Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

Abstract

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Fig 1. Generation of Ngly1- and Engase-deficient mice.

(A) Schematic views of the non-lysosomal degradation of free oligosaccharides (upper scheme) and the action of ENGase on glycoproteins (lower scheme). (B, C) A diagram of the targeting construct used to generate Ngly1-deficient mice (B) and Engase-deficient mice (C) (Figures Reproduced from ref. 27). In the Ngly1^−/− allele, from exon 11 to 12 were flanked by loxP site (filled triangles). Open triangles indicate FRT sites. In Engase^−/− allele, exon 5 was replaced with a PGK-Neo^r cassette. (D) Cytoplasmic fraction of MEF cells derived from Ngly1^−/− or Ngly1^+/+ (wild type, WT) embryos were immunoblotted with an antibody against mouse Ngly1. GAPDH was used as a loading control. Allow indicates Ngly1 and asterisks indicate non-specific bands.

Fig 2. Loss of Ngly1 causes ventricular septal defects (VSD) and the additional Engase deletion rescues the VSD phenotypes.

(A, B, G) Maximum intensity projection of heart μ-CT images of E16.5 embryo of wild-type (A), Ngly1^−/− (B), and Ngly1^−/−;Engase^−/− (G). White arrows in (B) indicates VSD. (C, D, H) Transverse section of wild-type (C), Ngly1^−/− (D), and Ngly1^−/−;Engase^−/− embryo (H) at E16.5 were stained with H&E. White arrows in (D) indicate VSD. Shown are representative sections (n = 3). Scale bar in (C), (D) and (H) indicate 200 μm. RA: right atrium, RV: right ventricle, LA: left atrium, LV: left ventricle. (E) Anemia was observed in Ngly1^−/− embryo at E16.5 (left panel). Right panel shows Ngly1^+/+ embryo at E16.5 (littermate of the left panel). (F) Edema was observed in Ngly1^−/− embryo at E16.5 (left panel). Right panel shows Ngly1^+/+ embryo at E16.5 (littermate of the left panel). Black arrowhead indicates edema. (I) Anemia was observed in Ngly1^−/−;Engase^−/− embryo at E16.5 (left panel). Right panel shows Ngly1^+/+;Engase^−/− embryo at E16.5 (littermate of the left panel). Representative images were shown.

Fig 3. Ngly1^−/−;Engase^−/− mice survive but still show several defects

(A) Survival curve of Ngly1^−/−;Engase^−/− male mice (n = 8), Ngly1^−/+;Engase^−/− male mice (n = 16), and Ngly1^+/+;Engase^−/− male mice (n = 7) in the C57BL/6 background. (B) Macroscopic comparison of 10 months-old Ngly1^−/−;Engase^−/− mice (upper panel) and Ngly1^+/+;Engase^−/− mice (lower panel). White arrow indicates a bent spine. (C) Hind-limb clasping of 5 months-old Ngly1^−/−;Engase^−/− mice. White arrowhead indicates clasped hind-limb. (D, E) Change of body weight of Ngly1^−/−;Engase^−/− mice, Ngly1^−/+;Engase^−/− mice, and Ngly1^+/+;Engase^−/− mice in the C57BL/6 background. (D) shows the results of male mice (n = 3~5) and (E) shows the results of female mice (n = 3~5). For statistical analysis, Student’s t-test was used. ***:p<0.001, **:p<0.01 (F) Ngly1^−/−;Engase^−/− mice developed coarse fur (n = 12). (G) Female Ngly1^−/−;Engase^−/− mice developed eye opacity (n = 8). Shown are representative appearances.

Fig 4. Engase-deletion improves the % survival and partially rescues the body weight loss of viable Ngly1^−/− mice.

(A) Survival curve for Ngly1^−/−;Engase^−/− mice (n = 9) and Ngly1^−/− mice (n = 29) in the C57BL/6 and ICR mixed background. (B, C) Change of body weight of Ngly1^−/+ mice and Ngly1^−/− mice in C57BL/6 and ICR mixed background. (B) shows the results of male mice (Ngly1^−/+:n = 3~4, Ngly1^−/−:n = 2~5) and (C) shows the results of female mice (Ngly1^−/+:n = 5~11, Ngly1^−/−:n = 2~5). (D) Hind-limb clasping of 4 weeks-old Ngly1^−/− mice in the C57BL/6 and ICR mixed background. (E, F) Change in body weight of Ngly1^−/+;Engase^−/− mice and Ngly1^−/−;Engase^−/− mice in the C57BL/6 and ICR mixed background. (E) shows the results of male mice (Ngly1^−/+;Engase^−/−:n = 4~8, Ngly1^−/−;Engase^−/−:n = 2~4) and (F) shows the results of female mice (Ngly1^−/+;Engase^−/−:n = 7~11, Ngly1^−/−;Engase^−/−:n = 2~3). For statistical analysis, Student’s t-test was used. ***:p<0.001. The number in panel B, C, E, and F indicates the relative ratio of weight of Ngly1^−/− mice to Ngly1^−/+ mice at 20 weeks of age.

Fig 5. N-GlcNAc hypothesis and the accumulation of N-glycoproteins in the cytoplasm of Ngly1^−/−;Engase^−/− MEF cells.

(A) Schematic representation of an N-GlcNAc hypothesis and therapeutic treatment of NGLY1-deficiency based on ENGase inhibition. In the absence of Ngly1, ENGase acts on some portions of unfolded glycoproteins to form N-GlcNAc proteins. The presence of an excess of N-GlcNAc proteins somehow results in detrimental effects on cells/mice. (B) Relative peak area intensity (%) of glycopeptides observed in Ngly1^−/−;Engase^−/− MEF cells. The peak area of each glycopeptide in wild-type MEF cells was identified as 100% and the relative ratio of the glycopeptide peak area in Ngly1^−/−;Engase^−/− MEF cells were calculated. Shown are the average values for two samples that were independently prepared.