Structures of N-Linked Oligosaccharides of Microsomal Glycoproteins from Developing Castor Bean Endosperms

Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns

“…PA-sugar chains were fractionated on a Jasco 880-PU HPLC apparatus equipped with a Jasco 821-FP Intelligent Spectrofluorometer by using a Cosmosil 5C18-P column (6 ϫ 250 mm; Nacalai Tesque, Kyoto) or an Asahipak NH2P-50 column (4.6 ϫ 250 mm; Showa Denko, Tokyo). Elution positions of sugar chains were compared with authentic sugar chains as prepared previously (30) or purchased (Wako).…”

“…Glycosidase digestions using ␤-N-acetylglucosaminidase (Diplococcus pneumoniae; Boehringer Mannheim) or ␣-mannosidase (jackbean; Sigma) were done by using about 1 nmol of PA-sugar chains (30). For ␤-galactosidase (D. pneumoniae; Boehringer Mannheim) and ␣1,2-mannosidase (Aspergillus saitoi; a kind gift from T. Yoshida, Tohoku University) digestions, 1 nmol of PA-sugar chain in 50 mM sodium acetate buffer (pH 5.5) was incubated with either 200 milliunits ␤-galactosidase or 60 g ␣1,2-mannosidase at 37°C.…”

“…The reactions were stopped by boiling, and an aliquot of the digests was analyzed by SF-HPLC. The molecular masses of the resulting digests were analyzed by IS-MS͞MS and͞or compared with authentic sugar chains (14,30).…”