Cloning and expression of mouse cytosolic alpha-mannosidase (Man2c1)

It is known that the neutral/cytosolic alpha-mannosidase (Man2c1) which can cleave alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannose residues, is stimulated by cobalt and is inhibited by furanose analogues swainsonine (SW) and 1,4-dideoxy-1,4-imino-d-mannitol (DIM). The enzyme is involved in the degradation of oligomannosides derived from dolichol intermediates and the degradation of newly synthesized glycoproteins. An immunological relationship has been demonstrated between the rat endoplasmic reticulum alpha-mannosidase and the cytosolic alpha-mannosidase. In fact antibodies raised against the soluble alpha-mannosidase recognized the membrane form of the ER alpha-mannosidase. A cDNA encoding the mouse cytosolic alpha-mannosidase was obtained by RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of 25 introns within the cytosolic alpha-mannosidase gene. The gene spans 11.5 kb from the major transcription initiation site to the RNA cleavage site. The transcription initiation site of mouse cytosolic alpha-mannosidase was mapped to 170 bases upstream of the ATG codon using 5' RACE. Northern blotting analysis revealed expression of a major transcript of 3.8 kb in all tissues examined. COS cells transfected with the cDNA showed a 20-fold increase in cytosolic alpha-mannosidase activity. This enzyme activity was stimulated by cobalt and inhibited by DIM and EDTA. Furthermore we demonstrated that the expressed enzyme was active towards the radiolabeled substrate Man9GlcNAc1 giving the final product Man5GlcNAc1 through the formation of Man8GlcNAc1 isomer C as intermediate.