Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous
fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex
and are required to generate complex N-glycans. However, lower eukaryotes such
Saccharomyces cerevisiae
contain
only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known
about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the
dimorphic fungus
Sporothrix schenckii
. Here, a membrane-bound α-mannosidase from
S. schenckii was solubilised
using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical
zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was
recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer
B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with
the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised
α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous
fungi,
S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases
is similar to that present in lower eukaryotes.
