Abstract
DM9 domain was first identified in Drosophila melanogaster, and it was subsequently found to integrate with or without other protein domains across a wide range of invertebrates and vertebrates. In the present study, a member of DM9 domain containing protein (DM9CP) family from marine invertebrate Crassostrea gigas (designated CgDM9CP-1), which was only composed of two DM9 domains, was taken as a protein model to study the biological functions of DM9 domain and its molecular determinants. CgDM9CP-1 was found to exhibit high binding specificity and avidity toward d-mannose residue. It served as a pattern recognition receptor (PRR) with a broad range of recognition spectrum to various pathogen-associated molecular patterns, including lipopolysaccharide, peptidylglycan, mannan, and β-1, 3-glucan in a d-mannose-dependent manner, as well as bacteria and fungi. In order to reveal the molecular mechanism underlying its pattern recognition activity, the crystal structures of wild-type and loss-of-function mutants were solved, and Asp22 and Lys43 were found to be the critical residues for ligand recognition. Moreover, CgDM9CP-1 protein was found to mainly distribute on the surface of C. gigas hemocytes, and it could be translocated into cytoplasm and colocalized with the engulfed microbes during hemocyte phagocytosis. The present result clearly indicated that CgDM9CP-1 was a PRR, and it provided an important clue for the better understanding of DM9CP function.
Highlights
DM9 is a novel protein domain originally identified in Drosophila melanogaster with no defined functions [1]
The d-mannose binding protein was analyzed by MALDI-TOF/TOF-MS, and the amino acid sequence was identified by searching ten tryptic peptides against the genome of C. gigas (Figures S1C,D and Table S1 in Supplementary Material)
Protein domain analysis using National Center for Biotechnology Information (NCBI)’s conserved domain database (CDD) and simple modular architecture research tool (SMART) showed that CgDM9CP-1 was only composed of two DM9 domains (Figure S2A in Supplementary Material), and they shared 33% sequence identity (Figure S2B in Supplementary Material)
Summary
DM9 is a novel protein domain originally identified in Drosophila melanogaster with no defined functions [1]. Magalhaes et al characterized a number of toxins, named natterins, from the teleost fish Thalassophryne nattereri [2, 3], which contained N-terminal DM9 domains fused to C-terminal Clostridium epsilon toxin/Bacillus mosquitocidal toxin (ETX/MTX2). DM9CPs were found in venomous fish T. nattereri and in viperid snake Bothrops jararaca, which could cause cell necrosis, edema, and even permanent disabilities in humans [10, 11]. These studies strongly suggest that DM9CPs are participated in the immune response. Their detailed biological functions and its underlying structural basis are still not well understood
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