Germline C1GALT1C1 mutation causes a multisystem chaperonopathy.

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Macromolecular Assemblies of the Mammalian Circadian Clock.

The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1δ (CK1δ) are present together in an ∼1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an ∼750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical ∼40-nm structures. In the cytoplasm, PERs, CRYs, and CK1δ were distributed into several complexes of ∼0.9-1.1 MDa that appear to constitute an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Single-particle EM of two purified cytoplasmic PER complexes revealed ∼20-nm and ∼25-nm structures, respectively, characterized by flexibly tethered globular domains. Our results define the macromolecular assemblies comprising the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.

Large-Scale and Site-Specific Mapping of the Murine Brain O-Glycoproteome with IMPa.

Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O-glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O-glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O-glycans. We developed two complementary approaches for the analysis of the total O-glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O-glycans on glycopeptides. We then applied these approaches to characterize the O-glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O-glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O-glycoproteome only partly overlap with those reported for the brain N-glycoproteome. These approaches will aid in identifying the novel O-glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O-glycosylation in the brain and other organs.

Antibodies from Lampreys as Smart Anti-Glycan Reagents (SAGRs): Perspectives on Their Specificity, Structure, and Glyco-genomics.

The repertoire of glycans expressed by individual cells and tissues is enormous, and various estimates indicate that thousands of different glycans and "glycan determinants" are critical for functional recognition by glycan-binding proteins. Defining the steady-state expression and functional impacts of the human glycome will require a concerted worldwide effort, along with the development of new immunological, genetic, chemical, and biochemical technologies. Here, we describe the generation of smart anti-glycan reagents (SAGRs), recombinant antibodies that recognize novel glycan determinants. The antibodies are generated by the sea lamprey (Petromyzon marinus), through immunization with glycoconjugates, cells, and even tissues. SAGRs represent a versatile immunological tool for defining the expression of glycans in cells and tissues. We also present a comparison of lamprey-derived anti-carbohydrate antibodies that have been characterized to date. Finally, we explore the unique glyco-genome of the lamprey itself as it compares to those of humans and mice and how it may relate to the lamprey's inherent capacity to produce antibodies to mammalian glycans.

Identification of distinct glycoforms of IgA1 in plasma from patients with immunoglobulin A (IgA) nephropathy and healthy individuals.

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and is histologically characterized by the deposition of IgA1 and consequent inflammation in the glomerular mesangium. Prior studies suggested that serum IgA1 from IgAN patients contains aberrant, undergalactosylated O-glycans, for example, Tn antigen and its sialylated version, SialylTn (STn), but the mechanisms underlying aberrant O-glycosylation are not well understood. Here we have used serial lectin separation technologies, Western blot, enzymatic modifications, and mass spectrometry to explore whether there are different glycoforms of IgA1 in plasma from patients with IgAN and healthy individuals. Although total plasma IgA in IgAN patients was elevated ∼ 1.6-fold compared with that in healthy donors, IgA1 in all samples was unexpectedly separable into two distinct glycoforms: one with core 1 based O-glycans, and the other exclusively containing Tn/STn structures. Importantly, Tn antigen present on IgA1 from IgAN patients and controls was convertible into the core 1 structure in vitro by recombinant T-synthase. Our results demonstrate that undergalactosylation of O-glycans in IgA1 is not restricted to IgAN and suggest that in vivo inefficiency of T-synthase toward IgA1 in a subpopulation of B or plasma cells, as well as overall elevation of IgA, may contribute to IgAN pathogenesis.

Identification of a novel protein binding motif within the T-synthase for the molecular chaperone Cosmc.

Prior studies suggested that the core 1 ß3-galactosyltransferase (T-synthase) is a specific client of the endoplasmic reticulum chaperone Cosmc, whose function is required for T-synthase folding, activity, and consequent synthesis of normal O-glycans in all vertebrate cells. To explore whether the T-synthase encodes a specific recognition motif for Cosmc, we used deletion mutagenesis to identify a cryptic linear and relatively hydrophobic peptide in the N-terminal stem region of the T-synthase that is essential for binding to Cosmc (Cosmc binding region within T-synthase, or CBRT). Using this sequence information, we synthesized a peptide containing CBRT and found that it directly interacts with Cosmc and also inhibits Cosmc-assisted in vitro refolding of denatured T-synthase. Moreover, engineered T-synthase carrying mutations within CBRT exhibited diminished binding to Cosmc that resulted in the formation of inactive T-synthase. To confirm the general recognition of CBRT by Cosmc, we performed a domain swap experiment in which we inserted the stem region of the T-synthase into the human ß4GalT1 and found that the CBRT element can confer Cosmc binding onto the ß4GalT1 chimera. Thus, CBRT is a unique recognition motif for Cosmc to promote its regulation and formation of active T-synthase and represents the first sequence-specific chaperone recognition system in the ER/Golgi required for normal protein O-glycosylation.

Acceptor specificities and selective inhibition of recombinant human Gal- and GlcNAc-transferases that synthesize core structures 1, 2, 3 and 4 of O-glycans.

BACKGROUND: Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior. METHODS: We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors. RESULTS: Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme-substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-d-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs. CONCLUSIONS: This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4. GENERAL SIGNIFICANCE: These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.

Tight complex formation between Cosmc chaperone and its specific client non-native T-synthase leads to enzyme activity and client-driven dissociation.

The interaction of the endoplasmic reticulum molecular chaperone Cosmc with its specific client T-synthase (Core 1 ß1-3-galactosyltransferase) is required for folding of the enzyme and eventual movement of the T-synthase to the Golgi, but the mechanism of interaction is unclear. Here we show that the lumenal domain of recombinant Cosmc directly interacts specifically in either free form or covalently bound to solid supports with denatured T-synthase but not with the active dimeric form of the enzyme. This leads to formation of a relatively stable complex of Cosmc and denatured T-synthase accompanied by formation of reactivated enzyme in an ATP-independent fashion that is not regulated by redox, calcium, pH, or intermolecular disulfide bond formation. The partly refolded and active T-synthase remains tightly bound noncovalently to Cosmc. Dissociation of T-synthase from the complex is promoted by further interactions of the complex with free forms of either native or non-native T-synthase. Taken together, these results demonstrate a novel mechanism in which Cosmc cycles to bind non-native T-synthase, leading to enzyme activity and release in a client-driven process.

Epigenetic silencing of the chaperone Cosmc in human leukocytes expressing tn antigen.

Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi ß3-galactosyltransferase that generates the core 1 O-glycan, Galß1-3GalNAcα-Ser/Thr, in glycoproteins. Dysfunctional Cosmc results in the formation of inactive T-synthase and consequent expression of the Tn antigen (GalNAcα1-Ser/Thr), which is associated with several human diseases. However, the molecular regulation of expression of Cosmc, which is encoded by a single gene on Xq24, is poorly understood. Here we show that epigenetic silencing of Cosmc through hypermethylation of its promoter leads to loss of Cosmc transcripts in Tn4 cells, an immortalized B cell line from a male patient with a Tn-syndrome-like phenotype. These cells lack T-synthase activity and express the Tn antigen. Treatment of cells with 5-aza-2'-deoxycytidine causes restoration of Cosmc transcripts, restores T-synthase activity, and reduces Tn antigen expression. Bisulfite sequencing shows that CG dinucleotides in the Cosmc core promoter are hypermethylated. Interestingly, several other X-linked genes associated with glycosylation are not silenced in Tn4 cells, and we observed no correlation of a particular DNA methyltransferase to aberrant methylation of Cosmc in these cells. Thus, hypermethylation of the Cosmc promoter in Tn4 cells is relatively specific. Epigenetic silencing of Cosmc provides another mechanism underlying the abnormal expression of the Tn antigen, which may be important in understanding aberrant Tn antigen expression in human diseases, including IgA nephropathy and cancer.

Identification and characterization of circulating immune complexes in IgA nephropathy.

The underlying pathology of immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is driven by the deposition of immune complexes containing galactose-deficient IgA1 [Tn(+)IgA1] in the glomerular mesangium. Here, we report that novel anti-Tn circulating immune complexes (anti-Tn CICs) contain predominantly IgM, representing large macromolecular complexes of ~1.2 megadaltons to several megadalton sizes together with Tn(+)IgA1 and some IgG. These complexes are significantly elevated in sera of patients with IgAN, which contains higher levels of complement C3, compared to healthy individuals. Anti-Tn CICs are bioactive and induce specific proliferation of human renal mesangial cells. We found that these anti-Tn CICs can be dissociated with small glycomimetic compounds, which mimic the Tn antigen of Tn(+)IgA1, releasing IgA1 from anti-Tn CICs. This glycomimetic compound can also significantly inhibit the proliferative activity of anti-Tn CICs of patients with IgAN. These findings could enhance both the diagnosis of IgAN and its treatment, as specific drug treatments are now unavailable.

The endoplasmic reticulum chaperone Cosmc directly promotes in vitro folding of T-synthase.

The T-synthase is the key beta 3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) in animal cell glycoproteins. Here we describe the novel ability of an endoplasmic reticulum-localized molecular chaperone termed Cosmc to specifically interact with partly denatured T-synthase in vitro to cause partial restoration of activity. By contrast, a mutated form of Cosmc observed in patients with Tn syndrome has reduced chaperone function. The chaperone activity of Cosmc is specific, does not require ATP in vitro, and is effective toward T-synthase but not another beta-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.

A novel fluorescent assay for T-synthase activity.

Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr ß3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.

Cosmc deficiency causes spontaneous autoimmunity by breaking B cell tolerance.

Factors regulating the induction and development of B cell­mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Cosmc controls B cell homing.

The molecular mechanisms regulating lymphocyte homing into lymph nodes are only partly understood. Here, we report that B cell-specific deletion of the X-linked gene, Cosmc, and the consequent decrease of protein O-glycosylation, induces developmental blocks of mouse B cells. After transfer into wild-type recipient, Cosmc-null B cells fail to home to lymph nodes as well as non-lymphoid organs. Enzymatic desialylation of wild-type B cells blocks their migration into lymph nodes, indicating a requirement of sialylated O-glycans for proper trafficking. Mechanistically, Cosmc-deficient B cells have normal rolling and firm arrest on high endothelium venules (HEV), thereby attributing their inefficient trafficking to alterations in the subsequent transendothelial migration step. Finally, Cosmc-null B cells have defective chemokine signaling responses. Our results thus demonstrate that Cosmc and its effects on O-glycosylation are important for controlling B cell homing.

The Cosmc connection to the Tn antigen in cancer.

The Tn antigen is a tumor-associated carbohydrate antigen that is not normally expressed in peripheral tissues or blood cells. Expression of this antigen, which is found in a majority of human carcinomas of all types, arises from a blockage in the normal O-glycosylation pathway in which glycans are extended from the common precursor GalNAcα1-O-Ser/Thr (Tn antigen). This precursor is generated in the Golgi apparatus on newly synthesized glycoproteins by a family of polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs) and then extended to the common core 1 O-glycan Galß1-3GalNAcα1-O-Ser/Thr (T antigen) by a single enzyme termed the T-synthase (core 1 ß3-galactosyltransferase or C1GalT). Formation of the active form of the T-synthase requires a unique molecular chaperone termed Cosmc, encoded by Cosmc on the X-chromosome (Xq24 in humans, Xc3 in mice). Cosmc resides in the endoplasmic reticulum (ER) and prevents misfolding, aggregation, and proteasome-dependent degradation of newly synthesized T-synthase. Loss of expression of active T-synthase or Cosmc can lead to expression of the Tn antigen, along with its sialylated version Sialyl Tn antigen as observed in several cancers. Both genetic and epigenetic pathways, in addition to potential metabolic regulation, can result in abnormal expression of the Tn antigen. Engineered expression of the Tn antigen by disruption of either C1GalT (T-syn) or Cosmc in mice is associated with a tremendous range of pathologies and engineered expression of the Tn antigen in mouse embryos leads to embryonic death. Studies indicate that many membrane glycoproteins expressing the Tn antigen and/or truncated O-glycans may be dysfunctional, due to degradation and/or misfolding. Thus, expression of normal O-glycans is associated with health and homeostasis whereas truncation of O-glycans, e.g. the Tn and/or Sialyl Tn antigens is associated with cancer and other pathologies.

WITHDRAWN: The Cosmc connection to the Tn antigen in cancer.

Ahead of Print article withdrawn by publisher. At request of the authors, this article will be published in the journal Cancer Biomarkers (ISSN 1574-0153).

Tn and sialyl-Tn antigens, aberrant O-glycomics as human disease markers.

In many different human disorders, the cellular glycome is altered. An interesting but poorly understood alteration occurs in the mucin-type O-glycome, in which there is aberrant expression of the truncated O-glycans Tn (GalNAcα1-Ser/Thr) and its sialylated version sialyl-Tn (STn) (Neu5Acα2,6GalNAcα1-Ser/Thr). Both Tn and STn are tumor-associated carbohydrate antigens and tumor biomarkers, since they are not expressed normally and appear early in tumorigenesis. Moreover, their expression is strongly associated with poor prognosis and tumor metastasis. The Tn and STn antigens are also expressed in other human diseases and disorders, such as Tn syndrome and IgA nephropathy. The major pathological mechanism for expression of the Tn and STn antigens is compromised T-synthase activity, resulting from alteration of the X-linked gene that encodes for Cosmc, a molecular chaperone specifically required for the correct folding of T-synthase to form active enzyme. This review will summarize our current understanding of the Tn and STn antigens in terms of their biochemistry and role in pathology.

Regulation of protein O-glycosylation by the endoplasmic reticulum-localized molecular chaperone Cosmc.

Regulatory pathways for protein glycosylation are poorly understood, but expression of branchpoint enzymes is critical. A key branchpoint enzyme is the T-synthase, which directs synthesis of the common core 1 O-glycan structure (T-antigen), the precursor structure for most mucin-type O-glycans in a wide variety of glycoproteins. Formation of active T-synthase, which resides in the Golgi apparatus, requires a unique molecular chaperone, Cosmc, encoded on Xq24. Cosmc is the only molecular chaperone known to be lost through somatic acquired mutations in cells. We show that Cosmc is an endoplasmic reticulum (ER)-localized adenosine triphosphate binding chaperone that binds directly to human T-synthase. Cosmc prevents the aggregation and ubiquitin-mediated degradation of the T-synthase. These results demonstrate that Cosmc is a molecular chaperone in the ER required for this branchpoint glycosyltransferase function and show that expression of the disease-related Tn antigen can result from deregulation or loss of Cosmc function.