| Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neu-trophil protein. We report that human PGRP-L is a Zn + -dependent N-acetylmu-ramoyl-L-alanineamidase, an enzyme that hydrolyzes the amide bond between MurNAc and L-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no directbacteriolytic activity. The other members of the human PGRP family, PGRP-I , PGRP-I , and PGRP-S, do not have the amidase activity. The C-termmal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal 1 343 deletion mutant) is reduced. The Zn + binding ammo acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the " active" configuration does not convert PGRP-S into an active amidase. This experiment is to detect whetger human PGRP-L is an N-acetylmuramoyl-L-alanine amidase.EXPERIMENTAL PROCEDURES一.PGRP Constructs and Proteins-Human PGRP-S, PGRP-L, PGRP-I,and PGRP-I and mouse PGRP-S were cloned in pcDNA3. 1 and expressed in COS-7 cells as described previously. Human PGRP-L deletion mutants were generated by PCR. Single amino acid substitution mutants of human PGRP-L, human PGRP-S, and mouse PGRP-S were generated using the site-directed mu-tagenesis kit from Clontech (Palo Alto, CA) with the primers. All constructs were subcloned into pcDNA3. 1 with C-terminal V5 and 6xHis tags and analyzed by restriction digests and by sequencing as described previously. Recombinant PGRP proteins were obtained from transiently transfected COS-7 cell lysates (PGRP-L, PGRP-I, and PGRP-I ) or lysates and supernatants (PGRP-S) by affinity chromatography using His-Bind kit (Novagen, Madison, WI) as recommended by the manufacturer, with binding, washing, and elution buffers all containing 20 mM Tris (pH 7.9) , 0.5 M NaCl, 1% Triton X-100, and 5 mM, 20 mM or 300 mM imidazole, respectively. The proteins were stored in the elution buffer with 10% glycerol at 8C.二. PGN Digestion and Amidase Assays-Soluble uncross-linked PGN was purified by vancomycin-affinity chromatography from Staphylococcus aureus Rb grown in the presence of penicillin G and analyzed as described previously. PGN was labeled with biotin on the N-terminal glycine of its interpeptide bridge, and its hydrolysis was measured as described previously. Complete loss of the biotin from PGN following digestion with lysostaphin ( which is specific for the glycine bridge of S. aureus PGN) confirms that biotin was indeed bound to the N-terminal glycine of this uncross-linked PGN. PGN-biotin (750 ng) was incubated for 3 days at 37 C with 10 50 ng of recombinant PGRP-L, PGRP-I , PGRP-I , and PGRP-S, or their mutants in 35 1 of 75% elution buffer with 1 mM ZnSO4 (unless otherwise indicated) , or with 10 200 ng of lysozyme ( grade I from chicken egg from Sigma St. Louis, MO) or lysostaphin ( affinity-purified from Staphylococcus staphylolyticus from Sigma) as positive controls, or trypsin (type II from bovine pancreas from Sigma) or buffer alone as negative controls. Enzyme-digested PGN-biotin were subjected to SDS-PAGE, blotted on Immobilon-P, and high molecular weight PGN-biotin was detected with streptavidin-peroxidase and enhanced chemiluminescence as described previously. Separate aliquots of the digests (2 1) were also analyzed by Western blot with anti-V5 antibodies as de-scribed for the presence of each PGRP protein.三. Immunofluorescence-COS-7 cells or HepG2/C... |
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