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Glycobiology
Glycobiology
glycob
Glycobiology
0959-6658
1460-2423
Oxford University Press

39216105
10.1093/glycob/cwae069
cwae069
Original Article
AcademicSubjects/SCI01000
Cosmc regulates O-glycan extension in murine hepatocytes
Aryal Rajindra P Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Noel Maxence Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Zeng Junwei Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Matsumoto Yasuyuki Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Sinard Rachael Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Waki Hannah Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Erger Florian Institute of Human Genetics, University Hospital Cologne, Faculty of Medicine, University of Cologne, Kerpenerstr. 34, Cologne 50931, Germany
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany

Reusch Björn Institute of Human Genetics, University Hospital Cologne, Faculty of Medicine, University of Cologne, Kerpenerstr. 34, Cologne 50931, Germany
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany

Beck Bodo B Institute of Human Genetics, University Hospital Cologne, Faculty of Medicine, University of Cologne, Kerpenerstr. 34, Cologne 50931, Germany
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany

https://orcid.org/0000-0002-8918-5034
Cummings Richard D Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States

Corresponding authors: Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 11087 - 3 Blackfan Circle, Boston, MA 02115, United States. Email: rparyal@bidmc.harvard.edu (R.P.A.) and rcummin1@bidmc.harvard.edu (R.D.C.)
Rajindra P. Aryal and Richard D. Cummings co-senior authors.

Present address: Medical Research Institute, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, 510080 Guangzhou, China.

Present address: Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States.

10 2024
31 8 2024
31 8 2024
34 10 cwae06924 6 2024
20 8 2024
29 8 2024
13 9 2024
© The Author(s) 2024. Published by Oxford University Press.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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.

Cosmc
glycoproteins
hepatocytes
liver
Tn antigen
National Institute of Health R01GM068559 U01CA168930
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pmcIntroduction

Hepatocytes provide many blood glycoproteins and synthesize an immense array of secreted and membrane glycoproteins (Schulze et al. 2019). Secreted glycoproteins include blood clotting factors, plasminogen, ceruloplasmin, α-fetoprotein, transferrin and receptors including the low-density lipoprotein receptor (LDLR) and other family members (Wang et al. 2019). Glycosylation represents a key post-translational modification, that in general is required for normal animal development and cellular functions (Reily et al. 2019; Riley et al. 2020; Thompson and Wakarchuk 2022; Varki et al. 2022; Cummings 2024). The importance of glycosylation of hepatocyte glycoproteins is evident in patients with liver diseases (Blomme et al. 2009), as well in patients with congenital disorders of glycosylation (CDG), which is often associated with steatosis and fibrosis (Lipinski et al. 2021). However, much remains to be learned about the functions of glycans in hepatocyte glycoproteins.

A common modification of membrane and secreted mammalian glycoproteins in liver and all organs is O-glycosylation of Ser/Thr residues (O-GalNAc glycosylation) (Bergstrom and Xia 2013; Steentoft et al. 2013; Bagdonaite et al. 2021; Brockhausen et al. 2022). Typically, such O-glycans are extended with galactose by the T-synthase (C1Galt1) to generate the common core 1 O-glycan (T antigen/CD176) Galβ1–3GalNAcα1-O-Ser/Thr (Ju et al. 2008a; Ju et al. 2011), that can be further extended to generate a vast repertoire of thousands of O-glycans found in mammalian glycoproteins (Xia et al. 2005; Jin et al. 2017; Gupta et al. 2020). The expression of the most common type of O-glycans, the core 1 O-glycans and the T-synthase responsible for its synthesis is under the control of a specific molecular chaperone Cosmc (C1Galt1C1) which is required for T-synthase folding and stability (Ju and Cummings 2002; Aryal et al. 2010; Wang et al. 2010; Aryal et al. 2012). Loss of Cosmc in cells leads to the loss of extended O-glycans and collapse of the O-glycome to the Tn antigen GalNAcα1-Ser/Thr (and in some cases co-expression of the sialyl-Tn antigen NeuAcα2-6GalNAcα1-Ser/Thr) (Wang et al. 2010; Steentoft et al. 2013), in a similar fashion to targeted loss of the T-synthase (Xia et al. 2004). We recently found patients with a hypomorphic mutation in the X-linked COSMC, which leads to production of Tn antigen and associated pathologies, including renal failure (Erger et al. 2023), thus further heightening our interest in the roles of O-glycans in different cells and tissues.

A major hepatocyte membrane glycoprotein is the LDL-receptor (LDLR), which contains multiple N- and O-glycans (Cummings et al. 1983; Pedersen et al. 2014). The O-glycans occur in the stem region and in the LA repeats in its LDL-binding domain (Pedersen et al. 2014; Wang et al. 2018). O-glycosylation is critical to the function of the LDLR, as the lack of O-glycans leads to dysfunctional surface expression and instability of the protein (Kingsley et al. 1986; Pathak et al. 1988; Seguchi et al. 1991; Wang et al. 2018). However, the roles of extended O-glycans are unclear. The ldlD mutant CHO cells are deficient in UDP-Gal/UDP-GalNAc 4-epimerase, and thus are deficient in both UDP-GalNAc and UDP-Gal, and cannot initiate O-glycosylation; as a consequence they are deficient in functional LDLRs (Kingsley et al. 1986). In those cells LDLR expression can be partly restored by feeding cells exogenous GalNAc, which can salvage UDP-GalNAc production, but function is more fully restored by a combination of both exogenous Gal and GalNAc. Such results suggest that extended O-glycan structures with galactose could be required for normal LDLR expression and function. However, a reservation in such studies is that addition of exogenous galactose to ldlD mutant CHO cells would also restore galactose on N-glycans and other classes of glycomolecules. In this regard, it is interesting that abnormal glycosylation of N-glycans on the LDLR can also cause its reduced expression in hepatocytes (Kamada et al. 2020).

In regard to overall protein O-glycosylation, alterations in expression of O-glycans through defects in specific members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GALNTs) enzymes that initiate O-glycosylation are associated with several liver-related dysfunctions. These enzymes are important in liver homeostasis, as their altered expression is associated with hepatocellular carcinomas and in regulating lipoprotein metabolism (Holleboom et al. 2011; Huang et al. 2015; Hussain et al. 2016; Khetarpal et al. 2016; Liu et al. 2017; Zilmer et al. 2020; Wang and Chen 2023).

To address the role of extended O-glycans in hepatocytes, we targeted deletion of Cosmc in hepatocytes (HEP-Cosmc-KO mice). Our studies indicate that loss of Cosmc leads to significant Tn expression in liver and in serum glycoproteins, and altered glycosylation of the LDLR but no significant change in LDLR expression. Interestingly, the phenotypes of the HEP-Cosmc-KO mice appear normal. Our studies provide novel information and raise new questions about the functions of extended O-glycans in hepatocytes.

Results

Generation and characterization of hepatocyte specific Cosmc-KO mice (HEP-Cosmc-KO)

We engineered a targeted deletion of Cosmc in murine hepatocytes by crossing the LoxP-flanked Cosmc (C1Galt1c1) mice (Wang et al. 2010) with Alb-Cre+/+ strain mice to generate HEP-Cosmc-KO mice. In such animals the albumin promoter, relatively specifically expressed in hepatocytes, was used to drive expression of Cre recombinase, resulting in deletion of Cosmc specifically in hepatocytes (Fig. 1A). We confirmed that the KO was efficient, as no significant Cosmc protein was detected in liver lysates from HEP-Cosmc-KO mice compared to WT animals (Fig. 1B). Immunochemical analyses described below also confirmed the specificity of the Cosmc deletion to 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.

In terms of phenotype the HEP-Cosmc-KO mice had no significant phenotypic difference from WT animals, either in overall weight, size, and appearance of their livers, lack of noticeable lipid (steatosis) in the livers of either strain, and no significant differences in WT versus HEP-Cosmc-KO mice in terms of fecundity, and longevity (Supplementary Fig. 1). Hepatic steatosis is a key feature of dysregulated cholesterol metabolism in hepatocyte-specific genetic lesions of HMG-CoA reductase (Nagashima et al. 2012). Blood counts (WBCs, RBCs, and platelets) for both WT and HEP-Cosmc-KO mice were in the normal range at all ages from 1 month to 1 year. Thus, remarkably, unlike other organs and tissues in mice for which Cosmc deletion results in observable phenotypes (Wang et al. 2010; Kudelka et al. 2016; Zeng et al. 2020; Zeng et al. 2021), loss of Cosmc in liver did not result in significant phenotypic differences under normal laboratory conditions.

Loss of T-synthase and Cosmc protein in HEP-Cosmc-KO mice

The loss of Cosmc in hepatocytes led to a significant loss of T-synthase enzyme activity in liver lysates compared to WT (Fig. 1C, left). As a control, the activity of β-hexosaminidase in such lysates was comparable in both extracts (Fig. 1C, right). Hepatocytes constitute the vast majority of cells in the liver and liver volume (>80%) (Gao et al. 2008). However, the remaining cells would not be affected by the targeted deletion of Cosmc, and these include hepatic stellate cells (HSCs), liver sinusoidal endothelial cells and resident macrophages such as Kupffer cells (KCs) (Endo and Makishima 2019), which also express Cosmc and T-synthase (see C1GALT1C1 in Human Protein Atlas). Thus, the results suggest that hepatocytes express a high level of T-synthase and the other cell types express disproportionately lower levels overall.

Expression of Tn antigen in liver glycoproteins in HEP-Cosmc-KO mice

To analyze the effect of Cosmc deletion on expression of O-glycans, we performed Western blotting with a variety of reagents. Analysis of liver lysates by SDS-PAGE and blotting with the plant lectin Vicia villosa agglutinin (VVA), which binds to the Tn antigen (Tollefsen and Kornfeld 1987), revealed staining of glycoproteins from the HEP-Cosmc-KO mice but not WT mice (Fig. 1D left; right- loading control). The expression of the Tn antigen was not significantly altered by the treatment of lysates with neuraminidase, which removes sialic acid from sialyl-Tn antigen (NeuAcα2-3GalNAcα1-Ser/Thr). Prior studies in multiple cell types have confirmed that neuraminidase can effectively remove the sialyl-Tn antigen and expose Tn antigen (Ju et al. 2008b). Additionally, our prior studies on deletion of Cosmc in intestinal epithelial cells of mice (IEC-Cosmc-KO) revealed no change in sialyl-Tn expression in epithelial cells in response to deletion of Cosmc (Kudelka et al. 2016). Together, these studies demonstrate that the sialyl-Tn antigen is not significantly expressed in hepatocytes of the HEP-Cosmc-KO mice. Overall protein expression as revealed in lysates were not significantly different between WT and HEP-Cosmc-KO mice based on SDS-PAGE analysis and Coomassie staining (Fig. 1E). We also analyzed liver lysates using blue native-agarose polyacrylamide gel electrophoresis (BN-APAGE) system, a method to analyze native protein complexes up to 6 MDa or more (Aryal et al. 2017). We did not observe any obvious differences between WT and HEP-Cosmc-KO mice in terms of native protein complexes as shown in BN-APAGE Coomassie staining (Fig. 1F). These results demonstrate that loss of Cosmc in hepatocytes results in expression of glycoproteins carrying the Tn antigen, but this loss of extended O-glycans does not appear to change overall protein homeostasis.

Expression of Tn antigen in serum glycoproteins and liver from HEP-Cosmc-KO mice

To further explore Tn antigen expression in liver, we analyzed serum glycoproteins from HEP-Cosmc-KO mice compared to WT. Multiple serum glycoproteins from HEP-Cosmc-KO mice were stained by VVA and this was not affected by neuraminidase or O-glycosidase treatments; the latter would remove the unmodified, core 1 disaccharide O-glycans (Brooks and Savage 1997). Additionally, VVA staining was not removed by PNGase F, which removes most of the N-glycans, confirming that the VVA binding is to the Tn antigen in O-glycoproteins (Fig. 2A and J). By contrast, staining with peanut agglutinin (PNA), which binds to the non-sialylated core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (Wu 1984), was observed in serum glycoproteins from WT mice, and the signal was removed with the use of O-glycosidase, thus demonstrating these are core 1 O-glycans (Fig. 2B and J). Interestingly, we also observed some PNA staining in HEP-Cosmc-KO mice serum analysis (Fig. 2B), indicating that those normal O-glycosylated proteins in sera were secreted from non-hepatocyte sources. Total protein staining in serum samples of both WT and HEP-Cosmc-KO mice were similar (Fig. 2C, left).

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.

To more directly explore the Tn-positive glycoproteins in serum, we enriched for those glycoproteins using immobilized Helix pomatia lectin (HPA), which tightly binds the Tn antigen (Do and Cummings 1992). Lectin blot analysis of these glycoproteins gave strong staining with VVA in glycoproteins of HEP-Cosmc-KO, but not those in WT (Fig. 2C, right). The sizes were discrete and ranged from ∼40 kDa to ∼270 kDa. These results demonstrate that a broad range of serum glycoproteins expressing the Tn antigen are present in the sera of HEP-Cosmc-KO mice but not WT animals.

We explored whether loss of Cosmc might affect other glycosylation pathways, e.g. N-glycosylation. To address this possibility, we stained total glycoproteins by lectin blotting with concanavalin A (ConA) and Sambucus nigra agglutinin (SNA); ConA binds to many high mannose-, hybrid- and complex-type N-glycans, and SNA binds to α2,6-sialylated N-glycans (Baenziger and Fiete 1979; Shibuya et al. 1987; Bojar et al. 2022; Cummings et al. 2022). We did not observe significant differences in expression of N-glycans or their sialylation as detected by these reagents in WT versus HEP-Cosmc-KO liver lysates; the N-glycans were appropriately sensitive to diminishment by PNGase F (Fig. 2D, E, and J; F- secondary control).

We further analyzed for Tn expression using immunohistochemical approaches of sectioned liver, lung, heart, spleen, and kidney, of WT and HEP-Cosmc-KO mice, with staining by ReBaGs6, a recombinant IgM that binds to the Tn antigen (Matsumoto et al. 2020), and by VVA (Fig. 2G and J). The insets of the liver sections stained with these reagents is expanded in Fig. 2H, showing brownish-red staining for Tn antigen. Both reagents demonstrated significant expression of the Tn antigen in hepatocytes, where regions of the visible central vein, hepatocytes, and sinusoids were observable (Fig. 2H). In contrast, no significant expression of the Tn antigen was found in non-liver specimens (Fig. 2G).

To observe the effect of Cosmc deletion on expression of normal core 1 O-glycans in the liver of HEP-Cosmc-KO mice, we used PNA in immunofluorescence studies of liver and other organs (Fig. 2I). As PNA recognizes the non-sialylated core 1 O-glycan, samples were treated with neuraminidase. In liver of HEP-Cosmc-KO mice we observed a diminished PNA staining compared to WT, consistent with loss of core 1 over a large percentage of cells, as would be expected if deletion occurred only in hepatocytes and not in other cell types. In contrast, we observed no significant differences in PNA staining of kidney, heart, and lung sections with or without neuraminidase. These results indicate widespread loss of normal core 1 O-glycans in liver compared to other tissues in the HEP-Cosmc-KO mice.

Altered O-glycosylation of the LDL-receptor without effect on its expression in HEP-Cosmc-KO mice

The LDLR is a major O-glycosylated membrane glycoprotein in hepatocytes and its O-glycans are required for normal functions (Kingsley et al. 1986; Kozarsky et al. 1988; Wang et al. 2018). During biosynthesis the LDLR is first observable as an N- and O-glycosylated glycoprotein of ~120 kDa in the ER, where the O-glycans are simply the Tn antigen and the N-glycans are high mannose-type, whereas the mature glycoprotein with complex-type N-glycans and extended O-glycans is >140 kDa (Cummings et al. 1983). We performed an analysis of the LDLR in lysates of liver from WT and HEP-Cosmc-KO mice. As shown in Fig. 3A, the size of the LDLR in WT liver lysates is ∼145 kDa, whereas its size is reduced to ∼120 kDa in lysates from HEP-Cosmc-KO mice. There was no significant difference in total LDLR from either strain as evidenced by similar intensities of staining in Western blots.

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.

A major consequence of dysregulated cholesterol metabolism in liver is elevation of the level of expression of HMG-Co A reductase, the rate limiting enzyme for cholesterol production (Schumacher and DeBose-Boyd 2021). Elevation in LDLR expression is associated with decreased expression of HMG-CoA reductase, and conversely, inhibition of the latter, e.g. as by statins, leads to elevations of expression of LDLR (Srivastava 2023). HMG-CoA reductase is an ER glycoprotein (∼100 kDa) (Edwards et al. 1980), with a high mannose-type N-glycan consisting of Man6-8GlcNAc2-Asn species (Liscum et al. 1983; Brown and Simoni 1984). We observed no significant changes in expression of HMG-CoA reductase in liver lysates from either WT or HEP-Cosmc-KO mice (Fig. 3B). Thus, the lack of extension and absence of core 1 O-glycans does not affect expression of either LDLR or HMG-CoA reductase.

To further assess glycosylation of the LDLR, we assessed how its size was influenced by N- and O-glycosylation using a variety of enzymes to deglycosylate the receptor. The murine LDLR has 3 potential sites of N-glycosylation (but perhaps only 2 sites are used), and has ∼20 sites for O-glycosylation (Pedersen et al. 2014). Removal of N-glycans by treatment with PNGase F reduced the size from ∼145 kDa to ∼130 kDa, whereas removal of N- and O-glycans by a combination of PNGase F, neuraminidase, and O-glycosidase reduced the size to ∼120 kDa (Fig. 3C). These results are consistent with the WT LDLR having multiple N- and O-glycans. In contrast, the LDLR in lysates from HEP-Cosmc-KO mice behaved as ∼130 kDa protein and removal of N-glycans reduced its size to ∼120 kDa. The size was unaffected by additional treatments with neuraminidase and O-glycosidase, consistent with the absence of extended O-glycans (Fig. 3C). We also examined the effect of these treatments on total liver lysates from WT cells using staining with ConA, a lectin that binds many types of N-glycans. Removal of N-glycans with PNGase F greatly reduced binding to liver lysates (a single band of the PNGase F remains) (Fig. 3D); whereas, treatments with neuraminidase and O-glycosidase were without significant effects. Together these results demonstrated that the only significant difference in LDLR in liver lysates between WT and HEP-Cosmc-KO mice was in expression of different O-glycans, as predicted. Additionally, the results indicate that altered O-glycosylation in HEP-Cosmc-KO mice does not impact the N-glycosylation pathway of the LDLR.

Serum glycoproteins from HEP-Cosmc-KO mice and patients with COSMC-CDG express the Tn antigen

Because in the above results we observed staining of glycoproteins by VVA using the sera of HEP-Cosmc-KO mice, we also examined the sera of individuals diagnosed with a deficiency of Cosmc, caused by heritable mutations in the X-linked COSMC gene. This genetic disorder is a congenital disorder of glycosylation (COSMC-CDG), which we recently described (Erger et al. 2023); females are carriers of this mutation, transmissible to male offspring. These male patients have a hypomorphic mutation leading to dramatic loss of Cosmc, but enough residual chaperone to allow a minor level of T-synthase capable of modifying many glycoproteins, though inefficiently. Interestingly, we also found that the mother of the children diagnosed with COSMC-CDG has some changes in O-glycosylation that are also found in offspring, perhaps due to X-skewing, such as loss of core 1 O-glycans on circulating IgA1 (Erger et al. 2023). However, we have not closely analyzed whether these patients generate serum glycoproteins with the Tn antigen.

To initiate these analyses of human sera we repeated the analysis of mouse sera from three individual mice. In serum from the three WT mice we observed a significant expression of glycoproteins expressing the core 1 O-glycan that binds to PNA, but only after desialylation by treatment with neuraminidase (Fig. 4A), confirming that the normal O-glycans in each mouse donor are sialylated core 1-type O-glycans. By contrast in serum from three different HEP-Cosmc-KO mice we observed a similar decrease in several of the PNA-staining glycoproteins (Fig. 4A). We observed no significant staining of sera from individual WT mice using the lectin VVA which detects the Tn antigen, either with or without neuraminidase; however, many glycoproteins in sera of individual HEP-Cosmc-KO mice were stained by VVA (Fig. 4B). These results further confirm that deletion of Cosmc in the hepatocytes of three different mice causes expression of serum glycoproteins carrying the Tn antigen, but not complete loss of glycoproteins with normal O-glycans stained with PNA. It is likely that those remaining PNA-positive glycoproteins in sera from HEP-Cosmc-KO mice were derived from non-hepatocyte sources.

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.

We next examined sera from two male patients (M1 and M2) with COSMC-CDG and the carriers of the X-linked mutation, mother (F1) and grandmother (F2). We observed that sera from M1 and M2, compared to F1 and F2, had less PNA staining (Fig. 4C). As expected, M1 and M2 but not F1 and F2 predominantly contained glycoproteins stained by VVA (Fig. 4D). Interestingly, the pattern of staining by VVA using sera of COSMC-CDG patients (M1 and M2) appeared to be dissimilar to that in the sera of HEP-Cosmc-KO mice (Fig. 4B compared to Fig. 4D). These results indicate that loss of Cosmc function, in either the HEP-Cosmc-KO mice or in individuals with COSMC-CDG, lead to expression of serum glycoproteins expressing the Tn antigen, as visualized by binding to VVA, although the pattern of staining is not highly similar.

In recent studies we found that patients with IgA nephropathy, in which IgA1 carries the Tn antigen, exhibit circulating immune complexes in their sera, in which anti-Tn IgM is complexed with the Tn-positive IgA1 (Matsumoto et al. 2022). Since HEP-Cosmc-KO mice produce Tn antigen-containing glycoproteins in serum, we explored the possibility of the formation of circulating immune complexes of IgG, IgM, or IgA with Tn antigen-containing glycoproteins in the serum of these mice using the BN-APAGE system. However, we observed no significant difference in circulating immune complexes of anti-Tn antibodies in sera of either WT and HEP-Cosmc-KO mice (Supplementary Fig. 2).

Discussion

Our studies demonstrate that Cosmc encodes a master regulator for the extension of O-glycans in hepatocyte-derived glycoproteins, as its loss in HEP-Cosmc-KO mice leads to expression of Tn-positive glycoproteins in liver and in sera. We also observe specific loss of extended O-glycans on the LDL-receptor in liver lysates of HEP-Cosmc-KO mice. Taken together, these data indicate that O-glycans in WT hepatocytes are largely core 1-type O-glycans and that loss of Cosmc leads to loss of their extension resulting in expression of the Tn antigen. Interestingly, loss of Cosmc in hepatocytes does not result in significant developmental changes in HEP-Cosmc-KO mice, including no apparent changes in cholesterol metabolism, as assessed by the levels of LDLR and HMG-CoA reductase, and appearance of the liver in aged animals.

We observed a significant change, however, in the molecular weight of the LDLR in liver lysates of HEP-Cosmc-KO mice, consistent with a loss of extended O-glycans. The functions of O-glycans on the biosynthesis and stability of the LDLR has been of considerable interest, as many studies have identified the essential nature of proper O-glycosylation for functional expression of LDLRs (Kingsley et al. 1986; Kozarsky et al. 1988; Wang et al. 2018). However, our study is the first to analyze the in vivo expression of LDLR in mice in which O-glycans expressed in hepatocytes are limited to only the Tn antigen. Prior studies indicated that many of the O-glycans on the LDLR are present in both the stem region and in the LA repeats important in binding LDL (Pedersen et al. 2014; Wang et al. 2018). Loss of GalNAc, the key monosaccharide for generating O-glycans, leads to loss of function in ldlD mutant CHO cells, which have a deficiency of UDP-Gal/UDP-GalNAc 4-epimerase activity essential in effective generation of UDP-GalNAc for O-glycan addition (Kingsley et al. 1986). However, the role of subsequent modification of O-GalNAc through galactose addition, was not completely clear, as addition of galactose supplementation can lead to restoration of galactose on both N- and O-glycans. Our results demonstrate that specific galactosylation of O-glycans and limitation of their structures to only the Tn antigen does not affect production or stability of the LDLR in hepatocytes of HEP-Cosmc-KO mice.

The lack of phenotypic consequences of Cosmc deletion on expression of the LDLR, suggest that the functions of O-glycans on the LDLR may be largely structural and limited. The most plausible interpretation is that the Tn antigen (GalNAc monosaccharide alone), which is present in specific regions or domains of the LDLR, is sufficient to maintain its protein conformational stability and efficient ligand binding. These results are consistent with normal expression of the LDLR in cell lines, e.g. Simple Cells, in which Cosmc has been deleted (Pedersen et al. 2014). However, in regard to a key role O-glycosylation of protein regarding presence or absence of O-GalNAc, differential effects are observed depending on the glycoprotein and the number or position of O-GalNAc residues. For example, there are many O-glycosylation sites in different glycoproteins that require the enzyme GalNAcT2 (Yang et al. 2023); heritable defects in this enzyme activity are associated with defects in high-density lipoprotein cholesterol (HDL-C) metabolism (Khetarpal et al. 2016). ApoC-III is derived from liver and has multiple O-glycosylation sites (Naber et al. 2023) and is partly regulated by GalNAcT2 O-glycosylation. Such roles of O-GalNAc have been noted in other proteins, such as fibroblast growth factor-23 (FGF23) (Topaz et al. 2004), and angiopoietin like-3 protein (Schjoldager et al. 2010), where O-GalNAc is required but its galactosylation does not appear to be required.

We observed that serum from HEP-Cosmc-KO mice, and from individuals with a mutation in COSMC (COSMC-CDG) contains glycoproteins with the Tn antigen and lacking galactose. Mammalian serum is known to contain an abundance of glycoproteins, e.g. up to ∼1500 in human serum (Dang et al. 2019). Many of these that are non-hormonal and non-immunoglobulin types that are derived from hepatocytes (Clerc et al. 2016). Thus, it is likely that Tn-positive glycoproteins in serum of HEP-Cosmc-KO mice arise from hepatocytes. In patients with COSMC-CDG, we observed a somewhat different pattern of Tn-positive glycoproteins in their sera, but nevertheless there were many present, similar in that regard to what we observed in the sera of HEP-Cosmc-KO mice. However, the cellular sources of the Tn-positive glycoproteins in these human sera specimens is unknown, but it is reasonable to speculate they may partly arise from the liver. The presence of circulating Tn-positive glycoproteins in sera of COSMC-CDG patients and HEP-Cosmc-KO mice raises several interesting issues concerning their potential clearance by carbohydrate-mediated recognition.

We did not observe circulating immune complexes in the sera of either WT or HEP-Cosmc-KO mice (Supplementary Fig. 2). We previously observed such immune complexes in patients with IgA nephropathy in which Tn-positive IgA1 is complexed with anti-Tn IgM (Matsumoto et al. 2022). While humans can generate antibodies to the Tn antigen, it is unlikely that HEP-Cosmc-KO mice would generate anti-Tn antibodies, as Tn would be seen as a normal self-antigen. Of course, we cannot rule out the existence of such immune complexes of low copy number of diverse sizes as well as complexes with very small glycoproteins which are beyond the detection limit of this system. Additionally, such immune complexes could be rapidly cleared by the liver or kidney from circulation. Alternatively, the HEP-Cosmc-KO mice may develop immune tolerance with Tn-antigen. These are interesting questions to be pursued in the future.

These results also raise the interesting question of potential clearance of serum glycoproteins through the Ashwell-Morell receptor (AMR) (asialoglycoprotein receptor) (Ashwell and Harford 1982; Grewal et al. 2008). This receptor is notable as an endocytic receptor for blood glycoproteins and platelets. The AMR recognizes glycans containing terminal β-Gal or β-GalNAc residues (Baenziger and Maynard 1980), which may be exposed by desialylation (Ashwell and Harford 1982), although it can also bind glycoproteins with terminal sialic acid linked to GalNAc in N-glycans (Siaα2,6-GalNAcβ1-R) (Park et al. 2005). Clearance of desialylated glycoproteins or platelets can occur either through infective processes, such as sepsis resulting in clearance of platelets (Grewal et al. 2008; Hoffmeister and Falet 2016), or through enzymatic removal by exogenous neuraminidases (Steer and Ashwell 1980). While the AMR has clear specificity for β-linked Gal/GalNAc residues over α-linked residues, there is limited information as to whether the receptor can recognize O-glycans with only GalNAc residues (Baenziger and Maynard 1980). Early studies indicated the possibility that the AMR could bind IgA1, which has exposed α-linked GalNAc residues in its hinge region (Stockert et al. 1982), but binding could also occur through the unusual N-glycans of IgA1 and IgA2 (Basset et al. 1999). In relation to this, GalNAc-containing RNA ligands, as for RNAi therapies, can be targeted to hepatocyte AMR (Springer and Dowdy 2018; Thangamani et al. 2021). However, these are typically multivalent GalNAc presentations, such as tri-GalNAc, and β-linked GalNAc, rather than α-linked GalNAc, may be important in those cases (Matsuda et al. 2015; Tai 2019).

A second receptor in liver is the C-type lectin CLEC4F expressed in Kupffer cells; this receptor is important in clearance of non-sialylated O-glycans on platelet glycoproteins (Liu et al. 2017). CLEC4F, like the AMR, binds glycans expressing terminal β-Gal or β-GalNAc residues (Yang et al. 2013). The macrophage galactose-type lectin MGL is also expressed by Kupffer cells and other immune cells, and recognizes GalNAc residues on glycoproteins (Deppermann et al. 2020; Tumoglu et al. 2023); however, unlike the AMR and CLEC4F, there is evidence that MGL can bind to the Tn antigen (Saeland et al. 2007; Singh et al. 2009). MGL is also important in the clearance of aged platelets (Deppermann et al. 2020). Together, the potential of AMR, CLEC4F, and MGL to recognize and bind Tn antigens on serum or membrane glycoproteins poses interesting questions to be studied in the future. However, the steady-state presence of such Tn-positive glycoproteins in sera of the HEP-Cosmc-KO mice and COSMC-CDG patients suggest that such receptors, even if capable of interacting with the Tn antigen to some extent, may not be efficient in removing Tn-positive glycoproteins from serum.

Nevertheless, there are alternative interpretations to explain the apparent stable presence of circulating Tn-positive glycoproteins in sera of Cosmc-KO mice and individuals with COSMC-CDG. Perhaps some glycoproteins are cleared from the circulation by AMR, CLEC4F, and MGL in hepatocytes or Kupffer cells, but the balance between synthesis and removal leads to the observed steady-state levels. Although possible, it should be recognized that clearance of their ligands by these receptors ordinarily is incredibly rapid and efficient, and in vivo studies reveal that serum levels of such glycoproteins disappear within a few minutes of entry into blood (Van Den Hamer et al. 1970; Park et al. 2003). In this regard it is noteworthy that mice generated to lack the AMR have normal circulating glycoprotein in sera, suggesting that the AMR is not greatly contributing to homeostasis and the balance of glycoproteins in serum under homeostasis (Tozawa et al. 2001). It is also possible that endogenously-produced Tn-positive glycoproteins in hepatocytes of HEP-Cosmc-KO mice are continuously binding to and inhibiting the AMR, thus preventing its ability to clear; this could result in serum accumulation of such Tn-positive glycoproteins. However, overall, based on protein staining, we did not find any differences in serum protein abundance in sera of WT mice compared to HEP-Cosmc-KO mice. As Tn-positive glycoproteins are continuously produced at high levels by hepatocytes, and despite their rapid clearance, their steady-state levels may be enough for us to observe them by Western blotting. Finally, the other possibility is that neither the AMR, CLEC4F, nor MGL have high affinity recognition and ability to clear circulating Tn-positive glycoproteins. In any case, the unusual presence of Tn-positive glycoproteins in sera of HEP-Cosmc-KO mice and COSMC-CDG patients warrants further studies on their potential interactions or clearance through liver receptor mechanisms.

In this regard that we have considerable evidence in cell-based studies that cells expressing the Tn antigen are not cleared by hepatocytes. For example, we generated B cell targeted deletion of Cosmc in mice (Zeng et al. 2020; Zeng et al. 2021), as well as targeted deletion in T cells (Cutler et al. 2019), and examined their presence in blood. For the B cell conditional Cosmc knockout mice (BC-CosmcKO), the blood contained circulating Tn-positive B cells with no evidence of their clearance by liver receptors (Zeng et al. 2020, Zeng et al. 2021). By contrast, in the T cell conditional Cosmc knockout (TCKO) mice we observed a quantitative loss of Tn-positive T cells in the periphery, though such cells were present in the thymus and released to the circulation (Cutler et al. 2019); thus, the clearance of Tn-positive T cells in those animals is enigmatic. It is likely that while circulating cells can access Kupffer cells that line the sinusoids, the AMR in hepatocytes is separated from the endothelial lining by the space of Disse (Burkel and Low 1966; Sawitza et al. 2009); thus, cellular access to hepatocytes is limited, although platelets can access to both cell types. The observation of circulating B cells that are Tn-positive in the BC-CosmcKO mice (Zeng et al. 2020, Zeng et al. 2021) suggest that their clearance by any Tn-antigen recognition pathway including the AMR in the liver is very limited.

Somewhat unexpectedly, we observed that loss of Cosmc in murine hepatocytes, which is required for expression of active T-synthase (Ju and Cummings 2002; Wang et al. 2010), results in a near complete loss of T-synthase activity in liver lysates, despite evidence that hepatocytes represent only ∼80% of the cells in liver (Bogdanos et al. 2013). The immunohistochemical data of liver from the HEP-Cosmc-KO mice (Fig. 2) indicate wide-spread expression of the Tn antigen and a lack of Sialyl-Tn antigen. Data in the Human Protein Atlas indicates that Cosmc (C1GALT1C1) is expressed at somewhat similar levels in all liver cells, including hepatocytes, Kupffer cells, and endothelial cells (https://www.proteinatlas.org/ENSG00000171155-C1GALT1C1/single+cell+type/liver). Such results suggest that the level of T-synthase protein and activity might be low in cells relative to hepatocytes, and perhaps the RNA expression levels do not strictly correlate with enzyme activity.

There are limitations in our study and issues to be addressed in future work. For example, the complete O-glycoproteomes of WT murine liver and that of HEP-Cosmc-KO mice might be interesting to define in the future and also as a comparison to the total plasma O-glycoproteome from both animal types. Such analyses could reveal perhaps those glycoproteins that might be missing or altered in expression in the blood of HEP-Cosmc-KO mice compared to WT, as well as providing a source for characterizing the sites of O-glycosylation using the Tn antigen as a simple marker of such modifications, as has been done for Simple Cell technologies (Steentoft et al. 2013). However, the complete glycoproteomics of serum glycoproteins is complicated by the wide differences in their abundance. In addition, it might be interesting to perform transfusion of serum from HEP-Cosmc-KO mice into WT mice and observe whether the transferred Tn-positive glycoproteins are differently cleared compared to WT serum glycoproteins in terms of their half-lives and uptake kinetics and kinetics. Finally, it might be interesting to examine the abilities of different receptors in liver, e.g. AMR, CLEC4A and MGL to bind to the Tn-positive glycoproteins in sera of the HEP-Cosmc-KO mice, and lysates of HEP-Cosmc-KO liver. It is also possible that the phenotype of the HEP-Cosmc-KO mice, while apparently normal during development, could be impaired in some way that would only be revealed by environmental challenge relative to innate or adaptive immune mechanisms. For example, we observed that people commonly contain circulating IgM to the Tn antigen, which can arise by infections with organisms that express the Tn antigen (Heimburg-Molinaro et al. 2013). It is possible that WT mice might mount a protective anti-Tn response to infections, whereas the HEP-Cosmc-KO mice might be unable to, as the HEP-Cosmc-KO mice can not generate protective anti-Tn antibodies in response to direct immunization, as was done earlier using Tn-positive cells and other materials (Avichezer et al. 1997). Other aspects of inflammation and disease responses could be altered in the HEP-Cosmc-KO mice. Clearly, our observations provide new insights into the roles of extended O-glycans in hepatocytes and serum glycoproteins and also raise many interesting questions about their recognition and functions of normal O-glycans in hepatocyte glycoproteins.

Materials and methods

Ethics approval

The Ethics Committee of the medical faculty of the University of Cologne has the study approval (ID 15-215) for the patients study as described (Erger et al. 2023).

Generation of hepatocyte-specific Cosmc-KO mice

Cosmcf/f female mice as defined (Wang et al. 2010), were crossed with male B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J (purchased from The Jackson Laboratory, JAX stock #003574) to generate Hepatocyte specific Cosmc-KO (HEP-Cosmc-KO) mice. The first generation of male mice were used in the study. HEP-Cosmc-KO mice line were co-housed with WT (floxed, y) under specific pathogen-free conditions (21.7 ± 0.6 °C, 45 ± 10% humidity, and 12-h light cycle 6 am–6 pm) at Harvard Medical School in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols (Beth Israel Deaconess Medical Center, Harvard Medical School).

Preparation of liver lysates and serum

Lysis buffer (10 mM HEPES pH 7.9, 100 mM KCL, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA pH 8.0, 1 mM DTT, 1.5 mM MgCl2, 20% Glycerol, 1 tablet of protease inhibitor (Roche, Ref#11836170001)/10 mL of lysis buffer). Each liver was perfused, followed by washing with 2X with chilled PBS; the liver was cut into small pieces with sharp blades on an ice-cold Petri dish. The product was washed 2× with 1 mL of cold PBS,. For each processed liver, 5 mL of chilled lysis buffer was used and dounced 20 times with Pestel A and B. The lysate preparation was further solubilized with Triton X-100 (0.5% final concentration), vortexed every 5 min for 30 min, and centrifuged at 21,000 × g for 10 min. The supernatant, referred to as “whole cell extract”, was collected, aliquoted, and snap-frozen at −80 °C.

Human serum was prepared and stored as described (Erger et al. 2023). For mice serum, collected blood (no heparin) was incubated at room temperature (RT) for 30 min and centrifuged at 12,000 rpm (14,000 × g) for 30 min. Carefully, the supernatant was collected, aliquoted, and stored at −20 °C or analyzed directly.

Immunoblot analysis

For SDS-PAGE immunoblots, whole cell extracts resolved on the SDS-PAGE system were transferred onto the membrane either using wet transfer or a Trans-Blot Turbo Transfer system (BioRad). Five percent non-fat milk in 1× TBST (50 mM Tris–HCl, pH 7.2, 150 mM NaCl containing 0.05% Tween-20) was used as a blocking reagent and blocked for 1 h at RT, followed by incubating the membrane overnight with primary antibody of interest prepared in 5% non-fat milk (1× TBST). Membranes were washed briefly and incubated with the HRP-conjugated secondary antibody (1:5000 in 5% milk in 1× TBST) for 1 h at RT. Signals were detected by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using an Amersham™ Imager 600 (GE Healthcare Life Sciences). Primary antibodies used in this study are the following- Cosmc (H-10, Santa Cruz #SC-271829, 1:200), RNA-Pol II-CTD-phospho-S2 (Abcam, ref# ab5095, 1:10,000), LDL receptor (Abcam, Ref# ab52818, 1:10 k), and HMG-CoA (Abcam, Ref# ab 174830, 1:1 K).

For SDS-PAGE lectin blots, we followed the manufacturer’s protocol (NEB). Briefly, lysates/serum were boiled in 1× Glycoprotein denaturing buffer (NEB) for 10 min. The boiled/denatured sample was either treated with mock or 1 μL of neuraminidase A (Neu A, Cat#P0722S) or a combination of Neu A and O-glycosidase (Cat#P0733S) or PNGase F (Cat#P0708S) for 2 h at 37 °C. SDS-PAGE lectin blot was performed by resolving ∼10 μg of the sample. For blocking, 5% BSA in 1× TBST was used for 1 h at RT followed by incubation with the following biotinylated lectins: peanut agglutinin (PNA, Cat#B-1075-5, diluted to 2 μg/mL), Concanavalin A (ConA, Cat#B-1005-5, diluted to 0.1 μg/mL), V. villosa agglutinin (VVA, Cat#B-1235-2, diluted to 1 μg/mL), in TBST overnight at 4 °C in a shaker. After washing two times for 5 min each with 1× TBST, HRP-labeled streptavidin (Cat#SA-5014-1, Vector Laboratories) was added at 1:10,000 dilution in 5% BSA 1× TBST and incubated for 1 h at RT. The membranes were washed 5 times for 5 min each and the signals were detected as described above.

Quantification of SDS-PAGE western blot

Western blot images were analyzed using ImageJ software. For normalized intensity calculation, intensities are divided by their respective loading controls.

Immunofluorescence analysis

Mice were euthanized with CO2 gas in accordance with AVMA guidelines and trans-cardially perfused with PBS containing 10,000 units/L of heparin (Sigma #3149) for 2 min (6 mL/min). Fixation of the tissues was performed with ice-cold 4% paraformaldehyde (PFA) (0.2 M PBS, pH 7.4) for 5 min at 5 mL/min. Organs were then removed, postfixed in 4% PFA overnight, transferred to PBS and stored at 4 °C until processing. Fixed organs were paraffin embedded and cut in 3 μm coronal sections with a microtome at the BIDMC Pathology Core Facility. Slides were then deparaffinized using a standard xylene/ethanol gradient protocol and placed in antigen retrieval solution (0.1 M citric acid and 0.1 M sodium citrate, pH = 6), incubated in a pressure cooker at boiling temperature for 3 min, and stored at 4 °C in TBS. Tissue sections were circled by hydrophobic PAP PEN and treated with denaturing buffer for 5 min at 95 °C. Sections were washed with TBS (20 mM Tris, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.2) 3× followed by 3 washes with TBS-tween 0.05% (TBST). After blocking with 3% BSA in TBS for 1 h at RT, tissues were washed three times with TBS and incubated with 25 μg/mL of PNA or VVA lectins-FITC conjugated (Vector Labs: VVA FL-1231; PNA FL-1071) in TBS-T for 1 h at RT in darkness with gentle shaking. For control glycosidase experiments, slides were pretreated with 200 U of NeuA in a final volume of 80 μL at 37 °C for 1 h prior to incubation with lectins. After the incubation with lectins, sections were washed with TBS-T 3 times. Sections were then washed 2 times with TBS-T, one time with TBS, and counterstained with Hoechst 33,342 diluted at 1:1000 in TBS for 10 min at RT in darkness. Sections were then washed 3 times with TBS and incubated with 0.1% Sudan Black B (Sigma, ref# 199664) in 70% ethanol for 5 min to reduce cell autofluorescence. After 2 washes with TBS, slides were mounted with a glass coverslip using Prolong Gold Antifade Mountant (ThermoFisher, P36930). After overnight curing the slides, image acquisition was performed on a VS120 Slide scanner from Olympus for low magnification. Images analysis was then performed using QuPath (v0.3.2) software.

Immunohistochemistry

Tissue sections were heated for 1 h using a 60 °C incubator, followed by deparaffinized with Xylene 3× for 10 min, dehydrated with 100% EtOH (2×), 95% EtOH (2×), and 70% EtOH (1×) for 2 min, and rehydrate in DW. Antigen retrieval was performed by heating slides in a pressure cooker for 10 min at 100 °C in citrate buffer (10 mM trisodium citrate, pH 6.0). After cooling down for ~1 h in the retrieval buffer, incubate the slides in 3% H2O2 in DW for 30 min. Tissue sections were blocked by 1% NGS in TBST overnight at 4 °C followed by washing with 1× TBST for 10 min each. Secondary reagents were prepared in 0.5% NGS in TBST and incubated with the slides for 1 h at RT, washed with TBST 3× for 10 min. The following primary and secondary reagents were used- ReBaGs6 and control mIgM (1 μg/mL), VVA (1 μg/mL), Goat anti-mIgM-HRP (1:200), and sAv-HRP (1:200).

Native complexes analysis

The blue native-agarose polyacrylamide gel electrophoresis (BN-APAGE)-immunoblot gels were prepared and processed as described (Aryal et al. 2017).

Enzyme assay

T-synthase activity and β-hexosaminidase activity were performed as described (Erger et al. 2023).

Necropsy analysis and hematology profile

Six mice (3 WT Cosmc-Floxed (male) and 3 hepatocyte specific Cosmc-KO (male), Hep-Cosmc-KO) ∼2 years of age were sacrificed and their blood was collected from the heart. Confirmed WT and Cosmc-KO mice were processed for necropsy. Briefly, the inner body part was exposed by cutting the skin, and mice were immediately placed into ∼20X volume of mouse body size 10% Formalin NB, and changed the formalin twice in 3–4 h intervals. After 36 h in the formalin, mice were ringed in water and placed in PBS; this item was delivered to RHC, Harvard Medical School. Blood cell analyses (WBC, RBC, and platelet cytograms) were conducted at the BIDMC Facility using animals of 1, 2, 3, 4, and 5 months of age for both WT Cosmc-Floxed (male) and Hepatocyte specific Cosmc-KO (male), Hep-Cosmc-KO).

BN-APAGE analysis for immune complexes on mice serum

BN-APAGE immunoblots were performed as described (Aryal et al. 2017). Four WT and parallel Hep-Cosmc-KO mice (∼6 weeks old) serum (2 μL each; stored at −20 °C) were thawed on ice and diluted to 398 μL of 1× PBS. Five μL of this preparation was mixed with blue native sample buffer just before running the gel. Transferred proteins onto PVDF membranes were blocked with 5% nonfat milk prepared in 1× TBST (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20) for 1 h at RT. Membranes were washed briefly with 1× TBST and incubated with the following secondary antibody conjugated with HRP, goat anti-mouse IgG (SouthernBiotech, 1:20,000), goat anti-mouse IgM antibody (SouthernBiotech, 1:10,000), goat anti-mouse IgA secondary antibody-HRP (62-6720, Thermo Fischer Scientific, 1:5000) prepared in 1×T BST 5% non-fat milk for 1 h at RT. Membranes were washed 5 × 5 min each and developed as described above.

HPA lectin enrichment for serum proteins

For enrichment, HPA-Ultralink beads were used, which were washed using washing buffer (50 mM Tris HCl (7.4), 1 M NaCl, 0.5% Triton X-100, 1.5 mM MgCl2). Beads were equilibrated in binding buffer (chilled PBS, 0.5% Triton X-100, 1 mM MgCl2, 1 mM CaCl2). Prepared beads were incubated with 13× diluted mice serum in binding buffer overnight at 4 °C (~30 rpm), and the beads were centrifuged at 500 × g for 2 min. Following centrifugation, beads were washed with an excess of binding buffer 5× and eluted with 50 μL of 1X sample buffer which was analyzed using SDS-PAGE-Lectin blot.

Mouse age analysis

Mice (at least 8 in each group of WT and Hep-Cosmc-KO) were maintained under normal conditions in mice facility (BIDMC-CLS, HMS), and explored for their life expectancy. The end date of the mice was noted, at which time either the mice needed to be euthanized or died. Mice age in days was counted and analyzed.

Supplementary Material

Aryal_et_al_Supplementary_cwae069

Acknowledgments and Funding

This work was supported by the National Institute of Health grants U01CA168930 and R01GM068559 to R.D.C., as well as by the HMS Center for Glycoscience. The authors thank the Neurobiology Imaging Facility (NIF) at Harvard Medical School, Boston, MA for Imaging. We thank Dr. Jamie Heimburg-Molinaro for manuscript editing and Sandra F. Cummings for resource management.

Author contributions

R.P.A. and R.D.C. contributed to the conception and design of the work, interpretation of data, and drafting and finalizing of the work. M.N., R.P.A., J.Z. handled the mouse work. R.P.A., M.N., J.Z., Y.M. contributed to the data acquisition, analysis, and writing the method section. R.S. contributed to acquiring the WB data (LDL receptor and Cosmc) and the activity assay. H.W. contributed to lectin enrichment and Western blot. F.E., B.R., and B.B.B. provided the COSMC-CDG patient serum. R.D.C. provided the funding. All authors reviewed and approved the manuscript for submission.

Rajindra Aryal (Conceptualization [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Supervision [equal], Writing—original draft [equal], Writing—review & editing [supporting]), Maxence Noel (Formal analysis [supporting], Investigation [supporting], Writing—review & editing [equal]), Junwei Zeng (Formal analysis [supporting], Investigation [supporting], Writing—review & editing [supporting]), Yasuyuki Matsumoto (Formal analysis [supporting], Investigation [supporting], Writing—review & editing [supporting]), Rachael Sinard (Formal analysis [supporting], Investigation [supporting], Writing—review & editing [supporting]), Hannah Waki (Formal analysis [supporting], Investigation [supporting], Writing—review & editing [supporting]), Florian Erger (Resources [equal], Writing—review & editing [supporting]), Björn Reusch (Resources [equal], Writing—review & editing [supporting]), Bodo Beck (Resources [equal], Writing—review & editing [supporting]), and Richard Cummings (Conceptualization [equal], Methodology [equal], Supervision [equal], Writing—original draft [equal], Writing—review & editing [equal], Funding).

Conflict of interest statement: The authors declare no competing interests.

Data availability

Data sets used and analyzed for this study will be made available upon reasonable request with no restrictions.
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