Human chitotriosidase CHIT1 cross reacts with mammalian‐like substrates

Abstract

a b s t r a c tHumans do not synthesize chitin, yet they produce a number of active and inactive chitinases. One of the active enzymes is chitotriosidase whose serum levels are elevated in a number of diseases such as Gaucher's disease and upon fungal infection. Since the biological role of chitotriosidase in disease pathogenesis is not understood we screened a panel of mammalian GlcNAc-containing glycoconjugates as alternate substrates. LacNAc and LacdiNAc-terminating substrates are hydrolyzed, the latter with a turnover comparable to that of pNP-chitotriose. Glycolipids or glycoproteins with LacNAc and LacdiNAc represent potential chitinase substrates and the subsequent alteration of glycosylation pattern could be a factor in disease pathogenesis.

A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Galβ1–4GlcNAc) and LacdiNAc (GalNAcβ1–4GlcNAc) Motifs Found on Vertebrate and Insect Cells

“…Further analyses are needed to elucidate which T. viride chitinase(s) is responsible for this activity and to evaluate the potential biological role. The general activity of chitinases toward a LacdiNAc-terminating substrate is a novel observation only reported previously for the human chitotriosidase (17).…”

“…In addition to the previous studies of a Salmonella chitinase and the human chitotrisidase by the authors of the present paper (14,17) only two studies have demonstrated such activity expressed by the chitinases in the bacterial species Amycolatopsis orientalis (32) and Bacillus circulans (33). The Amycolatopsis chitinase not only hydrolyzed chitobiose from pNP-(GlcNAc) 2 but also released free oligosaccharides and pnitrophenol from Gal␤1,4GlcNAc␤1,3Gal␤1,4GlcNAc␤-pNP GlcNAc␤1,3Gal␤1,4GlcNAc␤-pNP, and LacNAc-pNP (Gal␤1,4GlcNAc␤-pNP).…”

“…Because N-acetyllactosamine is a common component of mammalian glycans, it constitutes a possible chitinase target. We have also shown that human chitotriosidase cleaves LacNAc and LacdiNAc (GalNAc␤1-4GlcNAc) from LacNAc-TMR and LacdiNAc-TMR, respectively, with the latter having a turnover comparable to that of pNP-chitotrioside (17).…”

“…1. Initial screening was carried out by TLC analysis where 1 l of GlcNAc-TMR (1 mM), LacNAc-TMR (20 mM), LacdiNAc-TMR (20 mM), or Type I-TMR (Gal␤1-3GlcNAc-TMR, 1 mM) were incubated with 5 l of enzyme at ambient temperature for 2-24 h. Reaction progress was monitored by removing 1-l aliquots for thin-layer chromatography on silica gel plates developed with CHCl 3 / MeOH/H 2 O (65/35/5) as described previously (17). The TMRlabeled compounds, which are brightly colored red/purple, can be visualized by eye.…”

“…Assays were carried out in 4 l containing 50 mM sodium phosphate buffer, pH 6.0, 1 g of each enzyme, and 5 pmol of TMR-substrate. After overnight incubation at room temperature, a 1-l aliquot was removed, mixed with 300 l of buffer (10 mM ammonium formate, pH 4.5 (22%), acetonitrile (88%) (v/v)) for analysis by ultra-HPLC-MS as previously described (17).…”