Cosmc is an essential chaperone for correct protein O-glycosylation

Abstract

Cosmc is a molecular chaperone thought to be required for expression of active T-synthase, the only enzyme that galactosylates the Tn antigen (GalNAcalpha1-Ser/Thr-R) to form core 1 Galbeta1-3GalNAcalpha1-Ser/Thr (T antigen) during mucin type O-glycan biosynthesis. Here we show that ablation of the X-linked Cosmc gene in mice causes embryonic lethality and Tn antigen expression. Loss of Cosmc is associated with loss of T-synthase but not other enzymes required for glycoprotein biosynthesis, demonstrating that Cosmc is specific in vivo for the T-synthase. We generated genetically mosaic mice with a targeted Cosmc deletion and survivors exhibited abnormalities correlated with Tn antigen expression that are related to several human diseases.

Fig. 1.

Creation and characterization of ES cells with a null Cosmc and floxed Cosmc. (A) Schematic of targeted deletion strategy. (B) Southern blot confirmation of the genotypes of the constructs. (lanes 1 and 5) WT ES cells; (lanes 2, 3, 4, and 6) ES clones with deletion (Δ) of both Neo and Cosmc genes; (lane 7) ES clone with deletion of Neo gene only; (lane 8) ES clone with deletion of Cosmc only. (C) T-synthase activity assay of WT and Cosmc KO ES cells. Data show average of two replicates within one experiment. (D) IHC of ES cells with anti-Tn mAb (red represents Tn antigen expression). (Scale bar, 50 μm.) (E) RT-PCR of transcripts from WT and KO cells.

Fig. 2.

Creation and characterization of mosaic mice, Tn(+) cells in multiple tissues from mosaic mice. (A) Schematic of breeding strategies for generating Cosmc mosaic, heterozygous, and hemizygous KO mice. Mosaic genotypes are boxed in blue, heterozygous genotypes are boxed in green, and hemizygous genotypes are boxed in red. (B–D) IHC of the degree of Tn antigen expression from multiple organs, including heart (H), spleen (Sp), kidney (K), liver (Li), lung (L), small intestine (SMI), large intestine (LINT), stomach (S), and thymus (Th) for Group I Mosaics (B); Group II Mosaics (C); WT littermate controls (D). Brown represents Tn antigen expression in IHC. (Scale bar = 50 μm.) (E) T-synthase activity in splenocytes from WT animals and both Tn(+) and Tn(−) cells from mosaic animals. Data shows average of two replicates within one experiment.

Fig. 3.

Disruption of Cosmc in mice results in embryonic lethality and Tn antigen expression. (A) Embryos photographed at E9.5, E10.5, E11.5, E12.5, and E14.5 from WT, heterozygous, and hemizygous genotypes, demonstrating normal growth (WT) vs. hemorrhage and early death (heterozygous and hemizygous, respectively). (Scale bar, 1 mm.) (B) At E12.5, comparison of WT (healthy) vs. hemizygous (hemorrhagic, panel 2 and dead, panels 3 and 4). (Scale bar, 1 mm.) (C) T-synthase activity and β4 GalT activity in WT (2), heterozygous (3), and hemizygous (3) embryos. Data show the average of the number of noted embryos in parentheses ± 1 SD. (D and E) Embryonic extracts probed with PNA (D) and HPA (E) with and without Sialidase (S) treatment. The same amount of protein from the extracts of WT, heterozygous, and hemizygous embryos were analyzed in each lectin blot.

Fig. 4.

Whole-mount staining with Tn antibody (A) and lectins (B) revealed universal Tn antigen expression on the surface of every cell in Cosmc^−/y embryos at E10.5. (A1) Whole embryo from WT animal, (A2) whole embryo of Cosmc^−/y animal; green represents Tn antigen expression. (Scale bar, 500 μm.) (A3 and A4) Dissections of Cosmc^−/y embryo under fluorescent microscopy at 20× magnification. (Scale bar, 10 μm.) (B1–B6) Whole embryos photographed at E11.5 from WT (B1) and Cosmc^-/y (B2). E11.5 embryo sections from WT (B3 and B5) and Cosmc^−/y (B4 and B6) were stained with biotinylated lectins PNA (B3 and B4) and HPA (B5 and B6), detected with fluorescent streptavidin. (Scale bar, 1 mm.)