Determination of glycosylation sites using a protein sequencer and deglycosylation of native yeast invertase by endo-beta-N-acetylglucosaminidase

Abstract

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was tested for its capacity to release N-linked sugar chains from native yeast invertase. The enzyme liberated about 80% of the sugar chains from the native invertase. Deglycosylated invertase was digested by chymotrypsin or pepsin, and twelve N-acetylglucosamine-containing glycopeptides were isolated. The amino acid sequences of these glycopeptides were analyzed by a protein sequencer, and the elution position of 4-L-aspartylglycosylamine was directly identified by conventional sequencing. The endo-beta-N-acetylglucosaminidase was found to remove mainly nine sugar chains from native invertase.