Purification of a Peptidoglycan Recognition Protein from Hemolymph of the Silkworm, Bombyx mori

Abstract

A method was developed for obtaining a homogeneous silkworm hemolymph protein (peptidoglycan recognition protein, PGRP) which has affinity for peptidoglycan and the ability to trigger the prophenoloxidase cascade upon its binding to peptidoglycan. The purified PGRP had a molecular mass of about 19 kDa and is composed of a single polypeptide with an isoelectric point of 6.5. It bound to peptidoglycan in the absence of divalent cation, whereas its binding to beta1,3-glucan and chitin was not detected. N-Acetyl-D-glucosaminyl-(beta1-4)-N-acetylmuramyl-L-alanyl-D-isogluta mine did not inhibit purified PGRP to bind insoluble peptidoglycan, but fragmented soluble peptidoglycan did. PGRP seemed to require peptidoglycan as a possible ligand to keep its glycan portion consisting of at least two or more of the repeating unit. PGRP did not have any detectable lysozyme activity, and its amino acid composition and amino-terminal sequence of 20 amino acid residues were shown to be different from those of silkworm lysozyme. PGRP seems to be a hitherto unknown protein. In the absence of PGRP, the prophenoloxidase cascade in the plasma fraction of hemolymph could not be triggered by peptidoglycan, indicating that some type of activity, capable of activating the cascade, is generated upon their binding. However, the exact nature of this activity is not yet known. The purified PGRP bound to peptidoglycan did not hydrolyze significantly any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.

The road to Toll

“…In contrast to the rapid identification of TLRs and their ligands, the Drosophila molecules involved in microbial recognition remained unknown until recently. At present, we know that some aspects of microbial recognition in flies are mediated by peptidoglycan-recognition proteins (PGRPs) and Gram-negative-bacteria-binding proteins (GNBPs) 43,[56][57][58][59][60] -two protein families identified, initially in other insects, by their capacity to bind microbial components [61][62][63][64] (FIG. 3).…”

How Drosophila combats microbial infection: a model to study innate immunity and host–pathogen interactions

“…Peptidoglycan recognition protein (PGRP) was identified as a Grampositive-binding protein present in the moth Trichoplusia ni [54]. PGRP has also been implicated in the activation of the prophenoloxydase cascade in the silkworm Bombyx mori [55]. Interestingly, the Drosophila genome encodes 12 PGRP genes, and several of them are upregulated after septic injury [56].…”

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella

“…These structures are referred to as pathogenassociated molecular patterns (PAMPs), and the receptors recognizing these structures in innate immune system are called pattern recognition receptors (PRRs) (Medzhitov and Janeway, 1997;Janeway and Medzhitov, 2002). Peptidoglycan recognition proteins (PGRPs) belong to a family of PRRs that were first identified in insects and then in mammals such as Homo sapiens and Mus musculus (Yoshida et al, 1996;Kang et al, 1998;Liu et al, 2001;Dziarski and Gupta 2006). Despite the structural similarities, PGRPs in invertebrate and vertebrate differ in their distributions in tissues/ organs, and in functions.…”