JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M506385200 on August 29, 2005

J. Biol. Chem., Vol. 280, Issue 44, 37005-37012, November 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/44/37005    most recent
M506385200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumar, S.
Right arrow Articles by Boons, G.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumar, S.
Right arrow Articles by Boons, G.-J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Selective Recognition of Synthetic Lysine and meso-Diaminopimelic Acid-type Peptidoglycan Fragments by Human Peptidoglycan Recognition Proteins I{alpha} and S*{boxs}

Sanjay Kumar{ddagger}1, Abhijit Roychowdhury{ddagger}1, Brian Ember{ddagger}1, Qian Wang§, Rongjin Guan§, Roy A. Mariuzza§2, and Geert-Jan Boons{ddagger}3

From the {ddagger}Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and the §Center for Advanced Research in Biotechnology, W. M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850

Received for publication, June 13, 2005 , and in revised form, August 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The interactions of a range of synthetic peptidoglycan derivatives with PGRP-I{alpha} and PGRP-S have been studied in real-time using surface plasmon resonance. A dissociation constant of KD = 62 µM was obtained for the interaction of peptidoglycan recognition protein (PGRP)-I{alpha} with the lysine-containing muramyl pentapeptide (compound 6). The normalized data for the lysine-containing muramyl tetra- (compound 5) and pentapeptide (compound 6) showed that these compounds have similar affinities, whereas a much lower affinity for muramyl tripeptide (compound 3) was measured. Similar affinities were obtained when the lysine moiety of the muramyl peptides was replaced by meso-diaminopimelic acid (DAP). Furthermore, the compounds that contained only a stem peptide (pentapeptide, compound 1) and (DAP-PP, compound 2) as well as muramyldipeptide (compound 3) exhibited no binding indicating that the muramyltripeptide (compound 4) is the smallest peptidoglycan fragment that can be recognized by PGRP-I{alpha}. Surprisingly, PGRP-S derived significantly higher affinities for the DAP-containing fragments to similar lysine-containing derivatives, and the following dissociation constants were measured: muramylpentapeptide-DAP, KD = 104 nM; muramyltetrapeptide-DAP, 92.4 nM; and muramyltripeptide-DAP, 326 nM. The binding profiles were rationalized by using a recently reported x-ray crystal structure of PGRP-I{alpha} with the lysine-containing muramyltripeptide (4).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The innate immune system is an ancient evolutionary system of defense against microbial infections (1-4). It responds rapidly to highly conserved families of structural patterns, called pathogen-associated molecular patterns (PAMPs),4 which are integral parts of pathogens, and are perceived as danger signals by the host. Examples of PAMPs include bacterial cell wall structures that are absent from the host such as lipopolysaccharide of Gram-negative bacteria, lipoteichoic acid, mannans, DNA sequences containing unmethylated CpG dinucleotides, flagellin, and peptidoglycan (PGN) (2, 3). The recognition of PAMPs is mediated by sets of highly conserved pattern recognition receptors (5), each of which binds to a variety of PAMPs. Cellular activation by these receptors results in acute inflammatory responses that include the production of a diverse set of cytokines and chemokines, direct local attack against the invading pathogen, and the initiation of responses that activate and regulate the adaptive component of the immune response.

The discovery of Toll-like receptors (TLRs) less than a decade ago has advanced our understanding of the early events in microbial recognition and response and the subsequent development of an adaptive immune response (6-12). The Toll protein was first discovered in Drosophila, in which it has a pivotal role in embryonic development and microbial detection. Subsequently, a family of proteins structurally related to Toll was identified in higher organisms. Collectively, these transmembrane receptor proteins are referred to as TLRs. To date, eleven members of the mammalian TLR family have been identified, each potentially recognizing a discrete class of PAMP (13). For example, lipopolysaccharides are recognized by TLR4, bacterial flagellin by TLR5, double-stranded RNA by TLR3 (14), and bacterial DNA by TLR9. The most recently discovered member of this family, TLR11, plays a critical role in the recognition and control of uropathogenic bacteria, and two recent studies have demonstrated that TLR3 is involved in the recognition of single-stranded viral RNA. Although it was initially believed that TLR2 in combination with TLR1 or TLR6 recognizes PGN, recent studies with highly purified PGN indicate otherwise (15). Instead, it appears that NOD proteins (NOD1 and NOD2) (16, 17), and peptidoglycan recognition proteins (PGRPs) (18) are the pattern-recognition receptors that detect PGN.

PGRPs are a relatively new class of pattern-recognition receptors that are highly conserved from insects to mammals (19-21). Drosophila has 13 PGRP genes that are transcribed into at least 17 PGRPs (21). These PGRPs can be divided in extracellular (e.g. PGRP-SA), transmembrane (e.g. PGRP-LC), and intracellular or secreted (e.g. PGRP-LE) proteins (18). To date, four PGRPs have been discovered in humans, namely PGRP-S, PGRP-I{alpha}, PGRP-I{beta}, and PGRP-L (19, 20).

The different PGRPs may exhibit a selectivity for PGN derived from a particular group of microbes. In this respect, PGNs are large polymers composed of alternating {beta}(1-4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues, cross-linked by short peptide bridges (see Fig. 1) (22). Depending on the amino acid composition of position 3 of the peptide chain, PGNs are classified as either L-lysine-type (Lys-type) or meso-diaminopimelic acid-type (DAP-type). The lysine-type, typical for Gram-positive bacteria, is normally connected to the D-Ala of another peptide chain by a short bridge varying in length and amino acid composition, depending on the bacteria. In the case of Gram-negative bacteria and Gram-positive bacilli, DAP-type is normally found as the third amino acid and is directly connected to D-Ala of another peptide chain.

Drosophila PGRP-SA has been shown to interact with lysine-type PGN, activating the Toll receptor pathway (23). On the other hand, PGRP-LC and PGRP-LE recognize DAP-type PGN activating the Imd/Relish pathway (24-28). PGRPs have high homology with the T7 lysozyme, a type 2 N-acetylmuramoyl-L-alanine amidase that hydrolyzes the bond between MurNAc and L-Ala of PGN (29). In this respect, Drosophila PGRP-SC1b and PGRP-LB have been shown to possess amidase activity. (30)

Each of the four mammalian PGRPs (PGRP-L, PGRP-I{alpha}, PGRP-I{beta}, and PGRP-S) is able to bind peptidoglycan; however, possible selectivities for lysine or DAP-type PGN have either not been determined or remain controversial. In addition, the mode of cellular activation and bactericidal activity of these PGRPs is largely unknown. The limited data for PGRP-L indicates that this protein exhibits lytic activity (31, 32). The function of PGRP-I{alpha} and PGRP-I{beta} is unknown, and most research has thus far focused on PGRP-S. Mouse PGRP-S found in neutrophil tertiary granules participates in the intracellular neutralization of bacteria. Mice deficient in this PGRP are much more susceptible to intraperitoneal infections with low pathogenic Gram-positive bacteria. (33) However, bovine PGRP-S, located in neutrophil and eosinophil granules, has been shown to inhibit the growth of both Gram-positive and -negative bacteria (34).

To determine in the ligand requirements for various PGRPs, we have synthesized a range of partial structures of PGN (see Fig. 1) that contain lysine or DAP as the third amino acid. The interactions of these compounds with human PGRP-S and the C-terminal domain of human PGRP-I{alpha} containing two tandem domains have been studied in real-time using surface plasmon resonance (SPR).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Procedures for the expressing recombinant PGRP-I{alpha}C (residues 177-341) by in vitro folding from Escherichia coli inclusion bodies have been described previously (35).

Chemical Synthesis of PGN Part Structures
Compound 6: Sieber Amide resin (36) (100 mg, 42 µmol, Novabiochem) was swelled in dry dimethylformamide (DMF, ~120 min, 3 ml), treated with 20% piperidine in DMF (3 x 5 min, 3 x 2 ml), washed with freshly distilled DMF (3 x 3 ml), and then reacted with Fmoc-D-Ala-OH (26.12 mg, 84 µmol, Novabiochem) in DMF by using PyBOP (43.7 mg, Novabiochem), 1-hydroxybenzotriazole (11 mg, Aldrich), and N,N-diisopropylethylamine (29.2 µl, Alfa Aesar, Ward Hill, MA). Progress of the reaction was monitored by the Kaiser test. After completion of the coupling, the resin was washed with (3 x 3 ml), and the Fmoc protecting group was removed with 20% piperidine in DMF (3 x 5 min, 3 x 2 ml). The reaction cycle was repeated using Fmoc-D-Ala-OH (26.1 mg, 84 µmol), Fmoc-L-Lys(Mtt)-OH (52.4 mg, 84 µmol), Fmoc-D-isoglutamine (30.9 mg, 84 µmol), Fmoc-L-Ala-OH (26.12 mg, 84 µmol, Novabiochem), and, subsequently, 2-N-acetyl-1-{beta}-O-allyl-4,6-benzylidene-3-muramic acid (37) (35.4 mg, 84 µmol). The resulting resin-bound glycopeptide was washed with DMF (3 x 3 ml), dichloromethane (7 x 3 ml), and methanol (3 x 3 ml). The resin was dried in vacuo for 4 h, reswelled in dichloromethane (DCM) (~5 ml), and filtered. The glycopeptide was released by treatment of the resin with 2% trifluoroacetic acid in DCM (10 x 2 ml). The combined washings were concentrated under reduced pressure and co-evaporated with toluene (3 x 10 ml) to remove traces of trifluoroacetic acid. The crude product was subjected to 20% trifluoroacetic acid in DCM to ensure complete removal of the benzylidene protecting group. The resulting product was purified by Sephadex G15 size exclusion column (Amersham Biosciences) chromatography to give (allyl-2-N-acetyl-3-O-muramyl)-L-alanyl-D-isoglutamyl-L-lysine (23.4 mg, 70%) as a white amorphous solid. 1H NMR (500 MHz, D2O): {delta} = 5.87-5.93 (1H, m, OCH2CHCH2), 5.25-5.32 (2H, dd, OCH2CHCH2), 4.53 (1H, d, H1, J = 8.3 Hz), 4.13-4.35 (8H, m, {alpha} H-Ala x3, {alpha} H-Lys, {alpha} H-Glu, {alpha} H-lactic acid, OCH2CHCH2), 3.93 (1H, d, H6a, J = 12.2 Hz), 3.85 (1H, t, H2), 3.76-3.79 (1H, dd, H6b), 3.45-3.57 (3H, m, H3, H4, H5), 3.00 (2 H, t, {epsilon} CH2-Lys), 2.36-2.43 (2H, m, {gamma} CH2-Glu), 2.12-2.18 (1H, m, {beta} CH2-Glu), 1.94-2.03 (4H, m, {beta} CH2-Glu, NHAc), 1.67-1.82 (4H, m, {beta}, {delta}-CH2-Lys), 1.37-1.45 (14H, m, CH3-lactic acid, {gamma} CH2-Lys, CH3-Ala x 3) 13C NMR (75 MHz, D2O): 177.88, 175.98, 175.93, 175.31, 175.22, 174.92, 174.83, 174.26, 133.52 (OCH2CHCH2), 118.23 (OCH2CHCH2), 100.27 (C1), 82.98, 75.78, 68.84 (C3, C4, and C5), 70.66 (OCH2CHCH2), 60.86 (C6), 55.29, 54.30, 52.89, 50.14, 49.95, 49.60 ({alpha}-Cs), 39.30 ({epsilon} CH2-Lys), 31.46 ({gamma} CH2-Glu), 30.25 ({beta} CH2-Glu), 26.51 ({beta} CH2-Lys), 22.34, 22.21 ({delta} CH2-Lys, NHCH3), 18.91, 16.68, and 16.36. HRMS-MALDI-TOF calc. for C34H59N9O13 (M + Na): 824.4232, found 824.3087. The compound (10.6 mg, 12.4 µmol) was dissolved in a mixture of ethanol/acetic acid/water (EtOH/HOAc/H2O, 2:1:1, 0.8 ml), and 10% Pd on charcoal (9 mg) was added. After stirring at room temperature for 48 h, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was coevaporated from toluene (3 x 20 ml). The residue was subjected to Sephadex G15 size exclusion column chromatography to give the target compound 6 as a mixture of {alpha}/{beta} anomers (8.6 mg, 91%). 1H NMR (500 MHz, D2O): {delta} 5.04 (0.60H, d, H1-{alpha} anomer, J = 3.3 Hz), 4.56 (0.39H, d, H-1-{beta}-anomer, J = 8.4 Hz), 4.17-4.08 (6H, m, {alpha} H-Lys, {alpha} H-Glu, {alpha} H-Ala x 3, {alpha} H-3-propionic acid), 3.36-3.86 (6H, m, H2, H3, H4, H5, and H6), 2.87 (2H, t, {epsilon} CH2-Lys), 2.21-2.29 (2H, m, {gamma} CH2-Glu), 2.19-2.03 (1H, m, {beta} CH2-Glu), 1.82-1.87 (4H, m, CH2-Glu, NHAc), 1.54-1.67 (4H, {beta}, {delta}-CH2-Lys), 1.37-1.45 (14H, m, {gamma} CH2-Lys, CH3-lactic acid, CH3-Ala x 3). 13C NMR (75 MHz, D2O) 177.89, 176.14, 175.94, 175.32, 175.21, 174.93, 174.82, 174.38, 174.13, 95.07 (C1-{alpha}), 91.13 (C1-{beta}), 82.78, 79.87, 78.24, 77.90, 75.87, 71.65, 69.03, 68.81, 60.87, 60.68, 56.33, 54.30, 53.86, 52.89, 50.13, 49.96, 49.61, 39.30 ({epsilon} CH2-Lys), 31.42 ({gamma} CH2-Glu), 30.26, 27.08, 26.51, 22.38, 22.22, 22.15, 18.81, 16.73, 16.68, and 16.37. HRMS-MALDI-TOF calc. for C31H55N9O13 (M + Na): 784.8213, found 784.5895.

Compounds 5 and 6 were synthesized using similar protocol whereby suitable amino acids were chosen depending on the desired target. Analytical data for the glycopeptides is listed below.

MTrP_Lys (5)—Yield 47%, 1H NMR (500 MHz, D2O): {delta} = 5.04 (0.45H, d, H1-{alpha} anomer, J = 3.5 Hz), 4.55 (0.54H, d, H-1-{beta}-anomer, J = 8.0 Hz), 4.10-4.17 (5H, m, {alpha} H-Lys, {alpha} H-Glu, {alpha} H-Ala x 2, {alpha}-H-lactic acid), 3.34-3.81 (6H, m, H2, H3, H4, H5, and H6), 2.88 (2H, t, {epsilon} CH2-Lys), 2.25-2.28 (2H, m, {gamma} CH2-Glu), 2.10 (1H, m, {beta} CH2-Glu), 1.82-1.91 (4H, m, CH2-Glu, NHAc), 1.54-1.72 (4H, {beta}, {delta}-CH2-Lys), and 1.25-1.37 (11H, m, {gamma} CH2-Lys, CH3-lactic acid, CH3-Ala x 2). 13C NMR (HSQC, D2O) 95.07 (C1-{alpha}), 91.47 (C1-{beta}), 82.99, 80.06, 78.20, 75.81, 73.92, 71.95, 69.29, 60.91, 54.39, 53.99, 52.93, 39.34 ({epsilon} CH2-Lys), 31.63 ({gamma} CH2-Glu), 30.83, 27.10, 26.97, 26.57, 22.98, 22.19, 18.93, 16.71, and 16.41. HRMS-MALDI-TOF calc. for C28H50N8O12 (M + Na): 713.3548, found 713.4080.

MTP_Lys (4)—Yield 61%, 1H NMR (500 MHz, D2O): {delta} 5.16 (0.69H, d, H1-{alpha}-anomer, J = 3.3 Hz), 4.67 (0.31H, d, H1-{beta}-anomer, J = 8.1 Hz), 4.20-4.34 (4H, m, {alpha}-H, Lys, {alpha}-H, Glu, {alpha}-H, Ala, {alpha}-H, lactic acid), 3.50-4.00 (6H, m, H2, H3, H4, H5, and H6), 3.01 (2H, t, {epsilon}-CH2, Lys), 2.39-2.45 (2H, m, {gamma}-CH2, Glu), 2.15-2.23 (1H, m, {beta}-CH2, Glu), 1.65-2.00 (8H, m, {beta}-CH2, Glu, {beta}, {delta}-CH2, Lys, NHCOCH3), and 1.37-1.47 (8H, m, {gamma}-CH2, Lys, CH3, lactic acid, CH3, Ala). 13C NMR (75 MHz, D2O) 177.06, 176.02, 175.25, 174.14, 95.07 (C1-{beta}), 91.13 (C1-{alpha}), 82.77, 79.85, 78.46, 78.24, 77.89, 75.87, 73.51, 71.66, 69.05, 60.88, 60.67, 56.34, 53.86, 53.63, 52.87, 49.98, 49.03, 39.33 ({epsilon}-CH2, Lys), 31.57 ({gamma}-CH2, Glu), 30.53, 26.93, 26.42, 22.65, 22.37, 22.28, 22.15, 18.79, and 16.70. HRMS-MALDI-TOF calc. for C25H45N7O11 (M + Na): 642.3067, found 642.3777.

The DAP containing muramyl tripeptides 7-9 were synthesized similarly to procedures described above whereby the Lys(Mtt)-OH was replaced by a suitable protected DAP derivative (38) (38.7 mg, 42 µmol) to afford the desired protected DAP derivative. Once cleaved from the resin, the DAP-PGNs were treated with 20% trifluoroacetic acid to deprotect the tert Butoxycarbonyl and tert butyl protecting groups on the side chain of the DAP. The deprotected derivative was precipitated from cold diethyl ether to afford an off-white compound. To a solution of this compound in EtOH:H2O:1 (N) HCl (4:2:0.01, 0.6 ml), 10% Pd/C (5 mg) was added and stirred at room temperature for 16 h. The solution was filtered and purified by Sephadex G10 size exclusion column chromatography to afford the target compound as a mixture of {alpha}/{beta} anomers (9.3 mg, 30% overall).

MTP_DAP (7)—1H NMR (500MHz, D2O): {delta} 5.08 (0.16H, d, H1-{alpha}-anomer), 4.32 (0.84H, d, H1-{beta}-anomer, J = 8.4Hz), 4.07-4.23 (5H, m, {alpha}-H x 2, DAP, {alpha}-H, Ala, {alpha}-H, Glu, {alpha}-H, lac), 3.67-3.89 (3H, m, H2, H6ab), 3.38-3.50 (3H, m, H3, H4, and H5), 2.27-2.36 (2H, m, {gamma}-CH2, Glu), 2.10 (1H, m, {beta}-CHH, Glu), 1.65-1.93 (8H, m, {beta}, {delta}-CH2, DAP, {beta}-CHH, Glu, NHCOCH3), 1.42-1.45 (2H, m, {gamma}-CH2, DAP), and 1.29-1.35 (6H, m, CH3, Lac, CH3, Ala). 13C (HSQC): 102.18 (C1-{beta}), 91.74 (C1-{alpha}), 83.31, 78.93, 76.24, 69.16, 60.66 (C6), 60.65, 60.06, 58.04, 55.68, 55.01, 54.33, 53.66, 50.29, 32.09 ({gamma}-C, Glu), 30.75 (C-DAP), 27.71 ({beta}-C, Glu), 23.00 (NHCOCH3), 21.98, 19.29, and 17.27. HRMS-MALDI-TOF calc. for C25H45N7O11 (M + HCl): 700.1355, found 700.4058.

MTrP_DAP (8)—1H NMR (600MHz, D2O): {delta} 5.19 (0.16H, bs, H1), 4.48 (0.84H, d, H1, J = 7.2Hz), 4.23-4.39 (6H, m, {alpha}-H, e-H, DAP, {alpha}-H x 2, Ala, {alpha}-H, Glu, {alpha}-H, lac), 3.51-3.98 (6H, m, H2, H6, H3, H4, and H5), 2.40 (2H, m, {gamma}-CH2, Glu), 2.13-2.14 (1H, m, {beta}-CHH, Glu), 1.78-2.09 (8H, m, {beta},{delta}-CH2, DAP, {beta}-CHH, Glu, NHCOCH3), and 1.38-1.50 (11H, m, {gamma}-CH2, DAP, CH3, Lac, CH3 x 2, Ala). 13C (HSQC): 102.13 (C1-{beta}), 91.65 (C1-{alpha}), 81.82, 78.70, 77.49, 76.28, 72.13, 69.36, 61.22 (C6), 60.01, 59.15, 57.59, 57.42, 55.68, 54.79, 54.30, 53.13, 53.95, 49.97, 31.78 ({gamma}-C, Glu), 30.87 (C-DAP), 27.81 ({beta}-C, Glu), 22.92 (NHCOCH3), 21.24, 19.25, 18.95, 18.03, and 16.81. HRMS-MALDI-TOF calc. for C25H45N7O11 (M + HCl): 771.2135, found 771.8770.

MPP_DAP (9)—1H NMR (500MHz, D2O): {delta} 5.16 (0.41H, bs, H1), 4.40 (0.58 H, d, H1, J = 9.0 Hz), 4.22-4.29 (7H, m, {alpha}-H, e-H, DAP, {alpha}-H x 3, Ala, {alpha}-H, Glu, {alpha}-H, lac), 3.50-3.98 (6H, m, H2, H6, H3, H4, and H5), 2.39 (2H, m, {gamma}-CH2, Glu), 2.13-2.15 (1H, m, {beta}-CHH, Glu), 1.90-2.03 (8H, m, {beta},{delta}-CH2, DAP, {beta}-CHH, Glu, NHCOCH3), and 1.38-1.57 (14H, m, {gamma}-CH2, DAP, CH3, Lac, CH3 x 3, Ala). 13C (HSQC): 102.39 (C1-{beta}), 91.66 (C1-{alpha}), 83.35, 80.13, 78.52, 78.25, 76.11, 72.08, 69.40, 60.95 (C6), 60.88, 60.60, 57.40, 55.59, 54.61, 54.40, 53.77, 53.01, 50.01, 31.60, 30.48 (C-DAP), 27.05, 22.40 (NHCOCH3), 21.01, 19.06, and 16.83. HRMS-MALDI-TOF calc. for C25H45N7O11 (M + HCl): 842.2914, found 842.6710.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 1.
Structure of peptidoglycan (PGN) and synthetic compounds.

 
SPR Analysis of PGRP-Ligand Interactions
The biospecific interaction analysis was performed using BIAcore 3000 biosensor system (Biacore Inc., Uppsala, Sweden). The CM-5 research grade sensor chip, HBS-EP buffer, and immobilization reagents (1-ethyl-3-(3-N,N-dimethylaminopropyl)carbodiimide (EDC), N-hydroxy succinimide (NHS), and ethanolamine) were obtained from BIAcore Inc. phosphate-buffered saline buffer was purchased from Sigma, and MDP was purchased from Calbiochem. All solutions were filtered using a 0.22-µm PES membrane syringe filter and degassed prior to use. PGRP-I{alpha}C was covalently immobilized by a standard amine coupling procedure using the amine coupling kit supplied by the manufacturer. A fixed flow rate of 10 µl/min was used throughout the immobilization procedure with HBS-EP (pH 7.4, 0.01 M HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20) as the running buffer. The surface was activated using 70 µl of freshly mixed 1:1 100 mM NHS and 391 mM EDC for 7 min. Upon activation, a 60 µg/ml solution of PGRP-I{alpha} in 10 mM NaOAc (pH 4.5) was injected for 8 min. The remaining active esters on the surface were quenched using 70 µl of 1.0 M ethanolamine (pH 8.5) for 7 min. A ligand density of ~10,000 RU was achieved. Blocking of a control flow cell was accomplished by activation followed by an immediate quenching of the flow cell as described above. PGRP-S immobilization was accomplished using the same protocol as above with a change to 5 mM maleate buffer (pH 6.0) as the immobilization buffer. A ligand density of ~5,000 RU was achieved after 8 min of protein injection. Although a higher initial immobilization density was targeted, longer PGRP-S injections resulted in no change of protein density. Initial binding studies of the DAP containing compounds with PGRP-S suggested high affinity prompting the formation of a low density immobilization surface for kinetic analysis. A similar protocol was used with an injection of PGRP-S solution of 50 µg/ml in 10 mM NaOAc (pH 4.5) for 5 min to afford an immobilized surface of 2,700 RU.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 2.
Sensorgram representing the concentration-dependent binding of lysine-containing MPP (6) with immobilized PGRP-I{alpha} (10,000 RU). Steady-state binding analysis of MPP-Lys (6) at concentrations from the bottom to top of 20, 30, 40, and 50 µM with PGRP-{Theta}{alpha} resulted in a KD of 6.2 x 10-5 M. Inset plot shows non-linear steady-state affinity analysis.

 
For the binding studies, fixed flow rates of 5 µl/min for association and dissociation with a constant temperature of 25 °C were employed. The association and dissociation times were 5 min and 10 min, respectively. These surfaces had greater than 90% reproducibility if used within a 3- to 4-day period. PGRP I{alpha}: phosphate-buffered saline buffer (pH 7.4, 0.01 M phosphate, 138 mM NaCl, 2.7 mM KCl) was selected as both the running and dissociation buffer. The lysine containing analytes, pentapeptide (Lys-PP 1), muramyldipeptide (MDP 3), muramyltripeptide (MTP 4), muramyltetrapeptide (MTrP 5), and muramylpentapeptide (MPP 6), were passed over the surface at a concentration range from 20 to 60 µM using the kinetic wizard method. The surface did not require a regeneration injection for the Lys-PP, MDP, or MTP. For MTrP and MPP, a regeneration injection of 0.01% Tween-20 in water for 20 s at a flow rate of 30 µl/min followed by 15 min of stabilization time was required to achieve prior baseline status. The DAP-containing analytes, DAP-PP (2), MTP-DAP (7), MTrP-DAP (8), and MPP-DAP (9), were passed over the surface at concentrations ranging from 10 to 50 µM. For MTrP-DAP and MPP-DAP, the regeneration was performed as described for the lysine-containing fragments.

For PGRP-S, HBS-EP buffer was selected as both the running and dissociation buffers. Due to a greater variation in binding affinity, optimum concentration ranges for each analyte were established. For the lysine-containing compounds, MTP was examined from 500 to 1000 µM, MTrP from 200 to 500 µM, and MPP from 50 to 300 µM. Initial binding studies were conducted on a flow channel with 5000 RU density. The DAP-containing muramyl peptides showed high affinity biding with on and off rates in the measurable range for kinetic analysis. These studies were conducted on a flow channel containing 2700 RU of PGRP-S to minimize rebinding and mass transport effect. A concentration range of 10-1000 nM was selected for MPP-DAP and MTrP-DAP and for MTP-DAP, a concentration range of 100-1000 nM was selected. The kinetic studies were performed using wizard kinetic software with a flow rate of 10 µl/min and association and dissociation times of 5 and 10 min, respectively. The surface was regenerated by a 60-s injection of 10 mM NaOH, pH 11.4, at a flow rate of 30 µl/min.

Data Analysis
The responses near equilibrium (Req) for the comparative affinity and rate constants k1 and k-1 for kinetic analysis were obtained by fitting the primary sensogram data using the BIAevaluation 3.1 software. The dissociation rate constant is derived using,

(Eq. 1)
where Rt0 is the amplitude of the initial response, and k-1 is the dissociation rate constant. The association rate constant k1 can be derived from the measured k-1 values, using,

(Eq. 2)

where Rt is response at time t, Rmax is the maximum response, C is concentration of the analyte in the solution, and k1 and k-1 are association and dissociation rate constants, respectively. The ratio of k1 and k-1 yields the value of association constant KA (k1/k-1).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3.
Comparative binding analysis of lysine-containing MTP (4), MTrP (5), and MPP (6) with immobilized PGRP-I{alpha} (10,000 RU). Concentrations of 20, 30, 40, 50, and 60 µM of each analyte were passed over the surface for 5 min. The average RU from 270 to 300 s was divided by respective molecular weight of each analyte to achieve a normalized RU to allow a comparison of affinity through the bar diagram. MDP (3) and Lysine-pentapeptide (1) had no detectable binding at concentrations up to 1 mM.

 



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 4.
Comparative binding analysis of DAP-containing MTP (7), MTrP (8), and MPP (9) with immobilized PGRP-I{alpha} (10,000 RU). Concentrations of 10, 20, 30, 40, and 50 µM of each analyte were passed over the surface for 5 min. The average RU from 270 to 300 s was divided by respective molecular weight of each analyte to achieve a normalized RU to allow a comparison of affinity through the bar diagram. MDP (3) and DAP-pentapeptide (2) had no detectable binding at concentrations up to 1 mM.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The interactions of human PGRP-I{alpha}C and PGRP-S with a range of synthetic lysine- and DAP-containing PGN fragments were probed using surface plasmon resonance (SPR). SPR is a rapid and sensitive method for the evaluation of affinities of bimolecular interactions (39). A benefit of this method is that it relies exclusively on mass changes, thus allowing interactions to be studied in real-time without the need for external labels such as fluorophores, which in some cases can alter the nature of the interaction. In this study, recombinant human PGRP-I{alpha} and PGRP-S were immobilized on research-grade CM5 sensor chips, and the synthetic compounds 1-9 were employed as analytes. Collecting SPR data for low molecular weight analytes, such as compounds 1-9, is challenging, because the refractive index monitored during a binding event is relatively small, thus resulting in responses with much lower magnitudes than those observed in typical protein-protein interactions. Despite these challenges, the high sensitivity and reproducibility of modern instruments combined with proper experimental design permits the direct monitoring of the binding of low molecular weight analytes to immobilized proteins. (40) An alternative approach whereby the synthetic peptidoglycan part structures would be immobilized and the PGRPs employed as analytes is expected to lead to artifacts, because the immobilization may destruct vital functional groups of the small synthetic compounds.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5.
Comparative binding analysis of lysine-containing MTP (4), MTrP (5), and MPP (6) with immobilized PGRP-S (5,000 RU). Concentrations of 50, 100, 150, 200, 300, 500, 800, and 1000 µM of each analyte were passed over the surface for 5 min. The average RU from 270 to 300 s was divided by respective molecular weight of each analyte to achieve a normalized RU to allow a comparison of affinity through the bar diagram. MDP (3) and lysine-pentapeptide (1) had no detectable binding at concentrations up to 1 mM.

 



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 6.
Comparative binding analysis of lysine-containing MPP (6) and DAP-containing MPP (9) with PGRP-I{alpha} and PGRP-S at concentrations of 0. 50, 50, and 200 µM.

 
The compounds 1-9 (Fig. 1) were synthesized by polymeric support synthesis using a Sieber amide resin, Fmoc-protected amino acids, and a properly protected muramic acid derivative. The synthetic compounds were designed in such a manner that the significance of each amino acid and the muramic acid moiety could be addressed for binding. Furthermore, by employing two series of compounds that contain either lysine or DAP as the third amino acid, the importance of these residues could be studied.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 7.
Sensorgrams representing the concentration-dependent kinetic analysis of DAP-containing MPP (9), MTrP (8), and MTP (7) with immobilized PGRP-S (2,700 RU). Simultaneous kinetic analysis of: A, MPP-DAP (9) at concentrations of 10, 100, 200, 500, and 1000 nM resulted in ka, 4.13 x 104 M-1s-1; kd, 4.29 x 10-3 s-1; and KD, 1.04 x 10-7M; B, MTrP-DAP (8)at concentrations of 10, 100, 200, 500, and 1000 nM resulted in ka, 5.17 x 104 M-1s-1; kd, 4.78 x 10-3 s-1; and KD of 9.24 x 10-8M; C, MTP-DAP (7) at concentrations of 100, 200, 500, 750, and 1000 nM resulted in ka, 1.41 x 104 M-1s-1; kd, 4.61 x 10-3 s-1; and KD of 3.26 x 10-7M. Corresponding residual values are plotted below the individual sensorgrams.

 
A relatively high immobilization of ~10,000 RU of PGRP-I{alpha}C was accomplished on the N-hydroxy succinimide-activated groups of a CM-5 research grade sensor chip surface to achieve good signal-to-noise ratios. For all SPR studies, bulk refraction caused by the difference in refractive index of the running buffer and sample injection was negated by using a control cell that was functionalized by ethanolamine.

A representative sensogram for the interaction of the lysine-containing muramyl pentapeptide 6 with the immobilized PGRPI-{alpha}C is shown in Fig. 2. Fitting using steady-state conditions gave a dissociation constant KD = 62 µM (TABLE ONE). The compounds 4 and 5 showed binding at concentrations >50 µM, whereas peptide 1 (Lys-PP) and MDP (3) exhibited no binding at concentrations up to 1 mM. The latter results indicate that the muramyl moiety is required for complexation and that MTP 4 is the smallest fragment of PGN recognized by PGRP-I{alpha}. Equilibrium-based kinetic analysis was not possible for MTP 4 and MTrP 5 due to an apparent biphasic nature of the real-time sensogram slope shifts at higher concentrations. Given that SPR detection is based on changes in mass at the surface of a sensor chip, normalization of respective RUs by the molecular weight of each analyte allows a qualitative comparison of analyte sets. A prerequisite of such a comparison is that all variables are kept constant, most importantly the protein surface employed. Normalization of the binding data of 4-6 (Fig. 3) demonstrated that MTrP 5 and MPP 6 are significantly more potent ligands than MTP 3. Furthermore, the data suggest that MPP 6 has a slightly higher affinity than MTrP 5.


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Binding constants (KD) ofPGN fragments with human PGRPs

 
Normalized SPR data for the DAP-containing compounds 7-9 showed a trend similar to that of the lysine-containing derivatives 4-6, whereby PGRP-{Theta}{alpha} had the weakest affinity for the MTP-DAP, whereas MTrP-DAP and MPP-DAP had similar and significantly higher affinities (Fig. 4). A comparison of the normalized data for the DAP-containing compounds 7-9 and lysine-containing derivatives 4-6 indicated that the PGRP-I{alpha} has a low selectivity for the lysine- or DAP-containing compounds.

Next, the interactions of compounds 1-9 with a CM-5 research grade sensor chip, containing 5000 RU of PGRP-S, were studied. The normalized data for the binding of immobilized PGRP-S with lysine-containing muramyl peptides 4-6 showed that MPP 6 has a slightly higher affinity than MtrP 5, which are both significantly better ligands than MTP 4 (Fig. 5). Steady-state equilibrium analysis of MPP 6 gave a KD of 189 µM (TABLE ONE). The normalized data for the DAP-containing compounds 7-9 showed a similar trend to that observed of the lysine-containing derivatives 4-6 whereby MTrP-DAP 8 and MPP-DAP 9 were significantly better ligands than MTP-DAP 7. However, unlike the binding with PGRP-I{alpha}C, which displayed similar binding affinities for the lysine- and DAP-type part structures, PGRP-S demonstrated significantly higher affinities for the DAP-containing fragments. Normalized data for MPP 6 and MPP-DAP 9 with both proteins at concentrations from 0.50 to 200 µM illustrate the highly selective nature of PGRP-S for PGN part structures, whereas PGRP-I{alpha} does not discriminate between the lysine- and DAP-containing fragments (Fig. 6). Due to the high affinity for this set of analytes, a lower density surface containing 2700 RU of PGRP-S was prepared for kinetic analysis. The kinetic wizard method and simultaneous kinetic fitting gave the following dissociation constants: MPP-DAP KD = 104 nM, MTrP-DAP = 92 nM, and MTP-DAP = 326 nM (Fig. 7 and TABLE ONE).



View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 8.
Intermolecular contacts in the PGRP-I{alpha}C-MTP-lysine complex. Stereoview of interactions between PGRP-I{alpha}C and MTP-lysine (4) at the PGN-binding site. MTP-lysine is shown in purple, PGRP-I{alpha}Cin yellow, and contacting residues in green. Hydrogen bonds are shown as dashed lines; residues forming van der Waals contacts with MTP-Lysine are also highlighted.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although the structure of the carbohydrate backbone of peptidoglycan is preserved among all bacteria, considerable structural variability exists in the peptide moiety (22). The archetypal stem peptide of Gram-positive bacteria is L-alanine-{gamma}-D-glutamate-L-lysine-D-alanine. In the case of Gram-negative bacteria and Gram-positive bacilli, m-diaminopimelic acid (DAP-type) is normally found as the third amino acid. Drosophila PGRP-SA has been shown to interact with lysine-type PGN activating the Toll receptor pathway, (23) whereas PGRP-LC and PGRP-LE recognize DAP-type PGN activating the Imd/Relish pathway (24-28). Previous studies have shown that each of the four mammalian PGRPs is able to bind peptidoglycan. However, possible selectivities for lysine- or DAP-type PGN have either not been determined or remain controversial.

In this study, the complexation of human PGRP-I{alpha}C and PGRP-S with a range of synthetic PGN fragments, containing either lysine or DAP as the third amino acid, have been studied by SPR. A dissociation constant of KD = 62 µM for MPP-Lys 6 was determined by a fitting of steady-state conditions. Furthermore, the normalized data of the lysine-containing compounds 4-6 indicate that MTrP 5 and MPP 6 have similar affinities, whereas a much lower affinity for MTP 4 was measured. In addition, peptides 1 (Lys-PP) and 2 (DAP-PP) and MDP (3) exhibited no binding at 1 mM concentration.

Recently, the crystal structure of PGRP-I{alpha}C ligated with MTP 4 was reported at 2.3 Å (Fig. 8) (41). In this complex, the tripeptide stem of MTP was held in an extended conformation at the deep end of the binding groove, whereas the MurNAc moiety lies in a pocket in the middle of the groove, with the pyranose ring oriented perpendicular to the base of the pocket. The structure indicates that the protein can accommodate a fourth D-Ala residue making contacts with Gln-261, Tyr-266, and Asn-269. The SPR data reported here demonstrate that these proposed interactions contribute significantly to binding. By contrast, D-Ala at position 5 is expected to extend beyond the binding groove. Therefore this amino acid should contribute little to the binding, an observation consistent with the SPR data. The observation that the pentapeptide 1 does not bind with PGRPI{alpha}C indicates that the interactions with the muramic acid moiety are critical for binding. In this respect, the lactyl moiety forms a hydrogen bond with Tyr-242, whereas the acetamido of the saccharide moiety interacts with His-2231 and Arg-235. SPR experiments with DAP-containing fragments 7-9 revealed that they complex with similar affinities as compared with the Lys-containing compounds 4-6.

The complexation of PGRP-S with DAP-containing fragments 7-9 exhibited high affinities with dissociation constants in the nanomolar range. The affinity trend was similar to that observed for the binding of PGRP-I{alpha}C with DAP-containing compounds 7-9. Thus, MTP-DAP (7) was the smallest fragment to be recognized, and significantly higher affinities were observed for MTrP-DAP (8) and MPP-DAP (9). Interestingly, much lower affinities were measured for the interactions of PGRP-S with the lysine-type compounds 4-6. Thus, although only a minimal selectivity was observed for binding of PGRP-I{alpha}C with lysine- and DAP-containing compounds, PGRP-S displayed a significant preference for DAP-containing PGN fragments.

The PGRP-I{alpha}C-MTP complex shows that Asn-236 and Phe-237 form a number of van der Waals contacts with the side chain of L-lysine. Sequence alteration at these two positions may account for the discriminatory ability of different PGRPs toward Lys-type and DAP-type PGNs. For example, the corresponding sequence in Drosophila PGRP-LCx and PGRP-LE, which recognizes DAP-type PGNs, (1, 42) is Gly-Trp. PGRP-S also has Gly-89 and Trp-90 at these positions.

The structural difference between lysine and DAP is an extra carboxylic acid at the {epsilon}C of the side chain. This chemical difference does not allow the different PGRPs to exhibit absolute discriminatory ability toward one type of PGN. For example, a certain degree of cross-reactivity is also exhibited by Drosophila PGRP-SA and mouse PGRP-L, which hydrolyzes PGNs from both Gram-positive and negative sources (31). Similarly, in the case of PGRP-I{alpha}C, despite having Asn-236 and Phe-237, the protein can recognize both Lys- and DAP-type PGNs with similar affinities.

Selectivity for lysine- or DAP-containing peptidoglycan fragments has been observed for other pattern recognition receptors. For example, transfection studies have shown that NOD2 recognized MDP and muramyltripeptide containing lysine as the third amino acid. (43) On the other hand, NOD1 senses DAP-containing peptidoglycan (44, 45). The structure activity relationships determined for the NOD proteins shows a different profile from what has been determined for PGRP-I{alpha} and PGRP-S. In this respect, the NOD proteins do not respond to muramyl tetra- and muramyl pentapeptide structures. It appears that the different pattern recognition receptors for peptidoglycan have evolved in such a way that they can recognize different part structures.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GMO65248 (to G.-J. B.) and AI47990 and AI065612 (to R. A. M.). 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. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S17. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence may be addressed. Tel.: 240-314-6243; Fax: 240-314-6255; E-mail: mariuzza{at}carb.nist.gov.

3 To whom correspondence may be addressed: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602. Tel.: 706-542-9161; Fax: 706-542-4412; E-mail: gjboons{at}ccrc.uga.edu.

4 The abbreviations used are: PAMP, pathogen-associated molecular pattern; PGN, peptidoglycan; TLR, Toll-like receptor; PGRP, peptidoglycan recognition protein; DAP, meso-diaminopimelic acid; SPR, surface plasmon resonance; DMF, dimethylformamide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MDP, muramyldipeptide; RU, resonance unit(s); Lys-PP, pentapeptide; MTP, muramyltripeptide; MTrP, muramyltetrapeptide; MPP, muramylpentapeptide; HRMS, high resolution mass spectrometry; PES, polyethersulfone. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hoffmann, J. A. (2003) Nature 426, 33-38[CrossRef][Medline] [Order article via Infotrieve]
  2. Janeway, C. A., and Medzhitov, R. (2002) Annu. Rev. Immunol. 20, 197-216[CrossRef][Medline] [Order article via Infotrieve]
  3. Medzhitov, R., and Janeway, C. A. (2002) Science 296, 298-300[Abstract/Free Full Text]
  4. Beutler, B. (2004) Mol. Immunol. 40, 845-859[CrossRef][Medline] [Order article via Infotrieve]
  5. Van Amersfoort, E. S., Van Berkel, T. J. C., and Kuiper, J. (2003) Clin. Microbiol. Rev. 16, 379-414[Abstract/Free Full Text]
  6. Beutler, B., Hoebe, K., Du, X., and Ulevitch, R. J. (2003) J. Leukocyte Biol. 74, 479-485[Abstract/Free Full Text]
  7. Lien, E., and Ingalls, R. R. (2002) Crit. Care Med. 30, S1-S11[CrossRef][Medline] [Order article via Infotrieve]
  8. O'Neill, L. A. J. (2004) Science 303, 1481-1483[Abstract/Free Full Text]
  9. Check, W. (2004) Asm News 70, 317-322
  10. O'Neill, L. A. J. (2004) Trends Immunol. 25, 687-693[CrossRef][Medline] [Order article via Infotrieve]
  11. Schmitz, F., Mages, J., Heit, A., Lang, R., and Wagner, H. (2004) Eur. J. Immunol. 34, 2863-2873[CrossRef][Medline] [Order article via Infotrieve]
  12. Pasare, C., and Medzhitov, R. (2003) Curr. Opin. Immunol. 15, 677-682[CrossRef][Medline] [Order article via Infotrieve]
  13. Tsan, M. F., and Gao, B. C. (2004) J. Leukocyte Biol. 76, 514-519[Abstract/Free Full Text]
  14. S. N., Sarkar, K. L., Peters, C. P., Elco, S., Sakamoto, S., Pal, and Sen, G. C. (2004) Nat. Struct. Mol. Biol. 11, 1060-1067[CrossRef][Medline] [Order article via Infotrieve]
  15. L. H., Travassos, S. E., Girardin, D. J., Philpott, D., Blanot, M. A., Nahori, C., Werts, and Boneca, I. G. (2004) EMBO Rep. 5, 1000-1006[CrossRef][Medline] [Order article via Infotrieve]
  16. Inohara, N., and Nunez, G. (2003) Nat. Rev. Immunol. 3, 371-382[CrossRef][Medline] [Order article via Infotrieve]
  17. Chamaillard, M., Girardin, S. E., Viala, J., and Philpott, D. J. (2003) Cell. Microbiol. 5, 581-592[CrossRef][Medline] [Order article via Infotrieve]
  18. Dziarski, R. (2004) Mol. Immunol. 40, 877-886[CrossRef][Medline] [Order article via Infotrieve]
  19. Liu, C., Xu, Z. J., Gupta, D., and Dziarski, R. (2001) J. Biol. Chem. 276, 34686-34694[Abstract/Free Full Text]
  20. Kang, D. W., Liu, G., Lundstrom, A., Gelius, E., and Steiner, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10078-10082[Abstract/Free Full Text]
  21. Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H., and Hultmark, D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13772-13777[Abstract/Free Full Text]
  22. Schleifer, K. H., and Kandler, O. (1972) Bacteriol. Rev. 36, 407-477[Free Full Text]
  23. Michel, T., Reichhart, J. M., Hoffmann, J. A., and Royet, J. (2001) Nature 414, 756-759[CrossRef][Medline] [Order article via Infotrieve]
  24. Werner, T., Borge-Renberg, K., Mellroth, P., Steiner, H., and Hultmark, D. (2003) J. Biol. Chem. 278, 26319-26322[Abstract/Free Full Text]
  25. M., Ramet, P., Manfruelli, A., Pearson, B., Mathey-Prevot, and Ezekowitz, R. A. B. (2002) Nature 416, 644-648[CrossRef][Medline] [Order article via Infotrieve]
  26. Choe, K. M., Werner, T., Stoven, S., Hultmark, D., and Anderson, K. V. (2002) Science 296, 359-365[Abstract/Free Full Text]
  27. Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J. A., Ferrandon, D., and Royet, J. (2002) Nature 416, 640-644[CrossRef][Medline] [Order article via Infotrieve]
  28. Leulier, F., Parquet, C., Pili-Floury, S., Ryu, J. H., Caroff, M., Lee, W. J., Mengin-Lecreulx, D., and Lemaitre, B. (2003) Nat. Immunol. 4, 478-484[CrossRef][Medline] [Order article via Infotrieve]
  29. Cheng, X. D., Zhang, X., Pflugrath, J. W., and Studier, F. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4034-4038[Abstract/Free Full Text]
  30. Mellroth, P., Karlsson, J., and Steiner, H. (2003) J. Biol. Chem. 278, 7059-7064[Abstract/Free Full Text]
  31. Wang, Z. M., Li, X. N., Cocklin, R. R., Wang, M. H., Wang, M., Fukase, K., Inamura, S., Kusumoto, S., Gupta, D., and Dziarski, R. (2003) J. Biol. Chem. 278, 49044-49052[Abstract/Free Full Text]
  32. Gelius, E., Persson, C., Karlsson, J., and Steiner, H. (2003) Biochem. Biophys. Res. Commun. 306, 988-994[CrossRef][Medline] [Order article via Infotrieve]
  33. Dziarski, R., Platt, K. A., Gelius, E., Steiner, H., and Gupta, D. (2003) Blood 102, 689-694[Abstract/Free Full Text]
  34. Tydell, C., Yount, N., Tran, D., Yuan, J., and Selsted, M. E. (2002) J. Biol. Chem. 277, 19658-19664[Abstract/Free Full Text]
  35. Guan, R. J., Malchiodi, E. L., Wang, Q., Schuck, P., and Mariuzza, R. A. (2004) J. Biol. Chem. 279, 31873-31882[Abstract/Free Full Text]
  36. Sieber, P. (1987) Tetrahedron Lett. 28, 2107-2110
  37. Chowdhury, A. R., Siriwardena, A., and Boons, G. J. (2002) Tetrahedron Lett. 43, 7805-7807
  38. Chowdhury, A. R., and Boons, G. J. (2005) Tetrahedron Lett. 46, 1675-1678[CrossRef]
  39. Jonsson, U., Fagerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., Lofas, S., Persson, B., Roos, H., Ronnberg, I., Sjolander, S., Stenberg, E., Stahlberg, R., Urbanic-zky, C., Ostlin, H., and Malmqvist, M. (1991) BioTechniques 11, 620-627[Medline] [Order article via Infotrieve]
  40. Cannon, M. J., Papalia, G. A., Navratilova, I., Fisher, R. J., Roberts, L. R., Worthy, K. M., Stephen, A. G., Marchesini, G. R., Collins, E. J., Casper, D., Qiu, H. W., Satpaev, D., Liparoto, S. F., Rice, D. A., Gorshkova, I. I., Darling, R. J., Bennett, D. B., Sekar, M., Hommema, E., Liang, A. M., Day, E. S., Inman, J., Karlicek, S. M., Ullrich, S. J., Hodges, D., Chu, T., Sullivan, E., Simpson, J., Rafique, A., Luginbuhl, B., Westin, S. N., Bynum, M., Cachia, P., Li, Y. J., Kao, D., Neurauter, A., Wong, M., Swanson, M., and Myszka, D. G. (2004) Anal. Biochem. 330, 98-102[CrossRef][Medline] [Order article via Infotrieve]
  41. Guan, R. J., Roychowdhury, A., Ember, B., Kumar, S., Boons, G. J., and Mariuzza, R. A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 17168-17173[Abstract/Free Full Text]
  42. Kaneko, T., Goldman, W. E., Mellroth, P., Steiner, H., Fukase, K., Kusumoto, S., Harley, W., Fox, A., Golenbock, D., and Silverman, N. (2004) Immunity 20, 637-649[CrossRef][Medline] [Order article via Infotrieve]
  43. Girardin, S. E., Travassos, L. H., Herve, M., Blanot, D., Boneca, I. G., Philpott, D. J., Sansonetti, P. J., and Mengin-Lecreulx, D. (2003) J. Biol. Chem. 278, 41702-41708[Abstract/Free Full Text]
  44. Girardin, S. E., Boneca, I. G., Carneiro, L. A. M., Antignac, A., Jehanno, M., Viala, J., Tedin, K., Taha, M. K., Labigne, A., Zahringer, U., Coyle, A. J., Bertin, J., Sansonetti, P. J., and Philpott, D. J. (2003) Science 300, 1584-1587[Abstract/Free Full Text]
  45. Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y., Kawasaki, A., Fukase, K., Kusumoto, S., Valvano, M. A., Foster, S. J., Mak, T. W., Nunez, G., and Inohara, N. (2003) Nat. Immunol. 4, 702-707[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit