Endoplasmic Reticulum Kifunensine-Resistant α-Mannosidase Is Enzymatically and Immunologically Related to the Cytosolic α-Mannosidase

Abstract

Studies were undertaken to evaluate the relationship of the recently described (S. Weng and R. G. Spiro, 1993,J. Biol. Chem.268, 25656–25663) rat liver kifunensine (KIF)-resistant mannosidase (ER mannosidase II) to the mannose-trimming enzyme of cytosol. We observed that the ER mannosidase II manifests a large number of catalytic and immunological properties similar to those of the cytosolic α-mannosidase, which contrast with the quite different characteristics of the KIF-sensitive enzyme (ER mannosidase I). In addition to a mutual resistance to KIF inhibition, the cytosolic enzyme and ER mannosidase II have comparable susceptibility to blocking by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, and the latter agent was found to function effectively bothin vitroandin vivo.The cytosolic and ER II mannosidases were alike in specifically excising the terminal mannose of the α1,6-linked chain of Man9GlcNAc to yield Man8GlcNAc isomer C; in preferentially hydrolyzing polymannose–GlcNAc1over polymannose–GlcNAc2substrates; and in cleavingp-nitrophenyl α-D-mannoside. An immunological cross-reactivity between cytosolic mannosidase (Mr105 kDa) and ER mannosidase II (Mr82 kDa), neither of which is N-glycosylated, was established, suggesting that the latter is translocated posttranslationally into the lumen of the ER compartment in which we found it to be present as a soluble protein. Since antibodies directed against a sequence near the C-terminal end of the cytosolic enzyme reacted with ER mannosidase II while those against a sequence close to the N-terminus did not, it is likely that a proteolytic cleavage of the latter segment takes place during or after translocation. The absence in ER mannosidase II of the pronounced cobalt activation of the cytosolic enzyme suggests that the portion of the polypeptide chain removed during the 105- to 82-kDa conversion includes the binding domain for this ion.