1. Human peptidoglycan recognition protein S is an effector of neutrophil-mediated innate immunity
by Ju Hyun Cho, Iain P. Fraser, Koichi Fukase, Shoichi Kusumoto, Yukari Fujimoto, Gregory L. Stahl, and R. Alan B. Ezekowitz
Blood
Volume 106(7):
October 1, 2005
©2005 by American Society of Hematology

2. Expression and purification of recombinant human PGRP-S.
Expression and purification of recombinant human PGRP-S. (A) Cell culture supernatant and cell lysates were tested for expression by Western blot with anti-His(C-term) antibody. (B) Coomassie Blue-stained 12% SDS-PAGE gel showing affinity-purified rhPGRP-S. Molecular markers are shown on the left. (C) Gel filtration chromatogram showing elution of rhPGRP-S from a Superdex 75HR column at a flow rate of 0.5 mL/min. Based on elution volumes of molecular weight standards, rhPGRP-S elutes as a monomer of approximately 23 kDa. (D) fast protein liquid chromatography (FPLC) fractions containing rhPGRP-S were subjected to 12% SDS-PAGE.
Ju Hyun Cho et al. Blood 2005;106:
©2005 by American Society of Hematology

3. Human PGRP-S binds to S aureus CP5 and E coli K12.
Human PGRP-S binds to S aureus CP5 and E coli K12. Bacteria (2 × 106) were incubated with biotin-labeled rhPGRP-S (10 μg/mL) and BSA (40 μg/mL), respectively, at 37°C for 30 minutes and visualized with streptavidin-Alexa Fluor 488.
Ju Hyun Cho et al. Blood 2005;106:
©2005 by American Society of Hematology

4. Human PGRP-S binds to Lys-type and DAP-type PGNs
Human PGRP-S binds to Lys-type and DAP-type PGNs. Purified rhPGRP-S or rdPGRP-LC (0.5 μg) were incubated with insoluble Lys-type or DAP-type PGN, and bound protein on the insoluble PGN was separated from unbound protein, as described in “Materials and metho...
Human PGRP-S binds to Lys-type and DAP-type PGNs. Purified rhPGRP-S or rdPGRP-LC (0.5 μg) were incubated with insoluble Lys-type or DAP-type PGN, and bound protein on the insoluble PGN was separated from unbound protein, as described in “Materials and methods.” One tenth of unbound protein (lane U) and one fifth of bound protein (lane B) were analyzed by Western blot analysis using anti-His(C-term) antibody. Molecular markers are indicated on the left.
Ju Hyun Cho et al. Blood 2005;106:
©2005 by American Society of Hematology

5. Human PGRP-S inhibits the growth of S aureus CP5 and E coli K12.
Human PGRP-S inhibits the growth of S aureus CP5 and E coli K12. (A) Radial diffusion assay. Wells were bored into underlay gel (1% agarose in 0.01 × TSB, 10 mM NaPB, pH 7.4) impregnated with bacteria. Proteins were introduced into wells (0.1-5 μg/well), and the plates were overlaid with 1% agarose in 2 × TSB (6% wt/vol) and incubated overnight at 37°C. (B) Suspension assay. Bacteria were incubated in 1 × TSB (3% wt/vol) with 25 μg/mL rhPGRP-S, either alone (•) or supplemented with 100 μg/mL S aureus PGN or E coli PGN (▴), PGN only (▦) or no additives (⋄). Tubes were shaken at 300 rpm for 5 hours, and bacterial density was monitored by measurement of optic density (OD) at 600 nm at 1-hour intervals. Data represent the mean ± SD of 3 independent experiments.
Ju Hyun Cho et al. Blood 2005;106:
©2005 by American Society of Hematology

6. Human PGRP-S shows synergistic antibacterial effect with lysozyme against E coli K12.
Human PGRP-S shows synergistic antibacterial effect with lysozyme against E coli K12. (A) Lysis of lysozyme-treated E coli cells. Lysozyme-treated E coli cells (⋄) were incubated in 10 mM NaPB, pH 7.4, with rhPGRP-S (4 μg/mL, [▴]; 8 μg/mL, [•]) or PMBN (2.5 μg/mL, [▦]), and the change in OD at 600 nm was monitored for 10 minutes. Data represent the mean ± SD of 3 independent experiments. (B) Growth inhibition of E coli cells. E coli cells were incubated in 1 × TSB (3% wt/vol) with 50 μg/mL lysozyme (▦), 25 μg/mL rhPGRP-S (▴), 8 μg/mL rhPGRP-S and 50 μg/mL of lysozyme (•), or no protein (⋄). Tubes were shaken at 300 rpm for 5 hours, and bacterial density was monitored by measurement of optic density (OD) at 600 nm at 1-hour intervals. Data represent the mean of duplicate samples from 1 of 2 similar experiments.
Ju Hyun Cho et al. Blood 2005;106:
©2005 by American Society of Hematology

7. Colocalization of PGRP-S with lysozyme in the NETs
Colocalization of PGRP-S with lysozyme in the NETs. Neutrophils were activated with 100 nM PMA for 30 minutes and stained for DNA (A), PGRP-S (B), and lysozyme (C).
Colocalization of PGRP-S with lysozyme in the NETs. Neutrophils were activated with 100 nM PMA for 30 minutes and stained for DNA (A), PGRP-S (B), and lysozyme (C). For DNA detection, Hoechst (blue) was used. PGRP-S was detected with anti-human PGRP-S monoclonal antibody (mouse immunoglobulin G [IgG]) and Cy2-conjugated donkey anti-mouse IgG (green). Lysozyme was detected with anti-human lysozyme polyclonal antibody (sheep IgG) and Cy5-conjugated donkey anti-sheep IgG (red). The merged image (D) clearly shows colocalization of PGRP-S with lysozyme in the NETs.
Ju Hyun Cho et al. Blood 2005;106:
©2005 by American Society of Hematology