Abstract
Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A–B and C–D. The A–B and C–D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A–B and C–D contacts. Two ligand binding sites, one at C–D contact and another at A–B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both Gram-positive and Gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C–D contact (Site-1) while SA binds to it at the A–B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS formed 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2 Å) while SA formed 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN-γ due to LPS and SA decreased considerably upon the addition of CPGRP-S.
Highlights
The 19 kDa peptidoglycan recognition protein (PGRP-S) is one of the four mammalian Peptidoglycan recognition proteins (PGRPs) which were originally classified according to their molecular weights as PGRP-S (M.W., 20– 25 kDa), PGRP-Ia and PGRP-Ib (M.W., 40–45 kDa) and PGRPL (M.W. up to 90 kDa) [1]
We have shown that the components of bacterial cell wall molecules such as lipopolysaccharide (LPS) of Gram-negative bacteria, lipoteichoic acid (LTA) of Gram-positive bacteria and peptidoglycans (PGNs) of both Gram-positive and Gram-negative bacteria as well as mycolic acid (MA) and other fatty acids of the Mycobacterium tuberculosis [5,6,7] bind to camel PGRP-S (CPGRP-S) with affinities ranging from micromolar to nanomolar [8,9,10]
Binding Analysis The binding studies of CPGRP-S using SPR were carried out with both ligands, LPS and stearic acid (SA). It has been shown by previous structural studies of binary complexes of CPGRP-S with LPS and SA [9,10,11] that LPS bound to CPGRP-S in the binding Site-1 at the C–D contact while SA was found to bind the protein in the binding Site-2 at the A–B contact [19]
Summary
The 19 kDa peptidoglycan recognition protein (PGRP-S) is one of the four mammalian PGRPs which were originally classified according to their molecular weights as PGRP-S (M.W., 20– 25 kDa), PGRP-Ia and PGRP-Ib (M.W., 40–45 kDa) and PGRPL (M.W. up to 90 kDa) [1]. The previous studies have shown that LPS, LTA and PGN bind to CPGRP-S at Site-1 which is situated at the C–D contact while mycolic acid and other fatty acids were held at Site-2 at the A–B contact [9,10,11,12]. Having obtained these results, it was pertinent to determine whether CPGRP-S could bind to the components of multiple bacterial cell wall molecules simultaneously through its two independent binding sites or it would bind to only one kind of PAMPs at a time. This indicated the binding potential of CPGRP-S to interact with two independent PAMPs through its two separate binding sites, S-1 and S-2
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