Overexpression of the Golgi‐localized enzyme α‐mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl‐N‐acetylchitobiose to tetramannosyl‐N‐acetylchitobiose in the N‐glycan‐processing pathway

Abstract

Keywords: a-mannosidase II; a-mannosidase IIx; Golgi; HPLC; N-glycan.The major biosynthetic pathway of N-glycans in higher organisms has been established [1]. In the endoplasmic reticulum (ER), a lipid-linked glucosylated high-mannose oligosaccharide is transferred from the dolichol donor to a protein acceptor. High-mannose oligosaccharides are then processed by a-glucosidases and a-1,2 mannosidases (MI) to produce Man 5 GlcNAc 2 (M 5 Gn 2 ). A key conversion of this oligosaccharide to complex-type oligosaccharides occurs in the medial Golgi, where N-acetylglucosaminyltransferase-I (GnT-I) adds N-acetylglucosamine to the Mana133Manb13 terminal of M 5 Gn 2 to form Gn 1 M 5 Gn 2 [2]. The Golgi a-mannosidase II (MII) then removes two mannosyl residues from Gn 1 M 5 Gn 2 to form Gn 1 M 3 Gn 2 [3], which are further modified to complex oligosaccharides by N-acetylglucosaminyltransferase-II (GnT-II), galactosyltransferase and sialyltransferases.The presence of an MII-related enzyme was suggested by studies on a human genetic disease, congenital dyserythropoietic anemia type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test). Although the primary gene defect of HEMPAS is heterogeneous [4±8], the biochemical phenotype is characterized as a failure in N-glycan processing. Furthermore, mutant mice in which the MII gene was inactivated by homologous recombination showed strikingly similar phenotypes to those exhibited by HEMPAS patients [9]. Although cells from MII-null mice completely lack MII activity and accumulate hybrid-type oligosaccharides, MII-null splenocytes and fibroblasts were found to synthesize complex-type oligosaccharides. This finding indicates that complex oligosaccharides can be synthesized by an MII-independent pathway, leading to the proposal of an alternative pathway and the prediction of an a-mannosidase, designated a-mannosidase III (MIII). MIII hydrolyzes two a-mannosyl residues in M 5 Gn 2 and produces M 3 Gn 2 , which is further converted to Gn 1 M 3 Gn 2 by GnT-I. Bonay and Hughes [10] found a novel a-mannosidase in rat liver. This enzyme, in a cobalt iondependent manner, hydrolyzed a132, a133, and a136 mannosyl linkages of N-glycans. As the MIII activity was not observed in the tissues of the MII-null mouse until cobalt was added, MIII is most certainly the same activity Eur.

Essential and mutually compensatory roles of α-mannosidase II and α-mannosidase IIx in N -glycan processing in vivo in mice

“…5A), indicating that full-length MX can rescue the complex N-glycan deficiency in double-null cells. In vitro enzyme assays using the soluble catalytic domain of MX also detected activity toward a GlcNAc 1 Man 5 GlcNAc 2 substrate in contrast to prior studies using a recombinant form of human MX (17). We recently identified alternatively spliced forms and sequence polymorphisms of human MX and confirmed that several of these transcripts encode enzymes active toward GlcNAc 1 Man 5 GlcNAc 2 , whereas a splice variant used in our previous study (17) was not active toward high-mannose oligosaccharides or GlcNAc 1 Man 5 GlcNAc 2 (H. Singh, T.O.A., M.N.F., and K.W.M., unpublished data).…”

“…Previous studies suggest that human MX is catalytically active and plays a role in N-glycan biosynthesis (16)(17)(18). When mouse Man2a2, the orthologue of human MAN2A2, was disrupted, MX-nulls were apparently normal, except that mutant males were subfertile (18).…”

The role of N-glycans in spermatogenesis

“…The synthesis of N-glycans initiates from the so-called high-mannose-type oligosaccharide, which is then trimmed by the processing glucosidases and mannosidases. This figure illustrates the reactions surrounding MII, including MX (Akama et al, 2002;Oh-eda et al, 2001) and the putative enzyme MIII (Chui et al, 1997). or complex-type N-glycan, this result demonstrates that highmannose-type N-glycans alone can not support embryonic development beyond E9.5-10.5 in the mouse.…”

“…Overexpression experiments with MX suggest that MX hydrolyzes Man 6 GlcNAc 2 to Man 4 GlcNAc 2 in the Golgi ( Fig. 1) (Oh-eda et al, 2001). However, the substrate specificity of MX has not been defined unambiguously by in vitro experiments due to its weak enzymatic activity (Oh-eda et al, 2001).…”

“…1) (Oh-eda et al, 2001). However, the substrate specificity of MX has not been defined unambiguously by in vitro experiments due to its weak enzymatic activity (Oh-eda et al, 2001). Thus MX may be active only in the Golgi membrane in cells.…”