Poster: Enzymology

Abs # 233: An Arabidopsis Gene Encoding Endo-b-mannosidase Involved in Processing of N-Linked Sugar Chains

Presenter:	Ishimizu, Takeshi , txi@chem.sci.osaka-u.ac.jp
Authors	Ishimizu, Takeshi  (A)   Okutani, Satoshi  (A)   Sasaki, Akiko  (A)   Hase, Sumihiro  (A)
Affiliations:	(A): Department of Chemistry, Graduate School of Science, Osaka University

An enzyme hydrolyzing the Manb1-4GlcNAc linkage of N-linked sugar chains, endo-b-mannosidase, has been identified from flowers of lily (Sasaki et al., 1999). This enzyme was assayed using pyridylaminated (PA-) Mana1-6Manb1-4GlcNAcb1-4GlcNAc as a substrate in vitro. Recently, endo-b-mannosidase was purified to homogeneity from lily flowers as a heterotrimer. It consisted of three polypeptides which their molecular weights were 28K, 31K, and 42K. Amino acid sequences of each polypeptide of lily enzyme led to the identification of a candidate of arabidopsis gene encoding endo-b-mannosidase. This gene encoded a polypeptide consisted of 914 amino acid residues. Its molecular weight was 104355 corresponding to the mass of three polypeptides of lily enzyme. This gene had the homologous sequences of all the amino acid sequences derived from three polypeptides lily enzyme. These suggest that three polypeptides of lily enzyme were encoded by one gene. To evaluate that this gene product is an endo-b-mannosidase, the candidate gene was expressed in Escherichia coli and its activity was measured. This gene product expressed as a single polypeptide hydrolyzed Mana1-6Manb1-4-GlcNAcb1-4GlcNAc-PA to GlcNAcb1-4GlcNAc-PA, concluding that this arabidopsis gene encodes endo-b-mannosidase. Sasaki, A. et al., J. Biochem. 125, 363-367 (1999)