypeptides similar to regions of Bombyx mori GNBP (Lee et al., 1996). In this work, we isolated cDNA clones of the GNBP homolog, characterized the recombinant protein from a baculovirus expression system, and explored its immunological functions. We studied its transcriptional changes in larval hemocytes and fat body after an immune challenge. This protein, named M. sexta microbe binding protein (MBP), specifically bound to microbial cell wall components and triggered proPO activation. Together with other PRRs discovered in the same insect, it contributes to a surveillance mechanism by which the host can detect microbial incursions and stimulate defense reactions.
Section snippets
Insect rearing, immune challenge, and cDNA synthesis
M. sexta eggs were purchased from Carolina Biological Supply and the larvae were reared on an artificial diet (Dunn and Drake, 1983). Each of day 2, 5th instar larvae was injected with 30 μl H2O, formaldehyde-killed Escherichia coli (2 × 107 cells) or Micrococcus luteus (20 μg) (Sigma–Aldrich, M3770). Hemolymph samples were collected from cut prolegs of the larvae 24 h later and centrifuged at 5000 g for 5 min to remove hemocytes. Total RNA samples were isolated from hemocytes and dissected fat body
cDNA cloning and structural features of M. sexta MBP
Our analysis of immunity-related expressed sequence tags (Zou et al., 2008) revealed six M. sexta cDNA contigs encoding polypeptide sequences similar to regions of B. mori GNBP. To investigate a possible role of the putative GNBP in defense responses, we designed specific primers and amplified the cDNA fragment, which was labeled with [α-32P]-dCTP to screen 1.2 × 105 plaques of an induced fat body cDNA library. Each of the positive clones (Q2, Q3, and Q4) contained regions 75∼99% identical to the
Evolution of the superfamily of β-glucanase-related proteins
It is now clear that βGRPs, GNBPs and β-1,3-glucanases in insects are a superfamily of proteins that recognize and even hydrolyze polysaccharides from fungi and bacteria. While the naming of βGRPs and β-1,3-glucanases is supported by experimental data (Ochiai and Ashida, 2000, Ma and Kanost, 2000, Jiang et al., 2004, Pauchet et al., 2009), other members (e.g. D. melanogaster GNBP3, A. gambiae GNBPs) may have been incorrectly designated (Buchon et al., 2009). In our opinion, the entire
Acknowledgments
This work was supported by National Institutes of Health Grant GM58634 (to H.J.). This article was approved for publication by the Director of Oklahoma Agricultural Experimental Station and supported in part under project OKLO2450. We thank Drs. Michael Kanost, Jack Dillwith, Stephen Marek, and the three anonymous reviewers for their helpful comments on the manuscript. Dr. Marek also kindly provided a culture of B. bassiana for the binding assay.
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The nucleotide sequence reported in this paper has been submitted to the GenBank™/EBI Data bank with accession number HQ007028.