Structure And Function Of The Peptidoglycan Recognition Protein BbtPGRP3 From Branchiostoma Belcheri Tsingtauense

The innate immune system is the first line of defense for vertebrates to fight against invading microorganisms and the only defense system for invertebrates and plants. This system provides recognition of relatively invariable exogenous products of microbial metabolism, termed pathogen-associated molecular patterns (PAMPs), through a limited number of germ line-encoded pattern-recognition receptors (PRRs). One representative example of PAMPs is peptidoglycan (PGN), an essential and unique cell-wall component in both Gram-positive and negative bacteria. PGNs are normally recognized, and/or hydrolyzed (amidases, EC 3.5.1.28) in some cases, by a class of PRRs, termed peptidoglycan recognition proteins (PGRPs).Both invertebrates and vertebrates encode a large amount of PGRPs of varying functions. Resulting from extraordinary expansion of innate immune genes, the cephalochordate amphioxus, which is usually regarded as the proximate ancestor of vertebrates, harbors 17-18 PGRP genes, however, none of which has been reliably clustered with insect or mammalian PGRPs. Bioinformatic analyses indicated that all amphioxus PGRPs possess potential amidase activity; thus they should all function as effector or catalytic PGRPs. Three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1～3, are featured with a chitin-binding domain (CBD) fused at the N-terminus. CBDs are widespread across the animal and plant kingdoms and exhibit antifungal and/or antimicrobial activity, through specific binding to carbohydrates.Here we report the 2.7 A crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1～3 novel PGRPs of augmented catalytic activity and broad recognition spectrum.