Phylogenetic survey of endomannosidase indicates late evolutionary appearance of this N-linked oligosaccharide...

Endo-alpha-D-mannosidase is a processing enzyme which in contrast to other glycosidases involved in the trimming of N-linked oligosaccharides of glycoproteins acts at an internal position by cleaving the linkage between the glucose-substituted mannose and the internal portion of the polymannose unit and thereby provides an alternate deglucosylating pathway. In order to evaluate at what stage in evolution this unusual enzyme first emerged, we have carried out a phylogenetic survey of its distribution among a broad group of eukaryotes ranging from unicellular organisms to highly developed animals and plants, all of which are known to have the capacity to N-glycosylate proteins and subsequently trim the nascent glucosylated polymannose oligosaccharides. It became evident from enzyme assays and in vivo studies that endomannosidase is limited in its distribution to members of the chordate phylum, including placental and marsupial mammals, birds, reptiles, amphibians, and fish, with the single except of the Mollusca in which it was detected in three distinct classes. The enzyme's absence in all other invertebrates examined as well as in yeast, various protozoa and higher plants, stands in contrast to glucosidase II and alpha 1,2-mannosidase which were found to be present in all eukaryotes studied. The observation that endomannosidase activity was not present in insects was confirmed by radiolabeling experiments with Sf9 cells in culture. These cells, which are widely employed for the expression of mammalian genes, were in distinction to mouse cells unable to circumvent a castanospermine (CST)-induced glucosidase blockade. Moreover we observed that Tetrahymenae, which synthesize glycoproteins with truncated N-linked oligosaccharides, could not process these beyond the Glc3Man5GlcNAc2 stage in the presence of CST. The late appearance of endomannosidase during evolution suggests a need for an alternate deglucosylation route in higher animals which parallels the development of elaborate complex N-linked oligosaccharides. Such carbohydrate units are believed to carry out vital biological functions and deglucosylation is a prerequisite to the further processing steps required for their formation.