Cosmc regulates O-glycan extension in murine hepatocytes

Abstract

Hepatocytes synthesize a vast number of glycoproteins found in their membranes and secretions, many of which contain O-glycans linked to Ser/Thr residues. As the functions and distribution of O-glycans on hepatocyte-derived membrane glycoproteins and blood glycoproteins are not well understood, we generated mice with a targeted deletion of Cosmc (C1Galt1c1) in hepatocytes. Liver glycoproteins in WT mice express typical sialylated core 1 O-glycans (T antigen/CD176) (Galβ1-3GalNAcα1-O-Ser/Thr), whereas the Cosmc knockout hepatocytes (HEP-Cosmc-KO) lack extended O-glycans and express the Tn antigen (CD175) (GalNAcα1-O-Ser/Thr). Tn-containing glycoproteins occur in the sera of HEP-Cosmc-KO mice but not in WT mice. The LDL-receptor (LDLR), a well-studied O-glycosylated glycoprotein in hepatocytes, behaves as a ∼145kD glycoprotein in WT liver lysates, whereas it is reduced to ∼120 kDa in lysates from HEP-Cosmc-KO mice. Interestingly, the expression of the LDLR, as well as HMG-CoA reductase, which is typically altered in response to dysregulated cholesterol metabolism, are similar between WT and HEP-Cosmc-KO mice, indicating no significant effect by Cosmc deletion on either LDLR stability or cholesterol metabolism. Consistent with this, we observed no detectable phenotype in the HEP-Cosmc-KO mice regarding development, appearance or aging compared to WT. These results provide surprising, novel information about the pathway of O-glycosylation in the liver.

Keywords: Cosmc; Tn antigen; glycoproteins; hepatocytes; liver.

Fig. 1

Generation and characterization of hepatocyte specific Cosmc-KO mice (HEP-Cosmc- KO). A) Scheme for breeding the HEP-Cosmc-KO mice liver utilizing Cosmc f/f and Alb-Cre+/+ strains. Box indicates the first-generation male mice with Cosmc deletion in hepatocytes. B) SDS-PAGE- immunoblot probed for endogenous Cosmc by resolving equal amounts of both WT and Cosmc-KO mice liver tissue lysate, genotype labeled (top). SDS-PAGE of RNA pol II loading control (bottom). n = 3. C) Left, whole-liver tissue lysates were assayed for T-synthase activity (n = 12, three independent experiments of n = 4). Right, β-hexosaminidase activity was measured (n = 12, three independent experiments of n = 4). Data shows average of 12, +/− 1 standard deviation (SD). D) SDS-PAGE lectin blot showing Tn antigen expression. Left, liver whole tissue lysates, as indicated on top, treated with neuraminidase (removes sialic acid) or mock (untreated), resolved on SDS-PAGE, and probed with VVA lectin. n = 2. Right, stripped membrane from left probed for total protein which serves as loading control. E. Whole cell lysate, genotype labeled (top), was resolved on SDS-PAGE and stained with Coomassie. n = 3. F) Similar to E, whole tissue lysate resolved on BN-APAGE and stained with Coomassie. n = 3, MDa represents megadalton, error bars represent ±1 SD.

Fig. 2

Hep-Cosmc knock-out mice: Tn antigen on mice serum glycoproteins and the Cosmc-KO mice liver. A and B) SDS-PAGE-lectin blot probed with VVA (A) and PNA (B) utilizing mouse sera, genotype label (top), treated with or without NeuA (removes sialic acid), O-glycanase (removes core 1 O-glycans), and PNGase-F (removes all N-glycans) as indicated. C) SDS-PAGE-lectin blot showing Tn antigen as stained by VVA (right) on HPA lectin-purified materials from WT (control) and HEP-Cosmc-KO mouse sera. Left, Coomassie-stained material serves as input. n = 2. D and E) Similar to A and B, SDS-PAGE lectin blot probed with ConA and SNA. F) Similar to A–D, SDS-PAGE lectin blot probed for streptavidin-HRP (sAv) alone. G) IHC staining with ReBaGs6 antibody and VVA (both stain Tn antigen) for mouse tissue indicated (left), genotype labeled (top). n = 1. H) Liver IHC staining in G, boxed and numbered, shown in higher magnification, ReBags6 top and VVA bottom. IgM control and sAv show secondary reagents only. Scale bar on G represents 500 μm and H represents 100 μm. Dark spots indicates Tn staining. I) Immunofluorescence (IF) staining of mouse tissues, genotype labeled (left), treated with mock or neuraminidase (NeuA) as indicated (bottom) and probed with PNA. Scale bar represents 200 or 100 μm. NeuA treated, n = 2. J) Glycan structures and their binders and glycosidases are shown. Arrows show the point where the glycosidases release the glycans. Glycan symbols are provided (bottom). A and B, D–F) Untreated and PNGase F + NeuA treatment, n = 2. A) Untreated, n = 5, representative example of five WT and HEP-Cosmc-KO animals.

Fig. 3

LDL receptor and HMG-CoA reductase expression and glycosylation status. A) Mouse liver tissue lysates prepared from WT and Cosmc-KO mice, genotype labeled (top), were resolved on SDS-PAGE and probed for LDL receptor. Bottom, loading control probed for RNA-pol II. Right, ImageJ quantified and normalized with RNA-pol II, n = 3, average of 3, +/− 1 SD. B) Similar to A, SDS-PAGE immunoblot for HMG-CoA; bottom, RNA-pol II loading control. Right, ImageJ quantified and normalized with RNA-pol II, n = 3, average of 3, +/− 1 SD. C) SDS-PAGE immunoblots probed for LDL receptors for the liver lysates, as indicated (bottom), treated with enzymes as noted (top). Controls are untreated and refer to WT and hepatocyte specific Cosmc-KO mice liver lysates. Mock refers to untreated with enzyme. Bottom, RNA-pol II loading control. D) SDS-PAGE lectin blot for detection of complex N-glycans utilizing ConA showing functional glycosidase enzyme treatment. All experiments, 3 independent biological replicates (n = 3) except D (n = 1), representative examples shown.

Fig. 4

Serum sample analysis of hep-Cosmc-KO mice and COSMC-CDG patients. A and B) SDS-PAGE immunoblots of three mouse sera obtained from each genotype, treated with or without neuraminidase probed with PNA for normal O-glycans, and VVA for Tn antigen. Ponceau staining below serves as respective loading controls for A and B. Three biological replicates are shown. C and D) Equal amounts of serum obtained from COSMC-CDG patients, labeled on top, resolved on SDS-PAGE and probed with PNA (C) for normal O-glycans and VVA (D) for Tn antigen. M1 and M2 represent two male patients with COSMC-CDG, and F1 and F2 are their mother and maternal grandmother with heterozygous Cosmc mutation. n = 2.