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

Abstract

Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N-glycosylation. alpha-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 complex-type N-glycans, indicating the presence of an alternative pathway. Because alpha-mannosidase IIx (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N-glycan processing by generating MII/MX 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.

Fig. 1.

Survival of MII/MX double-nulls during embryonic and postnatal development and morphological analysis of double-nulls. (A) Survival rate of MII/MX double-nulls during embryonic and postnatal development. Man2a1(−/−)/Man2a2(+/−) mice were crossed, and embryos obtained from pregnant female mice and postnatal pups were genotyped. Each bar represents the relative numbers of surviving double-null embryos and neonates per total offspring. (B) Histological observation of neonatal mouse lung. Hematoxylin/eosin (H&E) staining of paraffin-embedded tissue sections demonstrated that the double-null neonatal lungs have less air space and thicker alveolar septa. (Scale bar, 200 μm.) (C) Electron micrographs of alveolar type II pneumocytes from wild-type and MII/MX double-null animals. Note the presence of large vacuoles and enlarged mitochondria in the double-nulls (mitochondria in both pictures are indicated by arrowheads). (Scale bar, 500 nm.) (D) Macroscopic observation of neonatal livers. Apparent abnormalities in the double-null liver are marked by asterisks. (E) Apoptosis analysis of cells from MII/MX double-null liver. Paraffin-embedded section of double-null E15 embryo displayed signs of massive apoptosis in liver, correlated with paler H&E staining. (F) Electron micrographs of hepatocytes from wild-type and MII/MX double-null embryos at E15. Mitochondria in both pictures are indicated by arrowheads. Note that the double-null hepatocyte contains large vacuoles and enlarged mitochondria, similar to observations of lung type II pneumocytes (C). (Scale bar, 2 μm.)

Fig. 2.

Lectin blot and lectin histochemistry of mouse embryos. (A) Lectin blot of membrane proteins from E15 embryos. Shown are an SDS/PAGE gel stained with Coomassie blue (CBB) for total protein and blots probed with lens culinaris agglutinin (LCA), leukocyte PHA, and E-PHA lectins. (B) Lectin histochemistry of E15 embryos. Paraffin sections of each embryo were probed with LCA or E-PHA lectins.

Fig. 3.

MALDI-TOF analysis of N-glycans from E15 embryos. (A) Wild type, (B) MX-null, (C) MII-null, and (D) MII/MX double-null. N-glycans were released from embryo homogenates by peptide N-glycosidase F digestion and were permethylated before MALDI-TOF analysis. Annotations are based on compositional information provided by MALDI molecular weights, complemented by collisional activation tandem MS and linkage analysis experiments (data not shown).

Fig. 4.

2D-HPLC analysis of N-glycans from E15 embryos. A summary of the quantitative glycan analysis is presented. The nomenclature of N-glycans has been described (19, 34). Original data of HPLC profiles are available upon request.

Fig. 5.

Enzymatic activity of recombinant MX in vivo and in vitro. (A) Rescue of complex-type N-glycan synthesis by α-mannosidases in MII/MX double-null fibroblasts. MII/MX double-null fibroblasts were transfected with vector alone (mock), vector encoding mouse MII cDNA (mMII), or vector encoding mouse MX cDNA (mMX). Transfected cells were probed with E-PHA lectin to detect complex-type N-glycans. (B) Enzymatic hydrolysis of N-glycan oligosaccharides with recombinant α-mannosidases. Each PA-tagged oligosaccharide, M5.1, M6.1, and H5.11 was incubated with a soluble form of the respective enzyme synthesized and secreted from COS-1 cells, and the digest was analyzed by amide-column HPLC. The elution position of the starting material and final product (100.2) is shown by the solid and dashed arrows, respectively.

Fig. 6.

The N-glycan processing pathway, including MX. This study demonstrates that MII and MX are isozymes hydrolyzing the hybrid-type H5.11 structure, and that either enzyme is required for formation of complex-type N-glycans. This study also excludes the likelihood of an alternative processing pathway catalyzed by a different enzyme capable of bypassing this critical processing step in MII/MX double-null mice.