Substrate specificity and molecular cloning of the lily endo-beta-mannosidase acting on N-glycan.
Endo-beta-mannosidase, which hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans, was recently purified to homogeneity from lily (Lilium longiflorum) flowers as a heterotrimer [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. Here, we describe the substrate specificity of the enzyme and cloning of its cDNA. The purified enzyme hydrolyzed pyridylaminated (PA-) Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc (n = 0-2) to Man(n)Manalpha1-6Man and GlcNAcbeta1-4GlcNAc-PA. It did not hydrolyze PA-sugar chains containing Manalpha1-3Manbeta and/or Xylbeta1-2Manbeta. The best substrate among the PA-sugar chains tested was Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA with a K(m) value of 1.2 mM. However, the enzyme displayed a marked preference for the corresponding glycopeptide, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-peptide (K(m) value 75 microM). These results indicate that the substrate recognition by the enzyme involves the peptide portion attached to the N-glycan. Sequence information on the purified enzyme was used to clone the corresponding cDNA. The monocotyledonous lily enzyme (952 amino acids) displays 68% identity to its dicotyledonous (Arabidopsis thaliana) homologue. Our results show that the heterotrimeric enzyme is encoded by a single gene that gives rise to three polypeptides following posttranslational proteolysis. The enzyme is ubiquitously expressed, suggesting that it has a general function such as processing or degrading N-glycans.
Sasaki
Akiko
A
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Ishimizu
Takeshi
T
Hase
Sumihiro
S
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D027762:Lilium / Q000737:chemistry / Q000235:genetics*; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D013379:Substrate Specificity
D008361:Mannosidases; C119276:endo-beta-mannosidase