SYNTHESIS OF NEOGLYCOPROTEINS USING OLIGOSACCHARIDE-TRANSFER ACTIVITYWITH ENDO-BETA-N-ACETYLGLUCOSAMINIDASE

We describe a novel method for the enzymatic synthesis of neoglycoprot eins. Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormia e (Endo-A) had high levels of transglycosylation activity. The enzyme activity of Endo-A was markedly increased by adding 4-L-aspartyl-glyco sylamine (GlcNAc-Asn) to the reaction mixture, Digesting (Man)(6)(GlcN Ac)(2) with the enzyme in the presence of GlcNAc-Asn gave a mixture of hydrolytic ((Man)(6)GlcNAc) and transglycosylic ((Man)(6)(GlcNAc)(2)A sn) products. By means of transglycosylation, (Man)(6)GlcNAc was trans ferred en bloc to the partially deglycosylated ovalbumin glycopeptide (EEKYN(GlcNAc) LTSVL) concomitant with the hydrolysis of (Man)(6)(GlcN Ac)(2)Asn. The structure of the transglycosylation product was designa ted as (Man)(6)(GlcNAc)(2)-peptide by amino acid composition and seque nce analysis as well as ion mass spectrometry. The enzyme also transfe rred oligosaccharide to partially deglycosylated ribonuclease B (GlcNA c-protein) during the hydrolysis of (Man)(6)(GlcNAc)(2)Asn. Native rib onuclease B had (Man)(5-9) (GIcNAc)(2) as its heterogeneous N-linked s ugar chains. High performance liquid chromatography showed that all of the N-linked sugar chains of the synthetic neoribonuclease of the pyr idylamino derivatives were modified to (Man)(6)(GlcNAc)(2).