Chapter 18Nucleocytoplasmic Glycosylation

Eukaryotic Monoglycosylation

Monoglycosylation, the addition of a single monosaccharide to the hydroxyl or amide moieties of amino acid side-chains, was first discovered in the form O-β-GlcNAc and is now known as a widespread modification of thousands of nuclear mitochondrial and cytoplasmic proteins throughout the animal and plant kingdoms and some protists, fungi, and bacteria (see Chapter 19). The addition of O-GlcNAc is linked to the side-chains of Ser or Thr and is mediated by a highly conserved lineage of O-GlcNAc transferases (OGT), a nonmembrane bound enzyme that resides in the cytoplasm and nucleus. A splicing variant is also found in animal mitochondria in which O-GlcNAcylation is found on respiratory enzymes. A highly related enzyme lineage, termed O-fucosyltransferase (OFT), mediates the addition of O-fucose (O-Fuc) to dozens of nucleocytoplasmic proteins in plants, numerous protists, and possibly bacteria. The OFT was first discovered genetically in plants as Spy, in which it controls transcriptional processes. In the protist pathogen Toxoplasma gondii, OFT modifies numerous proteins involved in transcription, mRNA biogenesis, nuclear transport, and cell signaling. Recently, Greb1, derived from another lineage of enzymes, was shown to O-β-GlcNAcylate estrogen receptor-α and potentially other nuclear proteins, which if verified is an interesting example of convergent evolution. Paralogs of these enzymes have not been described in the well-studied Saccharomyces cerevisiae, but biochemical data suggests that this yeast modifies intracellular proteins with mannose (Man) and even di- and tri-Man. Furthermore, several reports suggest that select intracellular proteins of mammalian cells are modified by N-acetylgalactosamine (GalNAc). The significance of these intriguing latter examples awaits identification of the responsible glycosyltransferases and evidence for their roles in cells. Examples in which the glycosyltransferases are known are represented schematically in Figure 18.1A and summarized in Table 18.1.

FIGURE 18.1.

Cellular topography of nucleocytoplasmic glycosylation. (A) Monoglycosylation (m) in the cytoplasm (e.g., O-GlcNAc and O-Fuc). (B) Complex (c) glycosylation in the cytoplasm by O-GlcNAc transferases (GTs) that extend the first sugar. (C) Cytoplasmic monoglycosylation (more...)

TABLE 18.1.

Examples of nuclear or cytoplasmic glycosylation events

Prokaryotic Monoglycosylation

Highly conserved homologs of OGT and OFT are found in numerous bacterial genomes, although studies of their functional roles are essentially nonexistent. An example of bacterial monoglycosylation that leads to a known function comes from the EF-P protein, the bacterial homolog of eukaryotic elongation initiation factor 5a. In some bacteria, EF-P suppresses translational stalling by a mechanism that involves oxidation of a critical lysyl residue, and its eukaryotic ortholog is regulated by hypusylation. A recent phylogenetic analysis of EF-P sequences revealed a subset of enzymes with an Arg in place of Lys, and a coevolving gene that was subsequently identified by biochemistry and mass spectrometry as an argininyl rhamnosyltransferase. Assembly of the Rha-Arg linkage activates EF-P and is required for pathogenicity of Pseudomonas, a Gram-negative, opportunistic human pathogen, and a number of other bacteria. The discovery of this linkage by a phylogenetic approach suggests that more noncanonical examples of glycosylation are yet to be discovered.

A directed proteomics search for glycoproteins in the Gram-positive intestinal bacterium Lactobacillus plantarum, biased only because potential glycopeptides were affinity enriched with the lectin wheat germ agglutinin (WGA), identified several glycoproteins with cytoplasmic localization and functions: the molecular chaperone DnaK, the PdhC E2 subunit of the pyruvate dehydrogenase complex, the DNA translocase FtsK1, the signal recognition particle receptor FtsY, FtsZ involved in cell division, and others of unknown function. Peptides from these proteins were modified with single HexNAc residues attached to Ser residues. Some of the sites were variably modified, hinting at a novel regulatory process, but nothing is known about the mechanism of this glycosylation.

Bacterial Glycosyltransferase Toxins

Pathogenic bacteria have developed a wide array of toxin and effector virulence factors that also monoglycosylate host-cell proteins (Figure 18.1C). By virtue of their glycosyltransferase domains, these impair the host's cytoplasmic or nuclear machinery and disrupt host-cell immune response. For example, small cytoplasmic G-proteins (GTP-binding proteins) of the Rho family are involved in regulating the cytoskeleton. Certain toxins from anaerobic bacteria were found to contain retaining glycosyltransferase activities that inhibit G-proteins by attaching a glycosyl moiety to a threonine residue (Thr-37) in their GTP-binding sites. These secreted toxins show the remarkable ability of translocating across the surface membrane into the cytoplasm of mammalian target cells. The enterotoxins from Clostridium difficile (ToxA) and Clostridium sordellii are α-glucosyltransferases from CAZy family GT44 that use UDP-glucose (UDP-Glc) as the donor. In contrast, a similar toxin from Clostridium novyi is an O-αGlcNAc transferase that uses UDP-GlcNAc as the donor. The C. novyi toxin has no primary sequence relationship to host glycosyltransferases. A distantly related toxin from Legionella pneumophila, also delivered by a type IV secretion system, installs an αGlc residue on elongation factor 1A in a broad range of eukaryotic host cells. The target residue, Ser53, is located in the G domain near the switch-1 region of the GTPase, and glucosylation inhibits its activity in vitro and in vivo. The recent discovery of a new effector glucosyltransferase, LtpM, shows that this is an expanding field of glycosyltransferase discovery. Interestingly, an unrelated toxin from Yersinia, which enters host cells via a phage tail-derived translocation system, was found to be an αGlcNAc-transferase that modifies Tyr-34 of RhoA. Typically, the sugar–amino acid linkage is not recognized by endogenous glycosidases, allowing the pathogen to irreversibly alter the function of the target—typically a GTPase domain.

Other Gram-negative bacteria use a type 3 secretion system to inject virulence factors that mediate a different form of monoglycosylation: GlcNAcα-Arg. This novel reaction is catalyzed by NleB-related glycosyltransferases that promote host-cell longevity during infection. NleB and NleB orthologs target arginine side-chains within death domains of death receptors and associated proteins TRADD, FADD, RIPK1, and TNFR1. Importantly, these factors all work to attenuate NF-κB signaling to dampen host-cell antibacterial and inflammatory responses. Furthermore, various components of glucose metabolism such as GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and HIF-1α are also targeted to reduced NF-κB signaling. Although these examples involve monoglycosylation mediated by exogenous glycosyltransferases (see Table 18.1), their existence reinforces the potential impact of glycosylation as a regulatory mechanism, and the diversity of glycosylation events that probably remain to be discovered.

Glycosylation of DNA

Although the focus of this chapter is on nuclear and cytoplasmic glycoproteins, it should be noted that specific bases in DNA have long been known to be a target of bacteriophage-encoded glycosyltransferases. Modification of T4-phage DNA hydroxymethylcytosine residues by βGlcTs or αGlcTs (Table 18.1) aids in the distinction of native and foreign DNA, rendering them resistant to host restriction enzyme digestion and impairing a subset of CRISPR/Cas systems. The use of Glc disaccharide, arabinose, and other sugar modifications of bases suggests that the full diversity of sugar modifications remains to be described. A related modification occurs in a protist, Trypanosoma brucei, in the form of base J. Using a mechanism resembling Skp1 glycosylation (vide infra), DNA thymidine residues are initially hydroxylated by an O₂-dependent nonheme dioxygenase generating hydroxymethyl moieties suitable for glycosylation by a novel nuclear β-glucosyltransferase, JGT, a CAZy GT2 glycosyltransferase (Table 18.1). Base J influences Pol II termination by recruiting a complex containing protein phosphatase 1 to dephosphorylate RNA polymerase II. Disruption in complex formation affects polycistronic transcription termination in the wider group of kinetoplastids.

Hydroxyproline-Linked Skp1 Glycans

The Skp1 glycoprotein, which bears a single linear pentasaccharide, was discovered in the nucleus and cytoplasm of a free-living soil amoeba, the cellular slime mold Dictyostelium discoideum. Skp1 is an adaptor in SCF (Skp1/Cullin1/F-box protein)-type E3-ubiquitin ligase complexes that function in the cytoplasm and nucleus of all eukaryotes. There they mediate the polyubiquitination and eventual proteasomal degradation of hundreds of proteins involved in cell cycle regulation, transcription, and signal transduction. Skp1 is subject to O₂-dependent hydroxylation at Pro143, which generates the substrate for five glycosyltransferase reactions. Based on mass spectrometric, sequential exoglycosidase treatments, and nuclear magnetic resonance (NMR) studies (Chapter 50), the glycan consists of a core trisaccharide equivalent to the type 1 blood group H structure, Fucα1-2Galβ1-3GlcNAc1α-, substituted at the 3-position of Fuc by a Galα1,3Galα- disaccharide (Figure 18.2B). In most other protists that modify Skp1, including the apicomplexan parasite T. gondii, a similar core trisaccharide is capped by a Galα1-3Glcα1- disaccharide (Figure 18.2A). The linkage of this glycan to protein, its localization in the cytoplasm, and the structures themselves are currently unique. Because the Pro residue is conserved in the Skp1 genes of plants, invertebrates, and unicellular eukaryotes, and gene sequences related to those of known Skp1 modification enzymes (see below) are present in the genomes of many protists, this modification appears to be widespread in aerobic unicellular eukaryotes and pathogenic fungi.

FIGURE 18.2.

Mechanism of glycosylation of Skp1 in the cytoplasm of protists. Glycosylation is enabled by prior hydroxylation of a single Pro-residue by the action of a cytoplasmic, O₂-dependent prolyl 4(trans)-hydroxylase that is homologous to the HIFα prolyl (more...)

The glycosyltransferases are all NDP-sugar-dependent and colocalize with Skp1 in the cytoplasm or nucleus (Figure 18.2A-C; Table 18.1). The Skp1 αGlcNAc-transferase (EC 2.4.1.229), which adds the first sugar, is related to the Thr/Ser polypeptide αGalNAc-transferases that initiate mucin-type O-glycosylation in the Golgi of animals, and therefore forms an anomeric linkage opposite to that of the aforementioned OGT (Chapter 19). However, unlike known polypeptide αGalNAc-transferases (Chapters 6 and 10), the Skp1 αGlcNAc-transferase lacks an amino-terminal signal anchor, consistent with its biochemical fractionation as a soluble cytoplasmic protein. Thus, the mechanism of initiation of Skp1 glycosylation is like that of mucin-type domains of secretory proteins, except that a different N-acetylhexosamine (GlcNAc vs. GalNAc) is attached to a distinct hydroxyamino acid (Hyp vs. Thr or Ser) in the cytoplasm versus the Golgi lumen. Additions of the second (βGal) and the third (α-L-Fuc) sugars are catalyzed by separate domains of the same soluble protein, PgtA. The β3-galactosyltransferase catalytic domain is most like bacterial lipopolysaccharide and capsular glycosyltransferases, which are also cytoplasmic, suggesting that cytoplasmic glycosylation in Dictyostelium has its evolutionary origins in bacterial glycolipid synthesis. The β-galactosyltransferase catalytic domain belongs to the inverting CAZy GT2 family, which includes numerous glycosyltransferases with catalytic domains exposed to the cytoplasm. In Dictyostelium, the fourth and fifth sugars are added by the two-domain glycosyltransferase AgtA, in which the α3-galactosyltransferase domain is fused to a β-propeller-like domain, which has a second function that may involve constitutive Skp1 sequestration activity. The catalytic domain is related to a large number of plant Golgi glycosyltransferases implicated in pectin biosynthesis and here catalyzes two successive additions. In contrast, most protists employ separate glycosyltransferases with independent evolutionary origins to catalyze the completion of the glycan (Figure 18.2A). In Toxoplasma and Pythium ultimum, a crop plant pathogen and agent for human pythiosus, addition of the αGlc residue is carried out by a CAZy GT31 enzyme most related to Golgi glycosyltransferases that extend mannose chains in yeast, and the terminal αGal residue is added by Gat1, a CAZy GT8 enzyme that is most related to the enzyme discussed below, glycogenin. Finally, in other protists, gene fusions consolidate the first two or final two enzymes as separate domains of the same protein (Figure 18.2C), which may facilitate efficient extension of the glycan to its final length.

Dictyostelium cells aggregate when starved and the resulting multicellular slug differentiates to form a fruiting body with stress-tolerant spores. This process is sensitive to the level of O₂, which is thought to help the cells determine whether they are below or above ground in their native soil environment. Considerable biochemical, genetic, and physiological evidence supports the model that O₂ levels regulate the rate of Skp1 hydroxylation, which in turn controls the rate of glycosylation. Glycosylation regulates the relative representation of different F-box protein substrate receptors in the Skp1 interactome (Figure 18.2D), by a mechanism that most likely controls the conformational profile of Skp1 rather than recognition by a glycan receptor. This may differentially regulates the lifetime of proteins that critically control or execute developmental progression in response to starvation.

Glycogenin

A second well-studied example is glycogen, a large branched homopolysaccharide used as a short-term storage form of glucose in bacteria, yeast, and animals. Glucose is added to and removed from the nonreducing termini per the availability of the sugar donor UDP-Glc and the nutritional needs of the cell. Glycogen stores in the liver are important in maintaining glucose homeostasis in the blood. Glycogen is assembled initially from a linear oligoglucoside that is extended by glycogen synthase and rearranged by the so-called branching enzyme. The initial oligoglucose is assembled by another enzyme, glycogenin, which attaches the first sugar to itself in an uncommon Glcα1-Tyr linkage, at Tyr-195 in human glycogenin-1. Thus, every glycogen molecule, which can contain up to 10⁵ Glc residues and 12 generations of branchpoints, is thought to have a single glycogenin protein molecule at its nonreducing end. Therefore, glycogen is a glycoprotein whose glycosylation is initiated by glycogenin, extended by glycogen synthase, and modified by branching enzyme. Free glucose-1-PO₄ is released, as needed, by the actions of debranching enzyme and glycogen phosphorylase.

Glycogenin naturally assembles as a homodimer under cellular conditions. Evidence indicates that the first Glc is transferred to the acceptor hydroxyl on Tyr across subunits, and extended up to a chain length of ∼10 by mixed inter- and intrasubunit additions of αGlc residues to the 4-position of underlying Glc residues. Interestingly, glycogenin shows triphasic kinetics in which there is a priming step typically with UDP-Glc; however, UDP-Gal can form this initial bond. After priming there is a short extension step again with some flexibility with substrate sugars, and finally a refining step catalyzed by the adjacent glycogenin that shows strong substrate specificity for UDG-Glc. Despite the in vitro evidence, aberrant levels of glycogen are still formed in mice lacking glycogenin-1 and its paralog glycogenin-2. Mutations in glycogenin-1 leading to decreased enzymatic function or loss of the protein cause glycogen storage disease type XV characterized by the presence of polyglucosan bodies of abnormally structured glycogen that cannot be digested by amylase. Interestingly, these polyglucosan bodies are derived from aberrant glycogen formation by glycogen synthase. These mutations contribute to respiratory distress, limb-girdle muscular dystrophy–like symptoms, and progressive weakness. However, patients do not show hypoglycemia, hepatomegaly, or hyperlipidemia as with other glycogen storage diseases. Crystallographic and mutational evidence for formation of a complex between glycogenin and glycogen synthase that affects glycogen synthase activity in vitro and in vivo offers a potential explanation for the mutational effects. Thus, levels of glycogenin may operate together with the better understood hormonally controlled mechanisms of glycogen formation that involve enzyme phosphorylation/dephosphorylation and that regulate glycogen elongation.

Glycogenin-like proteins are found in a wide range of plants, animals, and free-living single-celled eukaryotes. The Escherichia coli homolog is incapable of autoglucosylation. The most closely related sequence homolog in alveolates and stramenopile protists, which lack glycogenin, modifies Skp1 (vide supra). Glycogenin is a GT-A superfamily member from CAZy family GT8 (Carbohydrate-Active enZYmes sequence database) (Chapter 52), and more distantly related glycosyltransferases include bacterial lipopolysaccharide (LPS) glucosyl and galactosyl transferases and galactinol synthases—all cytoplasmic as for glycogenin—as well as other glycosyltransferases in the eukaryotic Golgi. The eukaryotic parasite Leishmania assembles mannogen, a polymer of β2-linked mannose polymers, instead of glucose polymers, but a protein primer is not involved for the cytoplasmically localized mannogen synthase. Interestingly, a novel Glc-Arg linkage has been described in a plant protein potentially associated with the synthesis of starch, which is related to glycogen, but this protein has no apparent sequence similarity to glycogenin. The function of this and the glycogenin-like proteins deserve further investigation for their potential to mediate other cytoplasmic glycosylation events.

Mitochondrial and Chloroplast Glycosylation

Mitochondria and chloroplasts are independently replicating organelles that reside in the cytoplasmic compartment. Because of their evolutionary origin from bacteria, which are now known to have substantial glycosylation capacity, these organelles can be expected to possess glycosylation machinery. Indeed, there is evidence for peptidoglycan-like networks between the inner and outer membranes of chloroplasts of certain algae. In other algae, glycosyltransferase MDR1 (mitochondrion dividing-ring 1), an integral membrane protein with a CAZy GT8 glycosyltransferase domain, assembles the mitochondrial dividing ring using α-linked polyglucose nanofilaments that have been visualized between the two membranes. A similar process occurs in chloroplasts, and the conservation of these genes suggests that this might be a heretofore unrecognized example of organelle glycosylation for many eukaryotes.

More recent cell biological studies emphasize intracellular connections between mitochondria and rough endoplasmic reticulum (rER) elements of the secretory pathway. This might explain a pioneering observation that two nuclear-encoded mitochondrial glycoproteins appear to be conventionally N-glycosylated in the rER based on pulse-chase labeling studies and susceptibility to N-glycanase (Figure 18.1J). Indeed, lectin-binding studies suggest that mitochondria contain complex glycoconjugates. In Ewing's sarcomas, the plasma membrane protein MRP-1 (multidrug resistance-associated protein 1) is glycosylated and localized to the mitochondrial outer membrane and could play a role in drug resistance. Several plant chloroplast proteins, including carbonic anhydrase-1, α-amylase, and pyrophosphatase/phosphodiesterase in “higher” plants and Glx, Syn, MutS in diatoms, might be transported from the rER as N-glycosylated proteins (Table 18.1). The presence of N-glycans highlights their potential biosynthetic origin in the conventional secretory pathway, and how these glycoproteins transit to these organelles warrants further investigation.

As noted above, mammalian mitochondria import OGT that O-GlcNAcylates some of their interior proteins. In addition, mono- and digalactolipids (MGDG: 1,2-diacyl-3-O-[β-D-Gal]-sn-glycerol and DGDG: 1,2-diacyl-3-O-[α-D-Gal-(1,6)-O-β-D-Gal]-sn-glycerol) are prominent lipids in chloroplast thylakoid membranes, and MGDG is essential for photosystem function (Chapter 24). Their synthesis is controlled by UDP-Gal-dependent galactosyltransferases residing in the inner and outer layer membranes of the chloroplast (Figure 18.1K). Under conditions of phosphate deprivation, the synthesis of these glycolipids is dramatically increased, becoming up to 70 mol% of glycerolipids. The glycolipids replace conventional phospholipids such as phosphatidylcholine in a variety of other cellular membranes including vacuoles, mitochondria, and the plasma membrane (Table 18.1). DGDG almost exclusively resides in the cytoplasmic leaflet of these membranes and oriented toward the cytoplasm, which is opposite of the conventional orientation of glycolipids as known from animal and yeast cells. Variants containing sulfoquinovose and GlcUA, which conserve the negative charge, are also produced. Little is known about the trafficking of these glycolipids. Given the functional importance of DGDG for cells based on genetic studies, it will be interesting to understand how the asymmetric distribution contributes to cytoplasmic functionality of the cellular membranes.

N-Glycan Types

The above examples describe well-documented examples from both prokaryotes and eukaryote of bona fide glycoproteins that function in the nucleus or cytoplasm and are glycosylated by residential nucleocytoplasmic glucosyltransferases (GTs). This section addresses other potential sources of nucleocytoplasmic glycoproteins. As suggested by the examples below, these proteins might be glycosylated by glycosyltransferases normally associated with the rER or Golgi, either by transient occurrence of the glycoprotein in the secretory pathway, or translocation of the secretory pathway glycosyltransferases into the nucleocytoplasmic compartment.

A sizable fraction of nascent proteins fail quality control of folding and assembly in the rER (Chapter 39) and are retrotranslocated to the cytoplasm as a part of ERAD (ER-associated degradation), as depicted in Figure 18.1H. Most of these are N-glycosylated and normally processed by a cytoplasmic N-glycanase (NGLY1) or an endo-β-N-acetylglucosaminidase (ENGase) to remove their N-glycans before their degradation by the 26S-proteasome or an autophagic vacuole. There are also N-glycan-dependent E3 ubiquitin ligases that assemble polyubiquitin chains that serve as a signal for proteasomal degradation. Interestingly, NGLY1 is essential for mitochondrial turnover via mitophagy, although evidence suggests it functions via a role other than deglycosylating mitochondrial proteins. If these processes do not operate on a given protein, because the N-glycan is modified in such a way that it is not recognized or is sequestered in a protein complex or the nucleus, then it may accumulate in the cytoplasm and potentially execute a novel function that might depend on its ER-derived glycan. In addition, there is evidence that endocytosed proteins, such as cholera toxin, can access the rER via retrograde transport and then enter the cytoplasm via retrotranslocation or a related process.

Other studies suggest that glycoproteins conventionally secreted or transported to the plasma membrane have the potential to later move to the nucleus (Figure 18.1I). This includes cytokines, growth factors, and sometimes their transmembrane receptors, some of which have been reported to exert direct effects on transcription. For example, fragments of the neural cell adhesion molecule (NCAM), which is modified by polysialic acid, appear to be internalized into cells in which they differentially impact transcriptional programs according to their polysialic acid content. These findings have been considered controversial because the mechanism of such translocation remains mysterious.

Different approaches support the occurrence of nucleus-associated glycoproteins. The sialic acid–specific lectin, Sambucus nigra agglutinin (SNA), was shown to bind to several proteins on the cytoplasmic face of the nuclear envelope, including two major nucleoporins: p62 and p180. Prior sialidase treatment blocked binding of SNA to these nuclear pore proteins and, based on peptide-N-glycosidase sensitivity, the sialic acids on p180 appeared to be on N-linked glycans. Furthermore, SNA blocked nuclear protein import in neuroblastoma cells, suggesting that the sialic acids might have functional importance on the nuclear pore proteins. Other notable reports of nuclear glycoproteins include a heat-shock-like nuclear chaperone protein with GlcNAc-binding activity, CBP70, which has been suggested to be N-glycosylated. A subpopulation of nuclear prion proteins may be N-glycosylated and possibly associated with the GlcNAc-binding lectin.

N-glycosylation of cytoplasmic proteins or protein domains has also been proposed. For example, the α-subunit of the dog kidney sodium pump (Na⁺, K⁺-ATPase), a transmembrane protein, was reported to contain traditional N-linked glycans with terminal GlcNAc residues in its cytosolic domain. This conclusion was based on the enzymatic attachment of radioactive Gal to GlcNAc residues by galactosyltransferase labeling of permeabilized right-side-out membrane vesicles. Peptide-N-glycosidase sensitivity of the radiolabeled products suggested that the acceptors are N-linked glycans (Chapters 9 and 50). However, this and other claims will be considered provocative until they are confirmed by direct site mapping of the putative glycans. Based on conventional principles of the separate cellular compartmentalization of the N-glycosylation pathway and cytoplasmic and nuclear proteins (not within the nuclear envelope, which is an extension of the rER) (Figure 18.1) and known pathways of translocation of proteins across membranes, there is not a simple explanation for how these modifications are acquired.

Finally, findings of cytoplasmic glycosylation in pathological tissues, such as tau N-glycosylation during neurodegeneration, raise the possibility that compartmental breakdowns result in the exposure of cytoplasmic proteins to normally latent Golgi glycosyltransferases.

Secretory Pathway O-Glycan Types

Glycosylation that is more reminiscent of secretory pathway–type O-glycans has also been reported. In plants, a nuclear pore–associated protein is recognized by the lectin WGA and could be labeled using tritiated UDP-Gal and β4-galactosyltransferase, which is specific for nonreducing terminal GlcNAc. The glycan, with an approximate size of five sugars, was released by mild alkaline degradation consistent with β-elimination from a Thr or Ser residue. The identity of the glycosyltransferases is unknown. More recently, evidence was presented for animal nuclear proteins, including lamins, ribonucleoproteins, and estrogen receptor-α, that are O-glycosylated by αGalNAc (Tn antigen) or core 1 O-glycans Galβ1,3-GalNAc-. Further data show that several intracellular proteins including p53 are enriched by the lectin VVA indicative of modification with O-GalNAc. Another potential example of a cytoplasmic glycan comes from a study on purified mammalian cytokeratin, which was reported to contain GalNAc and bind lectins that recognize α1,3-linked GalNAc in an N-acetylgalactosaminidase-sensitive fashion.

A distinct example of glycosylation has been described on parafusin, a cytoplasmic protein from the phosphoglucomutase family, in several eukaryotes. Evidence suggested that this protein possesses a Glcα1-PO₄-Man- linkage, and data were provided for glucose phosphotransferase and Glc-1-phosphate phosphodiesterase activities in cytosolic extracts. However, little is known about the enzymes that catalyze these modifications or the specific structures themselves.

Secretory Pathway Glycosyltransferases in the Nucleus?

A possible mechanism for the occurrence of nuclear O-glycans in the nucleus is if the enzymes that are normally in the secretory pathway gain access to the nucleus or cytoplasm (Figure 18.1F). For example, several reports describe glycosyltransferase activities in highly purified preparations of rat liver nuclei judged to be >99% pure by marker enzyme analysis. These studies documented that incorporation of GlcNAc was inhibited by low concentrations of the antibiotic tunicamycin (an inhibitor of formation of the N-glycan precursor), and later studies showed the direct transfer of chitobiose (GlcNAcβ1-4GlcNAc) from chitobiosyl-dolichol to endogenous nuclear acceptors by these mammalian nuclear preparations, suggesting a novel pathway of N-glycosylation. The products of these in vitro reactions were validated by their sensitivity to peptide N-glycosidase F and hydrazinolysis but insensitivity to alkali-induced β-elimination (Chapter 50). Supporting these observations, recent data suggests that both the core GalNAcT1 and GalNAcT2 can localize to the nucleus. Similar studies have documented the presence of nuclear mannosyltransferases. The finding that CMP (cytidine 5′-monophosphate)-sialic acid is synthesized in the mammalian nucleus offers further support for the concept of nuclear glycosyltransferases. Although these studies are provocative, they must also be interpreted with caution. The rER, which is the widely accepted site of N-glycosylation (Chapter 9), is functionally contiguous with the outer nuclear envelope, which also can fold into the interior. Even a minor contamination of nuclear envelope could lead to misinterpretation of enzyme assays. Also, it is very difficult to purify nuclei such that other cellular components do not nonspecifically adhere to the otherwise “pure” nuclei during their preparation. Given these potential problems, widespread acceptance of the existence of these nuclear glycosyltransferases must await independent confirmation by alternative methods.

Glycosaminoglycans

Another class of glycans traditionally thought to reside at the cell surface and the extracellular matrix are the glycosaminoglycans (or GAGs) (Chapter 17). Early studies provided evidence of GAGs in purified nuclei, and soon thereafter studies on sea urchin embryos indicated developmentally regulated effects of stage-specific heparan sulfate (HS) on transcription that were not observed for chondroitin sulfates or hyaluronan (Chapter 16). Given recent advances in the tools to study proteoglycans, these potentially exciting observations warrant a critical reexamination. Years later, subcellular fractionation of rat hepatocytes radiolabeled with ³⁵SO₄ revealed substantial enrichment of specific fragments of HS (after cleavage) in their nuclear fractions. Given the unique nature of these structures, it is hard to conceive how they could be derived by contamination from cell surface HS molecules, which lack these structures. Subsequent pulse-chase studies suggested that exogenous or cell surface HS is taken into the nucleus and modified to these unique nuclear molecular species. In comparison, others found dermatan sulfates, but not HSs, associated with the nucleus of a different cell type. Given the ability of HSs to influence gene transcription in vitro, these studies could eventually prove highly significant. However, all of the known enzymes involved in the biosynthesis of glycosaminoglycans have their active sites in luminal compartments (Chapter 17), and pathways for nuclear uptake of such large and negatively charged molecules have not been described.

Using another approach, immunocytochemical studies probing for a GPI-anchored HS proteoglycan (PG), glypican, have provided strong evidence for the nuclear accumulation of this PG in addition to its traditionally accepted presence at the cell surface. Although the mechanism of nuclear compartmentalization is not known, the glypican polypeptide harbors a nuclear localization sequence that is functional when grafted onto a neutral carrier expressed in the cytoplasm. In a separate study, overexpression of a chondroitin sulfate proteoglycan core protein in cultured cells resulted in accumulation of the core polypeptide in the cytoplasm and nucleus in addition to the secretory pathway, indicating, as has been suggested for other proteins, the possibility of dual compartmentalization. However, although these studies provide an explanation for how proteoglycan core proteins might accumulate in the nucleus, it is difficult to imagine how they have been modified by the glycosaminoglycan synthases residing in the Golgi lumen.

Cytochemical studies using naturally occurring proteins with high-affinity binding to hyaluronic acid have been used to document accumulation of hyaluronic acid in the nuclei of cultured cells. In response to inflammatory conditions, such as hypoglycemia, cells appear to synthesize hyaluronan in intracellular compartments. After cell division, the intracellular hyaluronan is extruded outside cells in cables that recruit inflammatory cells initiating a stress response. Mechanistically, this is likely caused by a redistribution of the hyaluronan synthases to intracellular membranes from the plasma membrane, across which hyaluronic acid is normally extruded (Figure 18.1N). Notably, hyaluronic acid–binding proteins naturally occur within the nucleus raising the possibility of a physiological role for nuclear hyaluronan via interaction with these hyaluronic acid–binding proteins.