Rajindra P. Aryal

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.

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.

The Endoplasmic Reticulum Chaperone Cosmc Directly Promotes in Vitro Folding of T-synthase

The T-synthase is the key β3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Galβ1–3GalNAcα1-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 β-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.

Epigenetic Silencing of the Chaperone Cosmc in Human Leukocytes Expressing Tn Antigen

Background: Tn4 B cells from a patient with Tn antigen-positive leukocytes lack transcripts of Cosmc. Results: The Cosmc promoter in Tn4 cells is methylated and 5-aza-2′-deoxycytidine treatment restores Cosmc transcription and normal O-glycans. Conclusion: Methylation-dependent epigenetic silencing of Cosmc occurs in Tn4 cells and results in Tn antigen expression. Significance: These findings provide a novel mechanism underlying aberrant expression of Tn antigen in human diseases. Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi β3-galactosyltransferase that generates the core 1 O-glycan, Galβ1–3GalNAcα-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.

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 Galβ1-3GalNAcα1-O-Ser/Thr (T antigen) by a single enzyme termed the T-synthase (core 1 β3-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.

A Novel Fluorescent Assay for T-synthase Activity

Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), 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.

Tight Complex Formation between Cosmc Chaperone and Its Specific Client Non-native T-synthase Leads to Enzyme Activity and Client-driven Dissociation

Background: Cosmc regulates protein O-glycosylation by regulating biosynthesis of its client T-synthase. Results: Cosmc forms a specific noncovalent complex with non-native T-synthase leading to enzyme activity and dissociation driven by additional free client. Conclusion: Both non-native and native T-synthase regulate release of T-synthase from complexes with Cosmc. Significance: This work gives insights into regulators of glycosyltransferases and endoplasmic reticulum/Golgi proteins. The interaction of the endoplasmic reticulum molecular chaperone Cosmc with its specific client T-synthase (Core 1 β1–3-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.

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.

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.

Identification of a Novel Protein Binding Motif within the T-synthase for the Molecular Chaperone Cosmc

Background: We hypothesize that ER chaperone Cosmc binds specifically to its single client T-synthase. Results: Using molecular and biochemical approaches, we demonstrate that Cosmc recognizes a specific peptide within unfolded T-synthase. Conclusion: Cosmc-T-synthase interaction requires unique peptide recognition in the unfolded T-synthase that becomes inaccessible to Cosmc upon final folding. Significance: This study describes a novel peptide-specific chaperone recognition that regulates protein O-glycosylation. Prior studies suggested that the core 1 β3-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 β4GalT1 and found that the CBRT element can confer Cosmc binding onto the β4GalT1 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.