Fig. 4

Zebrafish PGLYRP Proteins Are Enzymes with N-Acetylmuramoyl-L-Alanine Amidase Activity. (A) S. aureus soluble polymeric peptidoglycan, labeled with biotin on terminal Gly, was incubated in buffer alone or with the indicated PGLYRPs for 24 or 96 hr. High-molecular-weight polymeric biotin-peptidoglycan was detected on an immunoblot with streptavidin-peroxidase. Hydrolysis of the peptide from the glycan chain removes the biotin-labeled peptide. The results are from one of two similar experiments. (B and C) The muropeptides indicated in (B) were incubated in buffer alone (control) or in the presence of the indicated PGLYRPs, and the hydrolysis products were identified with MALDI-TOF MS; peaks for the indicated peptides and observed masses ([M + Na]⁺) are shown in (C). The vertical scale in the left-hand PGLYRP-5 and PGLYRP-2 panels was expanded so that minor peaks are revealed. The unlabeled peaks are either matrix or buffer adduct peaks that are the same in both digested and undigested samples or reflect natural isotopic distribution of the digestion products. K⁺ adducts of the same peptides were also detected (not shown). The hydrolyzed bond is indicated in (B) by an arrow. Identical hydrolysis products were generated by incubation of the same substrates with human PGLYRP-2, a proven N-acetyl-muramoyl-L-alanine amidase (Wang et al., 2003), as a positive control (not shown). The results are from one of two similar experiments.