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Demonstration of peptidoglycan-binding sites on lymphocytes and macrophages by photoaffinity cross-linking
1Northwest Center for Medical Education, Indiana University School of Medicine, Gary 46408.
Abstract:
One dominant binding site (70 kDa 6.5 pI protein) for bacterial cell wall peptidoglycan (PGN), a macrophage activator and polyclonal B cell mitogen, was demonstrated on mouse B and T lymphocytes and macrophages by photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis. This binding site was not present on erythrocytes. The binding was specific for polymeric PGN and was competitively inhibited by unlabeled PGN with IC50 = 48 micrograms/ml (0.38 microM). The binding was partially inhibited by O-acetylated PGN monomers (IC50 = 469 micrograms/ml, 521 microM), dextran sulfate (IC50 = 1024 micrograms/ml, 124 microM), and (GlcNAc)3 (IC50 = 6.6 mg/ml, 10 mM), and was not inhibited by non-O-acetylated PGN monomers and dimers, muramyl dipeptide, PGN pentapeptide, GlcNAc, teichoic acid, protein A, and gelatin. The cell surface location of the 70-kDa PGN-binding protein was indicated by the ability of PGN to bind to this protein in intact metabolically inactive cells (at 4 degrees C and in the presence of 0.1% NaN3) and by the ability to extract the 70-kDa PGN-binding protein from viable B lymphocytes by noncytotoxic concentration of n-octyl-beta-D-glucopyranoside.
Insights
Researchers identified a 70-kDa protein on mouse immune cells that binds bacterial peptidoglycan (PGN). This discovery sheds light on how immune cells recognize bacterial components and activate immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Bacterial cell wall peptidoglycan (PGN) is a potent immune activator.
- Understanding PGN-immune cell interactions is crucial for immunology.
Purpose of the Study:
- To identify and characterize the binding site for bacterial peptidoglycan on mouse immune cells.
Main Methods:
- Photoaffinity cross-linking
- Two-dimensional polyacrylamide gel electrophoresis
- Competitive inhibition assays
Main Results:
- A dominant 70-kDa protein with a 6.5 pI was identified as the PGN binding site on mouse B lymphocytes, T lymphocytes, and macrophages.
- This binding site was absent on erythrocytes.
- Binding specificity was demonstrated for polymeric PGN, with competitive inhibition by unlabeled PGN (IC50 = 48 µg/ml).
- Partial inhibition was observed with O-acetylated PGN monomers, dextran sulfate, and (GlcNAc)3.
- The protein's cell surface localization was confirmed through binding to intact cells and extraction from B lymphocytes.
Conclusions:
- A specific 70-kDa cell surface protein acts as a primary binding site for bacterial peptidoglycan on mouse immune cells.
- This interaction is specific and involves recognition of polymeric PGN structures.
- The findings contribute to understanding the molecular mechanisms of innate immune recognition of bacterial components.