Regulation of substrate specificity of plant alpha-mannosidase by cobalt ion: In Vitro hydrolysis of high-mannose type N-glycans by Co2+-activated Ginkgo alpha-mannosidase
2005
Woo, K.K. (Okayama Univ. (Japan)) | Kimura, Y.
In our previous study (Woo, K. K., et al., Biosci. Biotechnol. Biochem., 68, 2547-2556 (2004), we purified an alpha-mannosidase from Ginkgo biloba seeds; it was activated by cobalt ions and highly active towards high-mannose type free N-glycans occurring in plant cells. In the present study, we have found that the substrate specificity of Ginkgo alpha-mannosidase is significantly regulated by cobalt ions. When pyridyl-amino derivative of Man9GlcNAc2 (M9A) was incubated with Ginkgo alpha-mannosidase in the absence of cobalt ions, Mans-GlcNAc2-PA (M5A) having no alpha1-2 mannosyl residue was obtained as a major product. On the other hand, when Man9GlcNAc2-PA was incubated with alpha-manno-sidase in the presence of Co sup(2+) (1mM), Man8-5GlcNAc2PA were obtained as major products releasing alpha1-3/6 mannosyl residues in addition to alpha1-2 mannosyl residues. The structures of the products (Man8-5GlcNAc2-PA) derived from M9A by enzyme digestion in the absence of cobalt ions were the same as those in the presence of cobalt ions. These results clearly suggest that the trimming pathway from M9A to M5A is not affected by the addition of cobalt ions, but that hydro-lytic activity towards a 1-3/6 mannosyl linkages is stimulated by Co sup(2+). Structural analysis of the products also showed clearly that Ginkgo alpha-mannosidase can produce truncated high-mannose type N-glycans, found in developing or growing plant cells, suggesting that amannosidase might be involved in the degradation of high-mannose type free N-glycans.
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