Abstract
Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Among them, a recombinant protein purified from Escherichia coli expressing the candidate gene ORF1188 exhibited fucose-containing oligosaccharides-specific ENGase activity. The ENGase exhibited optimum activities at very acidic pHs (between pH 2.3–2.5). A BLAST search using the sequence of ORF1188 identified two fungal homologs, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.
Highlights
Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides
We identified four candidate ENGases in Sphingobacterium species, encoded by ORF1152, ORF1188, ORF3046 and ORF3750, collectively called here as Endo-SBs, and characterized the enzymatic properties of proteins expressed by these genes in detail
To further identify this strain, we performed a BLAST search based on its 16S rRNA gene sequence, and found that it belongs to the Sphingobacterium species
Summary
Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. A recombinant protein purified from Escherichia coli expressing the candidate gene ORF1188 exhibited fucose-containing oligosaccharides-specific ENGase activity. Endo-β-N-acetylglucosaminidases (ENGases, EC 3.2.1.96) are a class of enzymes, which can hydrolytically cleave β-1,4 glycosidic bonds within the N,N′-diacetylchitobiose moiety in the inner-core region of N-glycan and release glycan chain from their associated proteins, leaving one unit of N-acetylglucosamine (GlcNAc), with or without the core fucose, linked to the asparagine residue[1]. We identified four candidate ENGases in Sphingobacterium species, encoded by ORF1152, ORF1188, ORF3046 and ORF3750, collectively called here as Endo-SBs, and characterized the enzymatic properties of proteins expressed by these genes in detail. Recombinant proteins, purified from E. coli strains expressing the ORF1188, Beauveria and Cordyceps ENGase genes, catalyzed hydrolysis of fucose-containing complex type oligosaccharides
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