MUCIN GLYCOPROTEINS

About 150 years ago, E. Eichwald and E. Hoppe-Seyler noted that highly glycosylated proteins that contain hundreds of O-GalNAc glycans, which they termed mucins, are found throughout the body (Figure 10.1). Since then we have learned that O-GalNAc glycans are not only found as dense clusters on mucins, but also at single sites on most secreted and membrane-bound proteins. O-GalNAc glycans are involved in almost every aspect of biology, including cell–cell communication, cell adhesion, signal transduction, immune surveillance, epithelial cell protection, and host–pathogen interactions.

FIGURE 10.1.

A simplified model of a large secreted mucin. The PTS domain, rich in Pro, Thr, and Ser, is highly O-glycosylated on Ser/Thr. The mucin assumes an extended “bottle brush” conformation. The cysteine-rich regions form disulfide bonds generating (more...)

GalNAc O-linked to Ser/Thr is the initiating sugar of O-GalNAc glycans and is usually extended to form one of four common core structures (Table 10.1; Figure 10.1). Each core can subsequently be extended to give a mature linear or branched O-GalNAc glycan.

TABLE 10.1.

O-GalNAc glycan cores and antigenic epitopes of mucins

The great variety of O-GalNAc glycans often makes it very difficult to assign functions to individual O-GalNAc glycans at particular attachment sites, and most functions have historically been ascribed to the densely glycosylated mucins. In mucins, O-GalNAc glycans control chemical, physical, and biological properties. Because O-GalNAc glycans are hydrophilic, and usually negatively charged, they promote binding of water and salts and are major contributors to the viscosity and adhesiveness of mucins and the mucus they form. Mucins line the epithelial surfaces of the body, including the gastrointestinal, genitourinary, and respiratory tracts, where they shield epithelial cells against physical and chemical damage and protect against infection. Mucins can be antiadhesive and repel cell-surface interactions or, alternatively, promote adhesion by mediating recognition of glycan-binding proteins via their O-GalNAc glycans. A number of diseases are associated with abnormal mucin gene expression and abnormal mucin O-GalNAc glycans. These include cancer (Chapter 47), inflammatory bowel disease, congenital disorders of glycosylation (Chapter 45), and hypersecretory bronchial and lung diseases.

Humans have about 20 different mucin genes encoding both secreted and membrane-bound mucins, which vary considerably in their primary sequence and tissue-specific expression. Mucins are characterized by having densely glycosylated regions, previously termed “variable number tandem repeat” (VNTR) regions, but now called PTS domains (for the abundance of proline, threonine, and serine), which carry the majority of the O-GalNAc glycans on Ser and Thr residues (50%–80% of their molecular weight) (Figure 10.1). The O-GalNAc glycans expressed on a mucin are the result of the spectrum of glycosyltransferases active in the cell type producing the mucin. The first mucin polypeptide gene to be cloned, MUC1, encodes a transmembrane mucin that is ubiquitous in epithelium. The levels of MUC1 are high in certain tumors, in which its glycosylation is often abnormal. The dense and elongated O-glycans on membrane-bound mucins or other glycoproteins are thought to confer extended “bottle brush” conformations, lifting them above the surface of the cell (Figure 10.1), as well as providing protection from proteolytic cleavage. Additionally, dense O-GalNAc glycans on the large, secreted, gel-forming, intestinal mucin MUC2 allow it to adopt a hydrated, membrane-like structure that acts as the first line of defense to protect the underlying epithelium and mediate proper interactions with the microbiome. The importance of this mucin in intestinal health is illustrated by mice lacking Muc2, which spontaneously develop colorectal cancer.

O-GalNAc GLYCAN CORE STRUCTURES

The O-GalNAc glycans of mucins have four major core structures (cores 1–4; Table 10.1). Each core can be extended (Figure 10.1) by a variety of sugar residues to give linear or branched chains that resemble those on N-glycans (Chapter 9) and glycolipids (Chapter 11). Blood group determinants are commonly found in mucins at nonreducing termini of O-GalNAc glycans (Chapter 14). The extension of O-GalNAc with a β1-3Gal forms core 1, the most common O-GalNAc glycan. Core 2 is formed by the addition of β1-6GlcNAc to the GalNAc of core 1. Less common cores are core 3, in which β1-3GlcNAc is added to O-GalNAc, and core 4, in which core 3 is branched by the addition of β1-6GlcNAc (Table 10.1). Core 1 and 2 O-GalNAc glycans are found in glycoproteins and mucins produced in many different cell types. However, core 3 and 4 O-GalNAc glycans are more restricted to mucins and glycoproteins in gastrointestinal and bronchial tissues.

A single GalNAc residue attached to Ser/Thr forms the Tn antigen. As mentioned above, the core 1 O-GalNAc glycan (Galβ1-3GalNAc on Ser/Thr) forms the Thomsen–Friedenreich (TF or T antigen). Although Tn and T antigens are usually cryptic because they are extended by other sugars, they are found at increased levels in mucins from cancer cells. They can also carry Sia and form sialyl-Tn or sialyl-T antigens.

O-GalNAc cores are often extended to form complex O-GalNAc glycans that may include the ABO and Lewis blood group determinants (Chapter 14), polysialic acid, the linear i antigen (Galβ1-4GlcNAcβ1-3Gal), and the GlcNAc β1-6-branched I antigens (Table 10.1). Extensions by Type 1 (Galβ1-3GlcNAc) or Type 2 (Galβ1-4GlcNAc) units can be repeated and provide scaffolds for the attachment of additional sugars or functional groups. The termini of O-GalNAc glycans may contain Fuc and Sia in α-linkages, and Gal, GalNAc, and GlcNAc in both α- and β-linkages, and sulfate. Many of these terminal sugars are antigenic or recognized by lectins. In particular, the sialylated and sulfated Lewis antigens are ligands for selectins (Chapter 34), and Gal-terminating structures are ligands for galectins (Chapter 36). Some sugar residues, or their modifications, may mask underlying antigens or receptors. For example, O-acetyl groups on the Sia of the sialyl-Tn antigen prevent recognition by anti-sialyl-Tn antibodies. Gut bacteria may actively remove this mask. Dense O-glycosylation of mucin domains provides almost complete protection from protease degradation.

ISOLATION, PURIFICATION, AND ANALYSIS OF MUCIN O-GalNAc GLYCANS

The O-linkage between GalNAc and Ser/Thr residues is labile under alkaline conditions. Thus, O-GalNAc glycans can be released by a reaction termed β-elimination (i.e., treatment with 0.1 m sodium hydroxide). The hemiacetal GalNAc produced will undergo rapid alkali-catalyzed degradation under these conditions (called peeling), but can be reduced with sodium borohydride to yield stable N-acetylgalactosaminitol at the reducing end of the released O-glycan. β-Elimination is the method of choice to release O-glycans from glycoproteins that also have N-glycans, because the latter are not susceptible to cleavage under mild conditions. O-GalNAc glycans, as well as other Ser/Thr-linked glycans (Chapter 13), are released as alditols by β-elimination, but with losses of labile O-acetyl or sulfate esters. An alternative method that preserves the reducing end of O-GalNAc uses ammonia followed by boric acid. O-GalNAc that is not substituted with another sugar can be enzymatically released by a N-acetylgalactosaminidase. Another glycosidase, termed O-glycanase, releases core 1 (Galβ1-3GalNAc-) from Ser/Thr, provided the disaccharide is not further substituted. Thus, sialidase treatment followed by O-glycanase releases most simple, core 1 O-GalNAc glycans. Terminal Sia residues can also be easily removed with mild acid treatment. There are no known enzymes that can release more complex and extended whole O-GalNAc glycans, but mixtures of exoglycosidases can be used to sequentially remove sugars from O-GalNAc glycans on a glycoprotein. Glycoproteins with clusters of sialylated O-GalNAc glycans may be digested by an O-sialoglycoprotein endopeptidase.

Released, intact O-GalNAc glycans may be separated by different chromatographic methods, including high-performance liquid chromatography (HPLC). Chemical derivatization of GalNAc at the reducing end helps in the separation and subsequent analysis of sugar composition and linkages by gas chromatography and mass spectrometry (MS) (Chapter 50). Another tool to isolate O-glycans with specific epitopes is affinity chromatography using lectins. For example, Helix pomatia agglutinin binds to terminal GalNAc, whereas peanut lectin binds to unsubstituted core 1 (Table 10.1).

The structures of O-GalNAc glycans released from mucins and other glycoproteins may be determined by a combination of liquid or gas chromatography, MS, and nuclear magnetic resonance (NMR) spectroscopy. The anomeric linkage of each sugar can be determined using specific glycosidases that distinguish between α- or β-linked sugars, and by one- and two-dimensional NMR methods (Chapter 50).

The sites of O-GalNAc glycan modification in mucins are difficult to determine directly, but this has been achieved by sensitive MS methods and new enzymatic tools. A big step forward in understanding the extent of the O-GalNAc glycoproteome came with the proteome-wide mapping of O-GalNAc glycosylation sites. Several MS-based strategies have been applied including modifications of the endogenous glycan structures through chemical labeling, lectin enrichment of glycans derived from native or glycoengineered cells, or O-GalNAc-specific endo-peptidase treatment. Additionally, the use of mucin-type O-proteases (OpeRATOR and StcE) that specifically cleave N-terminal to an O-glycosylated Ser/Thr have aided in mapping sites of glycosylation. Based on these methodologies, we now know that >80% of the proteins passing through the secretory pathway are modified with O-GalNAc glycans, although the occupancy and the nature of an O-GalNAc glycan associated with a glycosylated site remains elusive.

BIOSYNTHESIS OF O-GalNAc GLYCANS

O-GalNAc glycans are added to Ser/Thr residues in proteins in the Golgi apparatus. The biosynthetic glycosyltransferases are type II transmembrane proteins with a short cytoplasmic tail at the amino terminus, a transmembrane domain, a stem region, and a catalytic domain in the lumen of the Golgi. The arrangement within Golgi membranes appears to be similar to an “assembly line” with early reactions occurring in the cis-Golgi and late reactions in the trans-Golgi (Chapter 4). Many of the enzymes, however, are diffusely distributed in Golgi compartments.

The subcellular localization, activity levels, and substrate specificities of glycosyltransferases involved in the assembly of O-GalNAc glycans play a critical role in determining the range of O-glycans synthesized by a cell (Table 10.2 and Figures 10.2 and 10.3). The glycosyltransferases that are involved in the assembly of O-GalNAc glycans are listed in Table 10.2. However, other enzymes that contribute to the synthesis of N-glycans and glycolipids also act on O-glycans, and some of these prefer O-glycans as acceptor substrates (Chapter 14). In vitro assays have shown that the activities of glycosyltransferases are controlled by factors such as metal ions and pH.

FIGURE 10.2.

Biosynthesis of core 1 and 2 O-GalNAc glycans as described in the text. Green lines are protein.

TABLE 10.2.

Glycosyltransferases that synthesize O-GalNAc glycans

Synthesis of O-GalNAc Glycan Cores

As mentioned above, O-GalNAc glycan synthesis begins with the transfer of GalNAc from UDP-GalNAc to Ser/Thr catalyzed by a GALNT. Although a single, unextended GalNAc linked to Ser/Thr (the Tn antigen) is uncommon in normal mucins, it is often found at increased levels in tumor mucins. This suggests that the extension of O-GalNAc glycans beyond the first sugar is blocked in some cancer cells. Sia added to GalNAc by the α2-6 sialyltransferase ST6GALNAC1 generates the sialyl-Tn antigen, which is common in advanced tumors. Other sugars are not known to be added to this O-glycan, but it can be O-acetylated by Sia O-acetyltransferase, which prevents detection by anti-sialyl-Tn antibodies.

The addition of one or two neutral sugars to O-GalNAc generates one of the different cores of O-GalNAc glycans (Table 10.1; Figure 10.2). Core 1 (Galβ1-3GalNAc-O-Ser/Thr) is generated by C1GALT1. This activity is present in most cell types but absolutely requires the molecular chaperone C1GALT1C1 (COSMC) during synthesis in the endoplasmic reticulum (ER) in mammalian cells to ensure proper folding and activity in the Golgi. Lack of core 1 synthesis in certain cell types can be due to either defective C1GALT1 or the absence of functional C1GALT1C1 and is reflected in high expression of Tn and sialyl-Tn antigens. For example, Jurkat T cells and colon cancer leukemic stem cells (LSCs) lack the C1GALT1C1 chaperone, and thus C1GALT1 activity, and have high expression of Tn and sialyl-Tn antigens.

A GlcNAc β1-6 branch added to the GalNAc residue of core 1 forms core 2 O-GalNAc glycans (Tables 10.1 and 10.2; Figure 10.2). Core 2 O-GalNAc glycans are more cell type–specific than the essentially ubiquitous core 1 O-GalNAc glycans, and their expression is highly regulated during activation of lymphocytes, cytokine stimulation, and embryonic development. Leukemia and cancer cells and other diseased tissues have abnormal amounts of core 2 O-GalNAc glycans. The enzymes responsible for core 2 synthesis are core 2 β1-6 N-acetylglucosaminyltransferases 1, 2, and 3 (GCNT1, GCNT3 and GCNT4). These glycosyltransferases do not require divalent cations as cofactors, and X-ray crystallography shows that positively charged amino acids replace the function of divalent metal ions. There are two different types of core 2 β1-6 N-acetylglucosaminyltransferases. One type synthesizes only core 2 O-GalNAc glycans (GCNT1, the leukocyte or L-type enzyme, and GCNT4), whereas the M-type enzyme (mucin type, GCNT3) synthesizes core 2 and 4 O-GalNAc glycans (Table 10.1; Figure 10.3). The L-type enzyme is active in many tissues and cell types, but the M type is found only in mucin-secreting tissues such as the intestine, stomach, and respiratory tissues. The expression and activity of both the L and M types are altered in certain tumors. The synthesis of core 2 O-GalNAc glycans has been correlated with tumor metastasis, possibly because of selectin ligands that are preferentially assembled on core 2 O-GalNAc glycans and that facilitate egress from the circulation (Chapter 34).

FIGURE 10.3.

Biosynthesis of core 3 and 4 O-GalNAc glycans as described in the text. Green lines are protein.

The synthesis of core 3 O-GalNAc glycans appears to be restricted mostly to mucus epithelia from the gastrointestinal and respiratory tracts and to the salivary glands. The enzyme responsible is core 3 β1-3 N-acetylglucosaminyltransferase 6 (B3GNT6) (Table 10.2, Figure 10.3). The enzyme has low in vitro activity but must be highly efficient in vivo because colonic mucins are rich in core 3 O-GalNAc glycans. The expression and activity of B3GNT6 is especially low in colonic tumors and virtually absent from tumor cells in culture. Overexpression of the enzyme in colon cancer cells decreases their ability to metastasize. Mice deficient in B3GNT6 show increased susceptibility to colitis and tumor development. The synthesis of core 4 by the M-type GCNT3 requires the prior synthesis of a core 3 O-GalNAc glycan (Figure 10.3). Transfection of colon cancer cells HCT116 with GCNT3 suppresses cell growth and invasive properties. In a xenograft model in nude mice, transfection with GCNT3 also suppresses tumor growth. Thus, both core 3 and 4 O-GalNAc glycans repress tumor progression, although the mechanisms of repression are not clear.

FUNCTIONS OF O-GalNAc GLYCANS

The functions of O-GalNAc glycans are many and varied, depending on their structure and density, as well on as the protein to which they are attached. As mentioned above, in densely glycosylated proteins such as mucins, O-glycans aid in hydration, structural support, interaction with the microbiome, and protection from proteolysis. In contrast, the function of single sites of O-GalNAc glycosylation vary widely and are still being determined. For example, O-GalNAc glycosylation at one or a few sites in certain proteins has been shown to regulate proprotein convertase cleavage, shedding of ectodomains, ligand binding, and cell–cell and cell–matrix interactions, influencing tissue formation and differentiation. Moreover, O-glycans, either in defined sites or clusters, may serve as carriers of terminal ligands such as blood type antigens like sialyl-Lewis x, which are important for host–pathogen interactions, and the circulation and homing of immune cells. The ligands for certain selectin-mediated interactions between endothelial cells and leukocytes require sialyl-Lewis x that is commonly attached to core 2 O-GalNAc glycans (Table 10.1). Finally, O-glycan terminal motifs on some immune cell subtypes can engage specific Siglecs and induce tolerance to self-antigens.

Methods to determine the function of O-GalNAc glycans are diverse and include the use of biosynthetic inhibitors, the construction of cell lines that lack or overexpress specific enzymes in O-glycosylation pathways, the use of O-GalNAc glycan-specific lectins or antibodies, removal of specific sugar residues by glycosidases, and deletion/mutation of specific GALNTs in model organisms. For example, a critical role of O-GalNAc glycans in selectin-mediated cell adhesion was revealed by treating cells with GalNAc-O-benzyl, which is a competitive inhibitor of core 1 and core 2 synthesis. GalNAc-O-benzyl acts as a decoy substrate for C1GALT1 and thereby reduces the synthesis of core 1 and core 2 O-GalNAc glycans on glycoproteins. Inhibitor-treated cancer cells lose the ability to bind to E-selectin and endothelial cells in vitro. Cancer cells often express sialyl-Lewis x and may thus use the selectin-binding properties of sialyl-Lewis x as a mechanism to invade tissues. Small molecule inhibitors that inactivate GALNTs are being developed to block the initiation of all O-GalNAc glycans by each GALNT.

Cell lines engineered to express altered O-GalNAc glycans, as well as mice with targeted mutations, are excellent models to identify roles for O-GalNAc glycosylation. Cells lacking C1GALT1C1 have been developed to identify a partial O-GalNAc proteome (i.e., the specific locations of core 1 or core 2 O-GalNAc glycans in the glycoproteome). The same approach has identified roles for core 1 and core 2 O-GalNAc glycans in cell transformation and cancer cell progression. Mice lacking C1GALT1, and thus lacking core 1 and core 2 O-GalNAc glycans, die during embryogenesis because of defective angiogenesis and hemorrhaging (Chapter 41). Moreover, a variety of phenotypes have been observed in mice with conditional deficiencies of core 1 O-glycans in specific tissues including spontaneous colitis, thrombocytopenia, defective lymphocyte homing, defects in podocyte function, and blood/lymphatic misconnections. Mice that lack core 1 and core 2 O-glycans within intestinal epithelial cells develop spontaneous duodenal tumors, illustrating the role of O-glycans in intestinal protection and homeostasis. Similarly, the influence of individual GALNTs has also been examined in model organisms and human tissue models. Loss or down-regulation of individual GALNTs have shown the importance for epithelial differentiation in human models. In Drosophila melanogaster, loss of GALNTs results in loss of viability, and defects in secretion and secretory vesicle formation, leading to epithelial cell damage and loss of cell–cell adhesion (Chapter 26). Mice deficient for GALNT1 display defects in cardiac development, hemostasis, immune cell homing, and ECM composition. Humans and animal models lacking GALNT2 result in a complex phenotype that includes dyslipidemia, hypercholesterolemia, and neurodevelopmental disorders. In humans, mutations in GALNT3 result in the disease hyperphosphatemic familial tumoral calcinosis, characterized by high blood phosphate levels and the development of calcified tumors. This disease results from the loss of GALNT3-mediated glycosylation that protects the phosphate regulating hormone FGF23, from inactivating cleavage. Mouse models of this disease display similar phenotypes, along with disruption of the oral microbiome. Finally, mice deficient for GALNT11 exhibit low-molecular-weight proteinuria, because of glycosylation changes in the proximal tubule endocytic receptor megalin that alter its ability to bind ligands.

Given the many potential functions of O-GalNAc glycans, there are surprisingly few direct medical and biotherapeutic applications. One of the most prominent examples includes the targeting of PSGL-1 in inflammatory conditions, either by antibodies or small molecule mimetics, which most recently has been successfully applied to children with sickle cell crisis. Cancer is another area in which disease-specific changes in O-GalNAc glycans can be exploited. Because of the association of increased levels of Tn, sialyl-Tn, and T antigens with cancer, several vaccine candidates based on these cancer-associated O-GalNAc-glycans are being investigated. An alternative and promising strategy is the use of high-affinity antibodies that are selective for cancer-associated O-GalNAc-glycans. Such antibodies may be especially effective with the new potent antibody-based therapeutic strategies, including Bi-specific T-cell engagers (BiTEs) and chimeric antigen receptors inserted in cytotoxic T cells (CAR-Ts). Indeed, CAR-Ts directed to glycopeptide epitopes in MUC1 have shown antitumor effects in preclinical animal models.

ACKNOWLEDGMENTS

The authors acknowledge the contributions of Harry Schachter to previous versions of this chapter.

FURTHER READING

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