Detailed Studies on Substrate Structure Requirements of Glycoamidases A and F

Abstract

Glycoamidases (peptide-N 4-(N-acetyl-␤-glucosaminyl)-asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated ␤-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH 2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal. While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The K m and V max values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 mol/min/mg, respectively. A natural glycodipeptide, Man 9 -GlcNAc 2 -Asn-Phe, was also completely hydrolyzed by glycoamidase A.

Identification and Characterization of a Novel Prokaryotic Peptide

“…Altmann et al (27) showed that the size of the carbohydrate moiety in the substrate has little influence on the enzymatic activities of N-glycosidase and that the hydrolysis rates of PNGase enzymes are primarily determined by the length of the peptide. Studies on the substrate requirements of PNGase F showed that Asn-linked GlcNAc occupied the most critical position for substrate binding and that PNGase F requires at least the di-N,NЈ-acetylchitobiosyl core unit and a tripeptide for its activity (28).…”

Imaging the glycome

“…Lectins are able to recognize structures as varied as the monosaccharides sialic acid (28) and fucose (29), and higher-order structures such as the Tn (30) and sialyl-Tn tumor antigens (31) and the conserved core region of N-glycans (32). However, lectins typically have low affinity for their glycan epitope and require multivalency for high-avidity binding (33).…”

Presence of a Glycosylphosphatidylinositol Lipid Anchor on Rose Arabinogalactan Proteins

“…Of these few predicted sites, only those in AGP-like glycoproteins of tobacco styles have been shown to be actually glycosylated, as judged by sensitivity to glycoamidase F (36,37). As a test for the presence of N-glycans, rose CM-AGPs were treated with glycoamidase A, which cleaves a broader range of substrates than glycoamidase F (38). For example, plant glycoproteins with N-glycans containing Fuc linked ␣1 3 3 to the innermost GlcNAc are cleaved by glycoamidase A but are highly resistant to glycoamidase F (39).…”