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

Abstract

Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N-glycosylation. ␣-Mannosidase II (MII) is a key enzyme converting precursor high-mannose-type N-glycans to matured complex-type structures. Previous studies showed that MII-null mice synthesize complextype N-glycans, indicating the presence of an alternative pathway. Because ␣-mannosidase IIx (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N-glycan processing by generating MII͞MX double-null mice. Some double-nulls died between embryonic days 15.5 and 18.5, but most survived until shortly after birth and died of respiratory failure, which represents a more severe phenotype than that seen in single-nulls for either gene. Structural analysis of N-glycans revealed that double-nulls completely lack complex-type N-glycans, demonstrating a critical role for at least one of these enzymes for effective N-glycan processing. Recombinant mouse MX and MII showed identical substrate specificities toward N-glycan substrates, suggesting that MX is an isozyme of MII. Thus, either MII or MX can biochemically compensate for the deficiency of the other in vivo, and either of two is required for late embryonic and early postnatal development.gene knockout ͉ mutation N -glycosylation is the major form of posttranslational modification of newly synthesized proteins through the secretory pathway. The major biosynthetic steps for N-glycans in vertebrates have been established (1, 2). A key conversion of highmannose to complex-type oligosaccharides occurs in the medial Golgi, where GlcNAc-transferase I (GlcNAc-TI) adds a GlcNAc residue to form a hybrid-type N-glycan, GlcNAc 1 Man 5 GlcNAc 2 (3). Golgi ␣-mannosidase II (MII) then removes two mannosyl residues to form GlcNAc 1 Man 3 GlcNAc 2 (4, 5), which is further modified by GlcNAc-transferase II (GlcNAc-TII) (6) to form GlcNAc 2 Man 3 GlcNAc 2 , the precursor of complex-type Nglycans.When the gene encoding GlcNAc-TI was disrupted in mouse, GlcNAc-TI-null embryos died between embryonic day 9 (E9) and E10 because of defects in morphogenic processes, including the establishment of left-right asymmetry, vascularization, and neural tube formation (7,8). The observation that GlcNAc-TInull embryos could synthesize only high-mannose-type Nglycans demonstrates that high-mannose-type N-glycans by themselves cannot support embryonic development beyond E9-E10 in the mouse. On the other hand, mice lacking GlcNAc-TII were born and synthesized hybrid-type N-glycans, implying that hybrid-type N-glycans can support embryogenesis (9). However, GlcNAc-TII-null mice showed severe gastrointestinal, hematopoietic, osteogenic, and neuronal dysfunction, with phenotypes similar to those seen in human congenital disorders of glycosylation IIa (10, 11). This evidence indicates that hybrid-type N-glycans are not sufficient to maintain normal postnatal development in the mouse and in humans (12).Although MII catalyzes the step after GlcNAc-TI and before GlcNAc-TII (1, 2), MII-n...

Respiratory Distress and Neonatal Lethality in Mice Lacking Golgi α1,2-Mannosidase IB Involved in N-Glycan Maturation

“…Inactivation of ␣-mannosidase II (22,23,25), and ␣-mannosidase IIx (24,25) individually reduces overall complex N-glycan synthesis significantly, but the mice are viable, albeit with different consequences, causing anemia or male infertility, respectively. However, ablation of both genes completely abrogates complex N-glycan formation with the accumulation of hybrid structures (25,38).…”

“…However, ablation of both genes completely abrogates complex N-glycan formation with the accumulation of hybrid structures (25,38). The substantial loss of typical N-glycans primarily results in postnatal death within 2 days, and less frequently in embryonic lethality, with only a few mice surviving postnatally (ϾP21).…”

“…However, ablation of both ␣-mannosidase II and IIx completely abrogates complex N-glycan biosynthesis with the accumulation of hybrid structures, and causes hepatic and pulmonary ultrastructural defects. Although a few of these mice survive (ϾP21), the majority die within 2 days of birth and some die in utero (25). These animal models have provided insights into critical functions mediated by N-glycans that cannot be observed in cultured cells.…”

Purification and characterization of a novel thermoacidophilic and thermostable α-mannosidase from the digestive fluid of oil palm weevil Rhynchophorus palmarum (Coleoptera: Curculionidae) larvae

“…They are involved in the processing of newly formed N-glycans by modifying oligosaccharide structures linked to appropriate asparagine residues of proteins and thus influence their properties and bioactivity (Varki, 1993;Moremen, 2000;Akama et al, 2006). In the pharmaceutical industry, -mannosidases are currently used for treatment of mannosidosis, a congenital disorder of glycosides (CDG), by enzyme replacement therapy (Sun et al, 1999;Hirsch et al, 2003).…”

T Cell Receptor Signaling Co-regulates Multiple Golgi Genes to Enhance N-Glycan Branching

“…These data implicate TCR signaling-mediated enhancement of GlcNAc branching as a critical regulator of CTLA-4 surface retention and autoimmunity. In addition to the Mgat enzymes, Golgi ␣-mannosidase I (MI) and II (MII) are required for GlcNAc branching (15,19,20). MI acts upstream and MII acts downstream of Mgat1, steps required for the action of Mgat2 and production of bi-, tri-, and tetra-antennary N-glycans (see Fig.…”