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Characterization of a Novel C-Type Lectin, Bombyx mori Multibinding Protein, from the B. mori Hemolymph: Mechanism of Wide-Range Microorganism Recognition and Role in Immunity¹

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan

To investigate the system used by insects to recognize invading microorganisms, we examined proteins from the larval hemolymph of Bombyx mori that bind to the cell surface of microorganisms. Two hemolymph proteins that bound to the cell surfaces of Micrococcus luteus and Saccharomyces cerevisiae were shown to be identical. This protein bound to all 11 microorganisms examined–5 Gram-negative bacteria, 3 Gram-positive bacteria, and 3 yeasts–and was consequently designated B. mori multibinding protein (BmMBP). The sequence of the cDNA encoding BmMBP revealed that it was a C-type lectin with two dissimilar carbohydrate-recognition domains (CRD1 and CRD2) distantly related to known insect C-type lectins. CRD1 and CRD2 were prepared as recombinant proteins and their binding properties were investigated using inhibition assays. Each domain had wide, dissimilar binding spectra to sugars. These properties enable BmMBP to bind to two sites on a microorganism, facilitating high-affinity binding to many types of microorganisms. The dissociation constants of BmMBP with M. luteus cells and S. cerevisiae were 1.23 x 10^–8 and 1.00 x 10^–11 M, respectively. rBmMBP triggered the aggregation of hemocytes from B. mori larvae in vitro and microorganisms recognized by BmMBP were surrounded by aggregated hemocytes in vivo, forming a nodule, which is the typical cellular reaction in insect immune responses. These observations suggest that BmMBP functions as a trigger for the nodule reaction and that the multirecognition characteristic of BmMBP plays an important role in the early stages of infection by a variety of microorganisms.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Sasagawa Scientific Research Grant from the Japan Science Society.

² Address correspondence and reprint requests to Dr. Ryoichi Sato, Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan. E-mail address: ryoichi{at}cc.tuat.ac.jp

³ Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; BmLBP, B. mori LPS-binding protein; GalNAc, N-acetylgalactosamine; CRD, carbohydrate-recognition domain; BmIML, B. mori immulectin; BmMBP, B. mori multibinding protein; GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; IPS, insect physiological saline; CBB, Coomassie brilliant blue; EST, expressed sequence tag.