Modeling human congenital disorder of glycosylation type IIa in the mouse: conservation of asparagine-linked glycan-dependent functions in mammalian physiology and insights into disease pathogenesis

Abstract

The congenital disorders of glycosylation (CDGs) are recent additions to the repertoire of inherited human genetic diseases. Frequency of CDGs is unknown since most cases are believed to be misdiagnosed or unrecognized. With few patients identified and heterogeneity in disease signs noted, studies of animal models may provide increased understanding of pathogenic mechanisms. However, features of mammalian glycan biosynthesis and species-specific variations in glycan repertoires have cast doubt on whether animal models of human genetic defects in protein glycosylation will reproduce pathogenic events and disease signs. We have introduced a mutation into the mouse germline that recapitulates the glycan biosynthetic defect responsible for human CDG type IIa (CDG-IIa). Mice lacking the Mgat2 gene were deficient in GlcNAcT-II glycosyltransferase activity and complex N-glycans, resulting in severe gastrointestinal, hematologic, and osteogenic abnormalities. With use of a lectin-based diagnostic screen for CDG-IIa, we found that all Mgat2-null mice died in early postnatal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors. Mice deficient in complex N-glycans exhibited most CDG-IIa disease signs; however, some signs were unique to the aged mouse or are prognostic in human CDG-IIa. Unexpectedly, analyses of N-glycan structures in Mgat2-null mice revealed a novel oligosaccharide branch on the "bisecting" N-acetylglucosamine. These genetic, biochemical, and physiologic studies indicate conserved functions for N-glycan branches produced in the Golgi apparatus among two mammalian species and suggest possible therapeutic approaches to GlcNAcT-II deficiency. Our findings indicate that human genetic disease due to aberrant protein glycosylation can be modeled in the mouse to gain insights into N-glycan-dependent physiology and the pathogenesis of CDG-IIa.

Truncated, InactiveN-Acetylglucosaminyltransferase III (GlcNAc-TIII) Induces Neurological and Other Traits Absent in Mice That Lack GlcNAc-TIII

“…Spectra from two wild-type and two homozygous mutants of each strain were distinguished only by the presence (in wild-type) and absence (in mutants) of glycans with masses predicted for bi-, tri-, or tetraantennary N-glycans with an unsubstituted HexNAc residue (Table I). Based on previous lectin blot analyses (7,12,19) and reports on similar analyses of kidney N-glycans from other mice (29,30), this "extra" HexNAc residue present only in wild-type N-glycans represents the bisecting GlcNAc. The spectra show a remarkable reproducibility in the profile of N-glycans synthesized by the kidneys of unrelated mice from the two strains (Table I) ⌬/⌬ mice lacking GlcNAc-TIII also did not have a growth-retarded phenotype in the genetic backgrounds examined (Fig.…”

“…The sections were acetylated twice in 0.1 M triethanolamine and acetic anhydride at room temperature for 10 min and washed twice with PBS, followed by dehydration in 30,50, 70, 90, 95, and 100% ethanol for 5 min each. After dipping in chloroform for 5 min at room temperature and in 0.2ϫ SSC for 1-2 min, prehybridization was carried out in a moist chamber with 50% formamide in 5ϫ SSC at 45°C for 3 h. For hybridization, 35 S-UTP-labeled antisense and sense RNAs encoding the Mgat3 gene coding region were prepared from a mouse Mgat3 cDNA (6) using T7 or SP6 RNA polymerase (Promega); probes were hydrolyzed; and hybridization, washing, and autoradiography were performed as described previously (26).…”

Inducing host protection in pneumococcal sepsis by preactivation of the Ashwell-Morell receptor

“…treatment with either AUS NA (5 U/kg) or PBS. Plasma was isolated for measurements of the abundance or activity of specific blood coagulation factors, as previously described (24,25). Measurements of glycoprotein abundance were obtained with anti-factor antibodies.…”

Creation and implications of a phenome-genome network

“…30,31 Mice homozygous null for Mgat2 show early post-natal lethality and a number of other defects. 32 Decreases in Tnnt1 and increases in Synj1 have been found to be markers for stem cell differentiation. 22,23 Our relations link these four genes and others to aging; it is possible subtle changes in expression of these genes may impact aging of an organism.…”