Characterization of natural human nucleotide-binding oligomerization domain protein 1 (Nod1) ligands from bacterial culture supernatant for elucidation of immune modulators in the environment.
Nucleotide-binding oligomerization domain protein 1 (Nod1) is an intracellular protein involved in recognition of the bacterial component peptidoglycan. This recognition event induces a host defense response to eliminate invading pathogens. The genetic variation of Nod1 has been linked to several inflammatory diseases and allergies, which are strongly affected by environmental factors. We have found that many of the bacteria that contain DAP-type peptidoglycan release Nod1 ligands into the environment. However, the structures of natural Nod1 ligands in the environment are not well understood. Herein, we report the isolation and structural elucidation of natural human Nod1 (hNod1) ligands from the Escherichia coli K-12 culture supernatant. The supernatant was fractionated with reversed-phase high performance liquid chromatography (RP-HPLC), resulting in the isolation of several hNod1 stimulatory fractions. Structural characterization studies demonstrated that the molecular structure of the most active fraction was the native hNod1 ligand GlcNAc-(beta1-4)-(anhydro)MurNAc-l-Ala-gamma-d-Glu-meso-DAP. We also found other peptidoglycan fragments using the 7-(diethylamino)coumarin-3-carbonyl labeling method to enhance sensitivity in mass spectroscopy studies. These results suggested that DAP-containing bacteria release certain hNod1 ligands to the environment, and these ligands would accumulate in the environment and regulate the immune system through Nod1.
Pradipta
Ambara R
AR
Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan.
Fujimoto
Yukari
Y
Hasegawa
Mizuho
M
Inohara
Naohiro
N
Fukase
Koichi
K
D001681:Biological Assay / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007107:Immune System; D007249:Inflammation; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D053474:Nod1 Signaling Adaptor Protein / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D008024:Ligands; D053474:Nod1 Signaling Adaptor Protein; D009711:Nucleotides; D010455:Peptides; D010457:Peptidoglycan