Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder

Abstract

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Keywords: MAN2C1; congenital disorder of deglycosylation; free oligosaccharides; neurodevelopmental disorder.

Figure 1

Metabolism of free oligosaccharides (fOSs) in mammalian cells fOSs are either generated during the ERAD pathway following the action of NGLY1 and/or via the hydrolysis of lipid-linked oligosaccharide by OST (LLO pathway). Once in the cytosol, ENGase cleaves the chitobiose core to form Gn1 fOSs with a single GlcNAc at their reducing end. These fOS species are then processed by the activity of the cytosolic α-mannosidase (MAN2C1) leading to the specific isomeric Man5GlcNAc1 structure. The catabolism of these species continues in the lysosomes by lysosomal α- and β-mannosidases.

Figure 2

Imaging features Brain MRI of individual 2 at age 5 years (A–D) showing a normal brain structure apart from minor hypoplasia of the inferior cerebellar vermis. Brain MRI of individual 5 at age 7 years (E–H) showing agenesis of the corpus callosum, extensive interhemispheric cysts type 2C (asterisk in E and G), polymicrogyria (arrows in G and H), and brainstem and cerebellar vermis hypoplasia. Brain MRI of individual 3 at 26 gestational weeks (I–K) showing partial agenesis of the corpus callosum (white arrows in I), 3^rd ventricular floor nodule compatible with hypothalamic hamartoma (white ar in I), cerebellar vermis hypoplasia (black arrow in I), Z-shaped brainstem (solid white arrow in I), irregular deep folding of the right frontal lobe (white arrow in J), bilateral ventricular dilation (asterisk in J), small cerebellum, subependymal heterotopia of the left occipital horn (black ar in K), and bilateral ventricular dilation (asterisks in K). Brain MRI of individual 4 at 29 gestational weeks (L and M) showing partial agenesis of the corpus callosum (white arrows in L), a large nodule in the floor of the 3^rd ventricle compatible with a hypothalamic hamartoma (white ar in L), hypoplasia of the cerebellar vermis (black arrow in L), a Z-shaped hypoplastic brainstem (solid white arrow in L), bilateral subependymal heterotopia (black ar in M), and a very abnormal bilateral gyration suggestive of polymicrogyria mainly in the fronto-parietal regions (box in M). Brain MRI of individual 6 at 1 month of age (N–P) showing bilateral perisylvian polymicrogyria (box in N and O) as well as diffusely abnormal white matter, thin corpus callosum, and inferior vermis hypoplasia (arrow in P).

Figure 3

Brain histology at 30 weeks gestation (individual 4) (A) Left cerebral hemisphere (internal view) with partial posterior corpus callosum agenesis (asterisk) and posterior radial sulci (black arrow). (B) Right cerebral hemisphere with marked gyration with excess of sulci. (C) Right frontal polymicrogyria (black arrows). (D) Lower view of the brain with hypothalamic hamartoma (black arrow), left arhinencephaly (asterisk), and arachnoid cyst (black ar). (E) Right predominantly occipital ventricular dilatation (asterisk) and subependymal nodules (black arrows). (F) Subependymal periventricular heterotopic nodules of gray matter (asterisk). (G) Hypoplastic and fragmented appearance of bulbar olives (black arrows). (H) Fragmented cerebellum deep nuclei (black arrows). (I) Sagittal section of the right eye: retinal coloboma (black arrow). (J) Amplification of retinal coloboma.

Figure 4

MAN2C1 variants localization and in silico analysis (A) Schematic representation of MAN2C1 variants (GenBank: NM_006715.3 and NP_006706.2) along the gene and predicted functional protein domains (not at scale). Exons are represented as white rectangles. Start and stop codons are indicated by black triangles. Each individual is represented by a unique colored triangle: red, 1 and 2; orange, 3 and 4; purple, 5; blue, 6. Domains are represented below respective coding exons, as colored rectangles: pink, glycoside hydrolase family 38 N-terminal domain (Glyco_hydro_38N); green, glycoside hydrolase family 38 central domain (Glyco_hydro_38cen); blue, glycoside hydrolase family 38 C-terminal domain (Glyco_hydro_38C); brown, glycoside hydrolase family 38 C-terminal beta sandwich domain (GH38C). Only domains predicted by the InterPro database were included in this figure. (B) Conservation analysis of amino acids surrounding each missense variant by the Clustal X color code (affected residues are identified above). In silico mutagenesis visualization of missense variants following protein structural modeling. MAN2C1 tetramer structure is represented with the functional domains colored as above (A). Mutated amino acids are superimposed. Further variant description can be found in Table S2.

Figure 5

Individuals with mutations in MAN2C1 show a defect in fOS processing (A) Representative cropped immunoblotting of MAN2C1 in control and proband-derived fibroblasts (I1, I2, and I5). β-actin staining was used as control for gel loading and for the quantification. (B) Quantification of Man2C1 protein expression in control and proband-derived fibroblasts. Values are means of three independent experiments. (C) HEK293T were transfected with the indicated constructs driven by two different promoters (pCMV and pEF6HisB) and subjected to MAN2C1 immunoblot (top). The indicated cells were lysed and then centrifuged, and the supernatant was used to perform enzymatic assays (middle and bottom) with 4-methylumbelliferyl α-D-mannopyranoside (4MUMan) as substrate. The release of the 4-methylumbelliferone (4MU) was measured at 460 nm for the indicated time-points. Data were normalized to WT MAN2C1 activity in pCMV. Values are means ± SEM of four independent experiments. (D) fOS HPLC analysis in fibroblast control cell line compared to the different proband-derived fibroblasts (I1, I2, and I5) after 1 h of radioactivity labeling with [2-³H] Mannose (left) and after a chase period of 2 h (middle) or 4 h (right). In these chromatograms, fOSGn2 species are shown in gray and fOSGn1 in white. The specific product of the MAN2C1 enzyme, the fOS M5Gn1, is in black. (E) Immunoblot analysis of Man2C1 protein abundance in HAP1 control and KO MAN2C1 cell lines. β-actin staining was used as control for gel loading and for the quantification. (F) fOS HPLC analysis in WT-HAP1 compared to the MAN2C1-KO HAP1 cells after 1 h of radioactivity labeling with [2-³H] mannose (left) and after a chase period of 2 h (right). In these chromatograms, fOSGn2 species are shown in gray and fOSGn1 in white. The specific product of the MAN2C1 enzyme, the fOS M₅Gn₁, is in black. G1M9 indicates oligosaccharides possessing one Glc and nine mannose residues. M9-M5 indicates oligosaccharides with five to nine mannose residues. Variants are annotated according the reference sequences GenBank: NM_006715.3 and NP_006706.2.

Figure 6

Pathogenicity of the MAN2C1 variants (A) Representative cropped immunoblot of MAN2C1 in HAP1 control and KO MAN2C1 cell lines infected by the empty vector and by different constructs containing WT MAN2C1 or the variant forms of MAN2C1 (p.Gly203Arg, p.Arg768Gln, and p.Cys871Ser) found in MAN2C1-deficient individuals. β-actin staining was used as control for gel loading and for the quantification. (B) Quantification of MAN2C1 protein abundance in infected HAP1 control and MAN2C1-KO cells. Values are means of three independent experiments. (C) fOS HPLC analysis after 1 h of labeling with [2-³H] mannose and after a chase period of 2 h on infected HAP1 cells lines: WT and MAN2C1-KO HAP1 cells infected by empty vector (respectively left and middle) and MAN2C1-KO HAP1 cells complemented with the WT form of MAN2C1 (right). (D) fOS HPLC analysis after 1 h of radioactivity labeling with [2-³H] mannose and after a chase period of 2 h on infected KO MAN2C1 HAP1 cell lines complemented with the different variants from the individuals under study: p.Gly203Arg (left), p.Arg768Gln (middle), and p.Cys871Ser (right). In all chromatograms, fOSGn2 species are shown in gray and fOSGn1 in white; fOS M₅Gn₁, the specific product of the MAN2C1 enzyme, is in black. In this experiment, MAN2C1 expression was under the control of the CMV promoter. G1M9 indicates oligosaccharides possessing one glucose and nine mannose residues. M9-M5 indicates oligosaccharides with five to nine mannose residues.