During the course of the structure analysis of sugar chains of sRNase from stylus of Japanese pear (Pyrus pyrifolia), the chitobiose structure was detected as a major N-linked sugar chain (1). As the middle size sugar chains such as Man ß 1-4GlcNAc ß 1-4GlcNAc were not detected, it is suggested that the glycoside bond of Man ß1-4GlcNAc was hydrolyzed with an endo-type glycosidase.

Two endo-type enzymes which hydrolyze the reducing end region of N-linked sugar chains of glycoproteins have been reported. One is peptide N-glycosidase, which hydrolyzes the bond between the reducing end GlcNAc residue and the asparagine residue of proteins, the other is endo- ß-N-acetylglucosaminidase, which hydrolyzes the chitobiose structure. On the other hand, ß-mannosidase, which hydrolyzes reducing end ß-linked mannose residue from the non-reducing ends, and ß-mannanase, which hydrolyzes ß-mannan, were reported. However, an enzyme which hydrolyzes the Man ß- linkage of N-linked sugar chains in an endo-manner has not yet been reported.
A crude enzyme solution was obtained from homogenates of lily (Lilium longflorum) and the enzyme was purified by ammonium sulfate precipitation, DEAE-Sephacel, Q-Sepharose anion exchange, Superdex 200 gel filtration, HA 1000 hydroxyapatide, Poros PE/M hydrophobic, Mono Q anion exchange, Superdex 200 gel filtration chromatography.

Enzyme products were analyzed by pulse amperometric detection using Man a1-6Man ß1-4GlcNAc2-PA, Man a 1-3Man ß 1-6Man ß 1-4GlcNAc2-PA, Man a 1-6(Man a 1-3)Man a 1-6Man ß1-4GlcNAc2-PA. Each substrate was hydrolyzed, and an oligomannose and GlcNAc2-PA were detected, indicating that only the Man ß1-4 linkge was hydrolyzed. The enzyme did not hydrolyze p-nitrophenyl ß-mannoside (Man ß1-4)6, indicating that the enzyme is not the reported ß-mannanase or ß-mannosidase. Based on the data obtained above the enzyme hydrolyzes Man ß1-4GlcNAc linkages of the N-linked sugar chains in an endo-manner (2).

The molecular weight of the enzyme is estimated to be 78,000 as analyzed by Superdex 200 gel filtration. The enzyme does not require metal cations. Among the substrates used, Man a1-6Man ß1-4GlcNAc2-PA was the best substrate. However, sugar chains containing the Mana1-3Man-structure were not hydrolyzed. Man ß1-4GlcNAc2-PA was hydrolyzed slowly.
Sumihiro Hase (Graduate School of Sciences, Osaka University)
References (1) T, Ishimizu Y, Mitsukami T, Shinkawa S, Natsuka S, Hase M, Miyagi F, Sakiyama S, Norioka : Presence of Asparagine-Linked N-acetylglucosamine and Chitobiose in Pyrus pyrifolia S-Rnases Associated with Gametophytic Self-incompatibility. Eur. J. Biochem. 263, 624-634, 1999
(2) A, Sasaki M, Yamaguchi T, Mega S, Norioka S, Natsuka S, Hase : Partial Purification and Characterization of a Novel Endo-ß-mannosidase Acting on N-linked Sugar Chains from Lilium longflorum Thumb.J. Biochem. 125, 363-367, 1999
Sep.15, 2001

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