Abstract
Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1–4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.
Highlights
Introduction αN-Acetylglucosaminidases (EC 3.2.1.50) are glycoside hydrolases (GH) that catalyze the cleavage of the terminal N-acetylglucosamine from α-linked N-acetylglucosaminides (GlcNAcαOR)
Cloning and expression of bacterial CAZy GH89 α‐N‐acetylglucosaminidases Protein structure-based alignment using UCSF Chimera (Pettersen et al 2004) and structural overlay using PyMOL (Yuan et al 2016) of CpGH89 (GenBank accession number ABG84150.1) and human α-N-acetylglucosaminidase (hNAGLU) reveal an extra loop in CpGH89 containing a tryptophan (W685) residue which was suggested to be important for the recognition of the GlcNAcα1–4Gal motif of its substrate (Fig. 1; Ficko-Blean and Boraston 2012)
This loop was hypothesized to restrict the type of the substrate that can enter the binding pocket and cause the high substrate selectivity of CpGH89, preventing the binding of heparan sulfate-type substrate that containing a terminal N-Acetylglu‐ cosamine (GlcNAc) α-linked to a β-D-glucuronic acid or α-L-iduronic acid (Birrane et al 2019; Ficko-Blean and Boraston 2012)
Summary
N-Acetylglucosaminidases (EC 3.2.1.50) are glycoside hydrolases (GH) that catalyze the cleavage of the terminal N-acetylglucosamine from α-linked N-acetylglucosaminides (GlcNAcαOR). They have been grouped in the Carbohydrate-Active enZYme (CAZY) database (www.cazy.org) (Henrissat 1991) GH89 family based on their protein sequence similarity. Among more than 1000 predicted GH89 family members (> 100 from eukaryote and > 900 from bacteria), only human α-N-acetylglucosaminidase (hNAGLU) (Weber et al 1996) and its homologues from bacteria Clostridium. HNAGLU is a lysosomal enzyme that catalyzes the hydrolysis of the terminal α1–4-linked N-acetylglucosamine (GlcNAc) at the non-reducing end of heparan sulfate (HS) (Birrane et al 2019; Valstar et al 2010). The crystal structure of the apo form of a recombinant human NAGLU (rhNAGLU, PDB ID: 4XWH) expressing high-mannose type N-glycans was reported recently (Birrane et al 2019)
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