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Maxwell Burch, Peter Lam, william byrd, Thomas redens, Marlyn P Langford; N-Acetylmuramyl-L-alanine amidase activity in human corneal epithelial cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2262. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mammals have four peptidoglycan (PGN) recognition proteins (PGRP). The mRNA of one mammalian PGRP, PGLYRP-2 (secreted by the liver) has been reported to be inducible in keratinocytes. PGLYRP-2 is an N-acetylmuramyl-L-alanine amidase (NAMAA) that hydrolyzes bacterial peptidoglycan and reduces its proinflammatory activity. The decreased susceptibility of the cornea to bacterial infection may be dependent in part upon the presence of N-acetylmuramyl-L-alanine amidase activity. The aim was to identify NAMAA activity in human corneal cells.
N-acetylmuramyl-L-alanine amidase activity in human corneal epithelial cells (HCoC), human retinal pigmented epithelial (ARPE-19), and rabbit kidney (RK13) cells was identified, quantified, and compared. Differential activity from lysozyme was detected by agarose gel electrophoresis of cleaved fluoresceinated muramyl dipeptide (FITC-MDP). To detect PGRPs, anti-PGRP specific antibodies were used to detect and inhibit PGRP activity. The relative amounts of cleavage muramyl-FITC and control FITC-MDP were determined densitometrically. An immunofluorescent antibody assay was used to visualize PGLYRP-1 and -2 in ethanol fixed slide cell cultures.
FITC-MDP cleavage by NAMAA (PGLYRP-2) activity, but not by lysozyme or trypsin, was detected in in HCoC, RPE and RK cell lysates. NAMAA activity was detected, but was not significantly increased by NOD ligand stimulation upon incubation for 24h. Antibody to PGRP-1 & 2 identified positive cells in all cultures, while anti-PGLYRP-2 antibody inhibited the NAMAA activity specifically. The NAMAA activity was slightly greater in RK13 cells (3.5 ng/h/mg protein) than in HCoC (3.1 ng/h/mg protein) and RPE cells (1.4 ng/h/mg protein).
A colorimetric assay for detection of NAMAA activity is presented. PGLYRP-1 and -2 and NAMAA activity were detected in HCoC, as well as RPE and RK13 cells. These results support PGLYRP-2/NAMAA activity as a non-inducible, constitutive enzyme in corneal cells that may act to limit bacterial infection and reduce localized inflammation.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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