Purification and characterization of N -acetylmuramyl- l -Alanine amidase from human plasma using monoclonal antibodies

Abstract

N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.

Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells

“…This action would antagonize the formation of biologically active molecules by lysozyme (Vanderwinkel et al, 1995). In contrast to the bacterial amidases described in E. coli, the eukaryotic amidase is capable of releasing peptides from macromolecular insoluble peptidoglycan (Vanderwinkel et al, 1990;Hoijer et al, 1996). Interestingly, a very recent report showed the presence of this amidase in intracellular locations of granulocytes, but not in monocytes (Hoijer et al, 1996).…”

“…In contrast to the bacterial amidases described in E. coli, the eukaryotic amidase is capable of releasing peptides from macromolecular insoluble peptidoglycan (Vanderwinkel et al, 1990;Hoijer et al, 1996). Interestingly, a very recent report showed the presence of this amidase in intracellular locations of granulocytes, but not in monocytes (Hoijer et al, 1996). It is possible that other eukaryotic cell types, such as epithelial cells, contain analogous N-acetyl-muramyl-L-alanine amidases that are able to act on the peptidoglycan of intracellular bacterial pathogens.…”

“…A eukaryotic N-acetyl-muramyl-L-alanine-amidase has been purified and characterized from human plasma (Vanderwinkel et al, 1990;Hoijer et al, 1996). Although at present its physiological function is still unknown, it has been speculated that it probably maintains adequate ratios between biologically active muropeptides and their inactive deglycosylated derivatives.…”

Phagocytes Containing a Disease-Promoting Toll-Like Receptor/Nod Ligand Are Present in the Brain during Demyelinating Disease in Primates

“…35,51 NAMLAA-containing cells were detected by staining with biotinylated monoclonal mouse anti-human NAMLAA-biotin IgG1 (AAA4) as described above. 27 mAb AAA4, raised against human serum amidase, specifically recognizes NAMLAA. 27,30 Lysozymecontaining cells were detected by staining with polyclonal rabbit anti-human lysozyme antibody (Ab-1; Neomarkers, Fremont, CA), followed by biotinylated donkey anti-rabbit antibody (Amersham Biosciences, Buckinghamshire, UK).…”

“…27 mAb AAA4, raised against human serum amidase, specifically recognizes NAMLAA. 27,30 Lysozymecontaining cells were detected by staining with polyclonal rabbit anti-human lysozyme antibody (Ab-1; Neomarkers, Fremont, CA), followed by biotinylated donkey anti-rabbit antibody (Amersham Biosciences, Buckinghamshire, UK). Incubations with secondary and tertiary reagents were done for 1 hour at room temperature.…”