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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 4th edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022. doi: 10.1101/glycobiology.4e.46
Many acquired changes in glycan synthesis, turnover/degradation, or recognition are involved in human diseases. Knowing these changes can improve disease diagnosis and/or therapies. This chapter lists a few examples, proposes mechanisms, and suggests novel therapies. Glycosylation changes in cancer and in inherited human genetic disorders of glycosylation are discussed in more depth in Chapters 47 and 45, respectively.
Vascular thrombotic events such as stroke and myocardial infarction, plus others including acute ischemic injury (e.g., trauma, hypovolemic shock), temporarily interrupt of blood flow that must be fixed quickly. Natural or medical interventions that restore blood flow suddenly reintroduce leukocytes to the traumatized tissue and can cause tissue damage called “reperfusion injury.” Selectins, cell adhesion molecules that are C-type lectins (Chapter 34), play major roles in this process. L-Selectin (CD62L) on leukocytes and P-selectin (CD62P) on the activated endothelium in the reperfused area initiate this cascade, and E-selectin (CD62E) participates later; however, among the selectins, the E-selectin-based leukocyte recruitment in humans (and primates, in general) is more prominent and sustained than in other mammals because the promoter region of the human E-selectin gene, but not that of P-selectin, contains elements responsive to inflammatory mediators tumor necrosis factor-α (TNF-α) and IL-1 (and to bacterial products like LPS). These increase transcription and expression of E-selectin (Chapter 34), whereas the P-selectin promoter in humans actually dampens gene transcription in response to such mediators. However, in mice, the promoters for both the P-selectin and E-selectin genes retain TNF/IL-1/LPS responsive elements, and mice thus display inflammation-driven inducible endothelial expression of both P-selectin and E-selectin. This regulatory distinction is critical in considering whether rodent models of P-selectin biology may reflect human pathophysiology. Nonetheless, animal models show that blocking initial selectin-based interactions ameliorates subsequent tissue damage. Pharmaceutical and biotechnology companies have made small-molecule inhibitors and biologics (e.g., monoclonal antibodies) to block these interactions in patients, and this approach has shown benefit and has achieved U.S. Federal Drug Administration (FDA) approval for clinical indications such as sickle cell vaso-occlusive crises (further described below; Chapters 55 and 57 also discuss selectin inhibitors). In addition, many preparations of the already-approved drug heparin can block P- and L-selectin interactions, but its dampening effects on reperfusion injury remain to be established.
Heart attacks, strokes, and other serious vascular diseases are associated with high levels of low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol. These molecules increase the risk of atherosclerotic lesions in the large arteries. Atherosclerotic lesions begin with the development of the “fatty streak,” in which monocytes enter into the subendothelial regions of the blood vessels. This process involves expression of P- and/or E-selectin on the endothelium, which recognizes glycosylated and sulfated P-selectin glycoprotein ligand-1 (PSGL-1) or sialyl Lewis x (“sLex”; CD15s) displayed on discrete glycoproteins and glycolipids of circulating monocytes. Indeed, P-selectin deficiency in mice delays progression of atherosclerotic lesions, which slows even more in combined P- and E-selectin-deficient mice. Oxidized lipids in LDL particles or inflammatory processes likely induces endothelial E-selectin in the early atheromatous plaque. Early intervention may be possible because these lesions develop slowly and relatively early in life. Retention of LDLs in the early plaque probably involves binding to proteoglycans and subsequent oxidation and uptake by macrophages and smooth-muscle cells. Clusters of basic amino acid residues in apolipoprotein B (the protein moiety of LDL) likely bind to the glycosaminoglycans. This binding also has a physiological function. Heparan sulfate (HS) found in the liver sinusoids regulates the turnover of lipoprotein particles. Reduced sialylation of LDL in patients also correlates with coronary artery disease. Although the mechanism(s) remain unclear, desialylated LDL may be more easily taken up and incorporated into atheromatous plaques. The nonhuman sialic acid Neu5Gc (enriched in red meats; see Chapter 15) can be metabolically incorporated into plaques, and recognition by circulating antibodies against Neu5Gc glycans may accelerate disease progression via chronic inflammation.
The oral cavity contains numerous host and microbial glycans and glycan-binding proteins (Chapter 21). Dental caries and gingivitis involve certain viridans group streptococci that have serine-rich bacterial proteins whose stability requires O-glycosylation. They also have a carbohydrate recognition domain that recognizes O-linked sialoglycans or oral mucins to allow attachment to cells in the mucosa. If these bacteria reach the bloodstream, particularly during dental procedures, the same binding proteins recognize platelet specific glycoproteins such as GPIbα, delivering the bacteria to damaged heart valves, and leading to the serious disease of bacterial endocarditis.
In humans, E-selectin is constitutively expressed on microvessels of the skin, recruiting both adaptive and innate immune effector cells. E-selectin is up-regulated in all inflammatory skin diseases, further promoting extravasation of circulating leukocytes containing sLex. Dermal lymphocytes are recognized by the monoclonal antibody “HECA452” that binds sLex. This “cutaneous lymphocyte antigen” is displayed on glycoforms of PSGL-1, CD43, and CD44 molecules (sLex-decorated CD44 is called hematopoietic cell E-/L-selectin ligand (HCELL)) (Chapter 34). In mice, both E-selectin and P-selectin are constitutively expressed on dermal microvessels. In humans, T helper 1 (Th1) lymphocytes access the skin via binding to E-selectin, suggesting that blocking this interaction could treat dermal inflammatory diseases.
Diabetes mellitus causes long-term vascular complications, in part by increasing nonenzymatic glycation (not to be confused with glycosyltransferase-mediated glycosylation), wherein the open-chain (aldehyde) form of glucose reacts with lysine residues generating reversible Schiff bases that can rearrange to generate “browning” (Maillard) reactions and permanent cross-linked advanced glycation end products (AGEs). These adducts impair protein and cellular functions, disrupting the function of extracellular matrix proteins (e.g., collagen), contribute to neurodegenerative diseases (e.g., Alzheimer's disease), and bind to receptors, such as the receptor for advanced glycation end products (RAGE) and also the macrophage scavenger receptor that participates in atherogenesis. Notably, glycation of hemoglobin generates “Hemoglobin A1c,” the biomarker for measuring long-term glucose control in diabetics.
Excess glucose increases UDP-GlcNAc through the glucosamine:fructose aminotransferase (GFAT) pathway, enhancing hyaluronan production (Chapter 16) and O-GlcNAcylation of multiple proteins that, in turn, alters their phosphorylation and functions (Chapter 19). In animal models, altered O-GlcNAcylation correlates with complications, such as diabetic cardiomyopathy (increased O-GlcNAcylation of various nuclear proteins) and erectile dysfunction (O-GlcNAcylation of endothelial nitric oxide synthase). Several cytoplasmic proteins involved in insulin receptor signaling and the resulting nuclear transcription changes are themselves O-GlcNAcylated and functionally altered in diabetes.
Kidney dysfunction is a very serious, potentially lethal, diabetic complication. Progressive albumin excretion eventually leads to nephrotic syndrome and to end-stage renal disease. This proteinuria correlates with reduced HS proteoglycan in the glomerular basement membrane. Reduced HS synthesis by glomerular epithelial cells may result from exposure to high glucose or increased porosity of the glomerular basement membrane. High glucose also increases plasminogen activator inhibitor-1 (PAI-1) gene expression in renal glomerular mesangial cells, via O-GlcNAc-mediated alterations in Sp1 transcriptional activity.
Numerous gastrointestinal pathogens or the products they secrete recognize and bind to gut mucosal glycans (Chapter 37). For example, cholera toxin (CT) secreted by Vibrio cholerae can bind GM1 ganglioside, and Helicobacter pylori, the cause of peptic ulcers and gastritis, binds Lewis type glycans in the stomach mucosa. Soluble glycan inhibitors could block binding of these gut pathogens. This could explain that a time-honored treatment for peptic ulcers (before the identification of H. pylori) was a combination of antacids and milk (which contains large amounts of free sialyloligosaccharides). Cholera infection outcomes are correlated with ABO(H) blood group expression. Blood group O(H) individuals are not more frequently infected, but experience more severe disease. Although GM1 is the primary functional ligand for CT, recent studies have suggested that CT also has a binding site for the H antigen, possibly explaining this association.
Protein-losing enteropathy (PLE) is the enteric loss of plasma proteins; however, its mechanisms are not well-understood. Some patients with congenital disorder of glycosylation type Ib (CDG-Ib or MPI-CDG) and CDG-Ic (ALG6-CDG) (Chapter 45) develop PLE, suggesting involvement of N-glycosylation. But other patients with normal N-glycosylation also develop PLE many years following (Fontan) surgery to correct congenital heart malformations. What causes this type of PLE? One concept is that environmental stress such as infections increase inflammatory cytokines, TNF-α and interferon-γ (IFN-γ), which, together with increased venous pressure resulting from the surgery, contribute to PLE. In both the N-glycosylation disorders and post-Fontan surgery patients, HS is lost from the basolateral surface of intestinal epithelial cells and returns when PLE subsides. HS binds cytokines, and its loss may increase the impact of inflammation at the cell surface and promote leakage. Combining increased venous pressure and cytokines along with localized HS depletion creates a downward spiral of disease. Traditional therapy for PLE includes albumin infusions, steroid hormones, or other anti-inflammatory drugs, but, interestingly, heparin injections can also reduce PLE, possibly by binding to and reducing circulating cytokines (Chapter 38).
Ulcerative colitis is an inflammatory disease typically affecting the superficial epithelial layer of the rectum and the distal colon. The primary cause of the disease is unknown, but both genetic and environmental factors, such as changes in the microbiome, are involved and remissions and exacerbations are common. Normally, colonic mucosal proteins display heavily O-acetylated sialic acids, but these acetyl groups are diminished in ulcerative colitis. Whether this contributes to the pathology is unknown, but O-acetylated sialic acids are more resistant to bacterial sialidases (Chapter 15). Decreased sialic acid O-acetylation is also a feature of colon carcinomas.
Heparin (a highly sulfated form of HS; Chapter 17) is a therapeutic agent extracted from porcine intestines or bovine lungs. It is a fast-acting and potent anticoagulant often used to avoid thrombosis, in procedures including dialysis and open-heart surgery. Its effectiveness relies on a specific sulfated heparin pentasaccharide that binds circulating antithrombin III and markedly enhances its ability to inactivate coagulation Factors Xa and IIa (thrombin) (Chapter 17). Animal-derived “unfractionated heparin” is now often replaced with low-molecular-weight heparins because they have fewer complications. One explanation is that the unfractionated heparin effects on thrombin require a long chain that interacts both with the antithrombin and with thrombin itself in a tripartite complex. In contrast, the shorter chains found in low-molecular-weight heparins only facilitate antithrombin inactivation of Factor Xa. Thus, low-molecular-weight heparins affect Xa but not thrombin activity. A synthetic pentasaccharide (fondaparinux) that binds and facilitates antithrombin inactivation of Factor Xa is an alternative to heparin. Although these improvements are valuable, unfractionated heparin has a variety of other biological effects besides anticoagulation. Thus, other beneficial effects of heparin, such as the blocking of P- and L-selectin, are reduced or even eliminated by the switch to low-molecular-weight heparins and the synthetic pentasaccharide.
An uncommon but feared complication of heparin treatment is heparin-induced thrombocytopenia (HIT). During HIT, complexes between heparin and platelet factor-4 form, and the de novo formed pathogenic antibodies against these complexes deposit on platelets, causing their aggregation and loss from circulation. Paradoxically, this process results in exaggerated thrombosis, rather than bleeding. The incidence of this complication is lower with low-molecular-weight heparins and significantly curtailed with the use of the pure pentasaccharide.
Sickle cell anemia is an inherited disorder of hemoglobin that leads to various acute and chronic painful complications. Symptoms were thought to be caused by vaso-occlusion due to abnormally shaped and/or membrane-modified hypoxic erythrocytes. Now, it is clear that abnormal adhesion of multiple cell types is responsible, and that this is partially mediated by selectins. Blocking selectin action with analogs of natural ligands such as sLex or heparin can restore blood flow in a mouse model of sickle cell disease. A pan-selectin inhibitor (GMI-1070, “rivipansel”) has undergone clinical trials for reduction of severity and duration of sickle cell crisis. Notably, glycopeptide mimetics of PSGL-1 and antibodies that block PSGL-1-P-selectin interactions (e.g., FDA-approved crizanlizumab) can mitigate sickle cell vaso-occlusive crises.
Blood transfusion medicine first identified the ABO blood group system, defined by differential expression of α-GalNAc and α-Gal transferases across populations (Chapter 14). These and other less prominent glycan antigens can cause hemolytic transfusion and rejection reactions, mostly because of errors in blood typing. Although anti-A and anti-B titers likely influence transfusion and transplantation outcomes, simple assessment of antibody levels fails to fully account for the clinical impact(s) of an ABO(H) incompatible transfusion. Efforts to generate “universal donor” red blood cells, via enzymatic conversion of blood group A and B antigens to the O state using bacterial enzymes, are underway.
Occasionally, patients with diseases such as cirrhosis and hepatocellular carcinoma spontaneously secrete a circulating anticoagulant and have an unusual coagulation test profile that makes it appear as if the patient has been treated with heparin. The anticoagulant activity can be purified from the plasma and has been identified as an HS glycosaminoglycan. Its source is unknown and therapy is difficult without correcting the underlying disease or transplanting the liver.
Plasma fibrinogen is heavily sialylated, and the sialic acids are involved in binding calcium. Certain genetic disorders of fibrinogen are associated with altered sialylation of its N-glycans, which causes altered function in clotting. Patients with hepatomas and other liver disorders can also sometimes manifest increased branching and/or number of N-glycans on fibrinogen, resulting in an overall increase in sialic acid content. This altered fibrinogen sialylation can present clinically as a bleeding disorder associated with a prolonged thrombin time. Patients with CDGs affecting N-glycan biosynthesis (Chapter 45) can also have thrombotic or bleeding disorders that are partly explained by altered glycosylation of plasma proteins and/or platelets involved in blood coagulation.
Paroxysmal nocturnal hemoglobinuria (PNH) is an unusual form of acquired hemolytic anemia (excessive destruction of red blood cells) that usually appears in adults and arises through a somatic mutation in bone marrow stem cells generating one or more abnormal clones. The defect is an inactivation of the single active copy of the PIGA gene, an X-linked locus involved in the first stage of biosynthesis of glycosylphosphatidylinositol (GPI) anchors (for details on GPI anchor biosynthesis, see Chapter 12). Although several blood cell types show abnormalities, the red cell defect is the most prominent, being characterized by an abnormal susceptibility to the action of complement. This is now known to be caused by the lack of expression of certain GPI-anchored proteins, such as the decay accelerating factor and CD59, that normally down-regulate complement activation on “self” surfaces. However, hypercoagulability also occurs, presumably because of loss of GPI-anchored proteins on other cells, such as monocytes and platelets. Interestingly, many of these patients had preceding, or later development of, bone marrow failure (aplastic anemia). Some of the latter also develop acute leukemia. Most normal humans already have a tiny fraction of circulating cells with a PIGA mutation, the PNH defect. These presumably represent the products of one or more bone marrow stem cells that develop this acquired defect because of a single hit on the active X chromosome but then do not become prominent contributors to the total pool of circulating red blood cells. In this scenario, the independent occurrence of a process damaging the proliferation of other (unaffected) stem cells allows the “unmasking” of the PNH defect.
Patients with the rare disorder paroxysmal cold hemoglobinuria have a cold-induced intravascular destruction of red cells (hemolysis). This hemolysis appears to be caused by a complement-fixing circulating IgG antibody directed against the red cell P blood group antigens that binds more efficiently at temperatures below the core body temperature of 37°C (such as within appendices) (Chapter 14). Biosynthesis of the P blood group antigens is dependent on the B3GALNT1 (β1-3-N-acetylgalactosaminyltransferase 1) and A4GALT (α1-4-galactosyltransferase) genes. The pathogenesis of this disorder is unknown, but it tends to occur in the setting of some viral infections and in syphilis. The IgG antibody presence is detected by the so-called “Donath–Landsteiner test,” in which the patient's serum is mixed either with the patient's own red cells or with those from a normal person and chilled to 4°C. Complement-mediated hemolysis occurs after warming to 37°C.
Cold agglutinin disease is caused by autoimmune IgM antibodies directed against glycan epitopes on erythrocytes. High titers of IgM agglutinins are present in serum and are maximally active at 4°C. The IgMs are presumed to bind to erythrocytes that are circulating in the cooled blood of peripheral regions of the body. The antibody fixes complement, which then destroys the cells when they reach warmer areas of the body. There are several variants of the syndrome. One affects young adults and follows infection with Mycoplasma pneumoniae or Epstein–Barr virus (infectious mononucleosis). This antibody is characteristically polyclonal, and is generally short-lived, disappearing when the infection subsides. An idiopathic variant of cold agglutinin disease affects older individuals, involves a monoclonal IgM, and can be a precursor or an accompaniment to a lymphoproliferative disease such as Waldenström's macroglobulinemia, chronic lymphocytic leukemia, or other lymphomas. These antibodies are typically directed against the “I” antigen (β1-6-branched poly-N-acetyllactosamine) present on glycolipids and glycoproteins of erythrocytes (Chapter 14). Fewer less common variants of cold agglutinin disease involve antibodies directed against sialylated N-acetyllactosamines. In some patients on chronic hemodialysis, the syndrome occurs because of the formation of antibody directed against the sialylated blood group antigen N.
In this condition a subset of bone marrow–derived blood cells express the Tn antigen (O-linked N-acetylgalactosamine, GalNAcα-O-Ser/Thr) and sialyl-Tn (NeuAcα2-6GalNAcα-O-Ser/Thr), thus becoming susceptible to hemagglutination by naturally occurring anti-Tn antibodies that exist in most normal humans. The underlying defect is a somatic stem cell–based loss of expression of the O-glycan core-1 β1-3 galactosyltransferase activity (also called the T synthase) (Chapter 10). This in turn is explained by acquired inactivation of Cosmc, a chaperone required for the biosynthesis of the T synthase. As with PNH, the existence of the C1GALT1C1 gene encoding Cosmc on the X chromosome allows a single hit on the active X chromosome to cause a glycosylation defect in a single bone marrow stem cell. Some patients are picked up simply because polyagglutinability of their red blood cells is detected when blood typing is performed for a possible transfusion. Others have varying degrees of hemolytic anemia and/or decreases in other blood cell types. Some subsequently progress into frank leukemia. It is unclear how the primary syndrome predisposes to development of malignancy. As with PNH, the possibility exists that an underlying bone marrow disorder simply allows the “unmasking” of preexisting minor stem cell clones along with the defect. In keeping with this notion, leukemic clones that arise later may not have the same defect.
The sialylation of platelets regulates their count and production (thrombopoiesis). Aging platelets lose sialic acid moieties and are consequently cleared by multiple Gal- and GalNAc-recognizing lectins, including the Ashwell–Morell receptor (AMR) (Chapter 34) and the macrophage galactose lectin (MGL). Some patients with immune thrombocytopenia (ITP) express sialidase NEU1 on their platelet surface. Influenza antiviral drugs including anamivir (Relenza), oseltamivir (Tamiflu), and peramivir (Rapivab) mimic the transition state to block viral neuraminidase (Chapter 55). Administration of oseltamivir increases platelet counts in a cohort of patients with ITP and in healthy human subjects, suggesting that platelet sialic acid regulates platelet count. Loss of platelet sialic acid and thrombocytopenia can occur in patients with genetic mutations in sialylation, such as GNE, which makes a precursor of sialic acids. Mutations in the CMP-Sia transporter gene SLC35A1 results in macrothrombocytopenia because of increased clearance. Mouse models such as sialyltransferase ST3GalIV knockout mice have profound thrombocytopenia. Crossing ST3GalIV-deficient mice with AMR-null mice results in platelet count recovery, implying that uncovering of galactose leads to AMR-mediated clearance. Multiple changes in O-glycans can also produce severe thrombocytopenia, including deficits in sialyltransferase ST3GalI, GalNT3, C1GalT1 or its chaperone, Cosmc.
IgG immunoglobulins are N-glycosylated, and those in the constant (CH2 or Fc) region of human IgG have several unusual properties. First, they are buried between the folds of the two constant regions. Second, the crystal structure of the protein shows that they are immobilized by glycan–protein interactions. Third, the IgG complex-type biantennary N-glycans are rarely fully sialylated, and instead have one or two terminal β-linked galactose residues (termed G1 and G2, respectively). IgG from patients with rheumatoid arthritis (RA) have even less galactose or none at all (termed G0). The severity of this inflammatory disease tends to correlate inversely with the level of galactosylation. Spontaneous clinical improvement occurs during pregnancy and correlates with restored galactosylation. The basis of the decreased galactosylation in RA is unknown. Some evidence points to less β-galactosyltransferase activity in patients’ lymphocytes, but whether altered glycosylation of IgG has a specific pathogenic role in rheumatoid arthritis is debatable, because G0 molecules are seen in other diseases including granulomatous diseases (e.g., tuberculosis) and Crohn's disease. The glycan changes are also seen to a lesser extent in osteoarthritis, a form of chronic degenerative arthritis with a different pathogenesis. One function attributed to the Fc N-glycans is to maintain the conformation of the Fc domains as well as the hinge regions. Other structural features are also necessary for effector functions such as complement and Fc receptor binding and Fc-dependent cytotoxicity. Nuclear magnetic resonance (NMR) studies show that the G0 N-glycans have an increased mobility resulting from the loss of interactions between the glycan and the Fc protein surface. Thus, it is thought that regions of the protein surface normally covered by the glycan are exposed in rheumatoid arthritis. Some studies suggest that the circulating mannose-binding protein recognizes the more mobile G0 N-glycan and activates complement directly. Rheumatoid arthritis patients also have increased circulating immune complexes consisting of antibody molecules (called rheumatoid factor) that recognize the Fc regions of other IgG molecules. However, the epitopes involved do not seem to be glycan-related. Another likely possibility is that the altered glycosylation changes interactions with Fc receptors.
Glycans and their binding proteins are key to the pathogenicity of essentially all infectious diseases, and this topic is covered in greater detail in Chapters 37, 42, and 43. Some key physiologic effects of glycan–host interactions are briefly covered here.
Many urinary tract infections (UTIs) are caused by Escherichia coli, which adhere to bladder epithelial cells via a mannose-binding lectin, FimH, located on the F-pilus of the bacterium. A simple and effective antibiotic-independent treatment and prevention for this very common infection is drinking D-mannose because it competes bacterial glycan binding to the urinary tract when it is excreted in the urine. Another alternative is optimized synthetic α-mannosides, which could treat and prevent UTIs.
Many pathogens initiate infection by binding to host cell-surface glycans (Chapter 37). Variable expression of these glycans may explain an individual's susceptibility to infection. For example, some pathogenic strains of E. coli infect the urinary tract using a lectin that binds the P blood group antigens (Chapter 14), with P negative individuals being immune. The E. coli P fimbriae lectins are also involved in spreading bacterial infections from the kidney to the bloodstream.
Binding of the spike glycoprotein of SARS-CoV-2, the virus causing COVID-19, to its ACE2 receptor on host epithelial cells first involves its initial binding to neighboring HS glycosaminoglycan chains, which induces a conformational change to enhance binding to ACE2. Importantly, mutations that alter spike glycosylation sites (e.g., variant B.1.1.7 (N501Y)) impact infectivity.
Some pathogenic microorganisms secrete sialidases (neuraminidases), which usually remain at the site of infection. However, in some severe cases (e.g., Clostridium perfringens–mediated gas gangrene), sialidases reach the plasma where they desialylate red cells, resulting in clearance and anemia. Measuring plasma sialidase may aid diagnosis and prognosis. Similarly, the action of viral (e.g., influenza and dengue) or bacterial (e.g., Streptococcus pneumoniae) sialidases causes loss of platelet sialic acid and contributes to increased platelet clearance. Sialidase-producing S. pneumoniae can also cause hemolytic uremic syndrome, and selectively inhibiting sialidase could have therapeutic value. Paradoxically, the thrombocytopenia resulting from desialylation of platelets predisposes to bleeding but may serve to protect against sepsis-induced disseminated intravascular coagulation.
Nephrotic syndrome occurs when the kidney glomerulus fails to retain serum proteins during initial filtration of plasma, allowing these proteins to leak into the urine. The epithelial/endothelial mucin molecule called podocalyxin on the foot processes (pedicles) of glomerular podocytes helps maintain pore integrity and excludes proteins from the glomerular filtrate. Sialic acids on podocalyxin are critical for this function. Loss of sialic acid is seen in spontaneous minimal-change renal disease in children and in the nephrotic syndrome that follows some bacterial infections. Animal models seem to mimic this situation with proteinuria and renal failure developing in a dose-dependent manner after a single injection of V. cholerae sialidase, which correlated with loss of sialic acids from the glomerulus. This was accompanied by effacement of foot processes and the alteration of tight junctions between podocytes. The anionic charge returned to endothelial and epithelial sites within two days, but the foot process loss remained. Another model is aminonucleoside nephrosis, induced in rats by injection of puromycin. Again, defective sialylation of podocalyxin and glomerular glycosphingolipids is seen in this model. A genetic mouse model with impaired sialic acid synthesis (UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase [GNE] deficiency) dies soon after birth because of kidney dysfunction and undersialylated podocalyxin.
In humans, all immunoglobulin classes contain N-glycans in the Fc domain, but only IgA1 and IgD contain O-glycans (localized within the hinge regions). The IgA1 O-glycan chains are thought to stabilize the three-dimensional structure of the molecule. IgA nephropathy is a form of glomerulonephritis caused by the deposition of aggregated IgA1 molecules within the glomerulus, and patients with this condition harbor circulating IgA1 with O-glycan truncations. Underglycosylated IgA1 has the tendency to both self-aggregate as well as trigger immune reactions leading to IgG-IgA complexes, and both processes lead to glomerular IgA1 deposits. The primary mechanism of underglycosylation remains unknown. One possible scenario is a defect in the C1GALT1C1 gene that encodes Cosmc, similar to that found in the Tn polyagglutinability syndrome (see above). Instead of affecting a bone marrow stem cell, the defect would presumably involve a clone of B cells that specifically expresses underglycosylated IgA1.
In chronic kidney disease, genome-wide association studies (GWASs) have indicated that deficiency in polypeptide GalNAc-transferase 11 (GALNT11) may be etiologic. GALNT11 adds O-GalNAc to LRP2 (megalin), a major endocytic receptor within renal proximal tubules. Studies with GALNT11 knockout mice have shown a critical role of LRP2 O-glycans in mediating its protein resorption function and, also, in preventing its loss with age.
Systemic lupus erythematosus (SLE) is an autoimmune disorder in which antigen–antibody complexes accumulate in various organs, especially the skin and kidney. How SLE begins is unknown, but the pathology may involve both cytokines and HS. HS is reduced on the glomerular basement membrane, and this was thought to result from masking of HS by complexes of nucleosomes and antinuclear antibodies, but the actual mechanism(s) is likely to be more complex. Even though anti-double-stranded DNA antibodies are the hallmark of SLE, circulating antibodies to HS strongly correlate with disease activity. In some studies, HS injections into dogs induce SLE symptoms within several weeks, and elevated HS is found in the urine of SLE patients, especially in severe cases. Some SLE patients also develop protein-losing enteropathy (PLE), perhaps as a consequence of misplaced or degraded HS and elevated cytokines, creating the appropriate environment for PLE (see above).
A variety of diseases are associated with circulating antibodies directed against specific glycan molecules enriched in the nervous system, resulting in autoimmune neural damage. Such antibodies can arise via distinct pathogenic mechanisms. In the first situation, patients with benign or malignant B-cell neoplasms (e.g., benign monoclonal gammopathy of unknown significance [MGUS], Waldenström's macroglobulinemia, or plasma cell myeloma) secrete monoclonal IgM or IgA antibodies that are specific for either gangliosides or, more commonly, for sulfated glucuronosyl glycans (the so-called HNK-1 epitope). These antibodies react with glycolipids bearing the epitope 3-O-SO3-GlcAβ1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer (3′sulfoglucuronosylparagloboside) and against the N-glycans on a variety of central nervous system (CNS) glycoproteins (MAG, P0, L1, N-CAM) that bear the same terminal sequence. The resulting peripheral demyelinating neuropathy can sometimes be more damaging than the primary disease itself. Therapy consists of attempts to treat the primary disease with chemotherapy or to remove the immunoglobulin by plasmapheresis. Both approaches are usually unsuccessful at lowering the immunoglobulin to a level sufficiently to diminish the symptoms. The second situation is an immune reaction to molecular mimicry of neural ganglioside structures by the lipo-oligosaccharides of bacteria such as Campylobacter jejuni. Following an intestinal infection with such organisms, circulating cross-reacting antibodies against gangliosides such as GM1 and GQ1b appear in the plasma. These are associated with the onset of symptoms of peripheral demyelinating neuropathy involving the peripheral or cranial nerves (the Guillain–Barré and Miller Fisher syndromes, respectively). The third situation is a human-induced disease arising from attempts to treat patients with stroke using intravenous injections of mixed bovine brain gangliosides. Although some evidence for benefits was seen, several cases of Guillain–Barré syndrome were reported as a likely side effect. One explanation is that the presence of small amounts of gangliosides with the nonhuman sialic acid, N-glycolylneuraminic acid (Neu5Gc), facilitates formation of antibodies that cross-react with gangliosides containing the human sialic acid Neu5Ac.
Alzheimer's disease is a common primary degenerative dementia of humans, with an insidious onset and a progressive course. The ultimate diagnosis is made by postmortem histological examination of brain tissue, which shows characteristic amyloid plaques with neurofibrillary tangles that are associated with neuronal death. Several types of glycans have been implicated in the histopathogenesis of the lesions: O-GlcNAc and HS glycosaminoglycans. Paired helical filaments are major component of the neurofibrillary tangle. These are primarily composed of the microtubule-associated protein Tau, which is present in an abnormally hyperphosphorylated state. This hyperphosphorylated Tau no longer binds microtubules and self-assembles to form the paired helical filaments that may contribute to neuronal death. Normal brain Tau is known to be heavily modified by Ser(Thr)-linked O-GlcNAc, the dynamic and abundant posttranslational modification that is often reciprocal to Ser(Thr) phosphorylation (Chapter 19). The hypothesis currently being investigated is that site-specific or stoichiometric changes in O-GlcNAc addition may modulate Tau function and may also play a part in the formation of paired helical filaments by allowing excessive phosphorylation. Inhibitors of O-GlcNAcase that cross the blood–brain barrier are now in clinical trials.
HS proteoglycans may also have an important role in amyloid plaque deposition as investigators have shown high-affinity binding between HS proteoglycans and the amyloid precursor, as well as with the A4 peptide derived from the precursor. In addition, a specific vascular HS proteoglycan found in senile plaques bound with high affinity to two amyloid protein precursors. Further studies to determine the pathological roles of HS proteoglycans in Alzheimer's disease are needed. Recently, GWASs showed a strong correlation between Alzheimer's disease and the higher expression of a truncated form of CD33 (Siglec-3), which is expressed in brain microglia and may be suppressing clearance of amyloid.
Altered glycosylation is a universal feature of cancer cells, but only certain specific glycan changes are frequently associated with tumors (for details, see Chapter 47). Findings include (1) increased β1-6GlcNAc branching of N-glycans; (2) changes in the amount, linkage, and acetylation of sialic acids; (3) truncation of O-glycans, leading to expression of Tn and sialyl Tn antigens, as well as N-glycan truncation yielding paucimannosidic glycans; (4) expression of the nonhuman sialic acid, Neu5Gc, incorporated from dietary sources; (5) expression of sialylated Lewis structures and selectin ligands; (6) altered expression and enhanced shedding of glycosphingolipids; (7) increased expression of galectins and poly-N-acetyllactosamines; (8) altered expression of ABH(O) blood-group-related structures; (9) alterations in sulfation of glycosaminoglycans; (10) increased expression of hyaluronan; (11) increased expression of the enzyme that attaches GPI anchors to proteins; and (12) increased O-GlcNAcylation on many proteins. Some of these changes have been shown to have pathophysiological significance in model tumor systems, and some are targets for diagnostic and therapeutic approaches to cancer. For example, the principal serum diagnostic/prognostic indicator of adenocarcinomas of the pancreas and gastrointestinal tract is the biomarker known as “CA19-9” which is the tetrasaccharide sialyl Lewis a (sLea), a binding determinant for E-selectin; its isomer, sLex, is displayed on bone marrow progenitors and by engagement with marrow microvessel E-selectin mediates hematopoiesis and leukemogenesis.
Asthma is characterized by sporadic recurrent hyperresponsiveness of the tracheobronchial tree to various stimuli, resulting in widespread narrowing of the airways. The two dominant pathological features are airway wall inflammation and luminal obstruction of the airways by inflammatory exudates, consisting predominantly of mucins. Most cases are due to antigen-specific IgE antibodies, which bind to mast cells as well as to basophils and certain other cell types. Antigens can cross-link adjacent IgE molecules, triggering an explosive release of vasoactive, bronchoactive, and chemotactic agents from mast cell granules into the extracellular milieu. Eosinophils also contribute to the pathogenesis of asthma in several ways, by synthesizing leukotrienes, stimulating histamine release from mast cells and basophils, providing a positive feedback loop, and releasing major basic protein, a granule-derived protein that has toxic effects on the respiratory epithelium. Underlying all this, it appears that CD4+ Th2 cells are responsible for orchestrating the responses of other cell types. Recent evidence indicates that the selectins are intimately involved in recruitment of eosinophils and basophils (and possibly T lymphocytes) into the lung, raising the hope that small-molecule inhibitors of selectin function and/or heparin can be used to treat the early stages of an asthmatic attack. Likewise, chemokine interactions with HS are important in leukocyte trafficking. Evidence from Siglec-F knockout mice also indicates that the functionally equivalent human paralog Siglec-8 is a good target for reducing the contributions of eosinophils to the pathology (Chapter 35). Finally, the large increase in mucus production is at least partly mediated by an up-regulation of synthesis of mucin polypeptides, under the influence of various cytokines that stimulate the goblet cells of the airway epithelium.
Shock, trauma, or sepsis can all cause acute respiratory distress syndrome. Diffuse pulmonary endothelial injury causes pulmonary edema because of increased capillary permeability. Selectins and integrins help arrest neutrophils on the injured endothelium where they release injurious oxidants, proteolytic enzymes, and arachidonic acid metabolites, resulting in endothelial cell dysfunction and destruction. Bronchoalveolar lavage is rich in neutrophils and their secreted products documenting the inflammatory response. Here again, giving small molecule selectin inhibitors and/or heparin before serious lung damage and respiratory failure is a possible therapy.
Cystic fibrosis is a common genetic disorder caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR). This causes defective chloride conduction across the apical membrane of involved epithelial cells. Cystic fibrosis is associated with increased accumulation of viscous mucins in the pancreas, gut, and lungs, which leads to many symptoms of the disease. There are known to be widespread increases in sialylation, sulfation, and fucosylation of mucin glycoproteins. One possible explanation is that the primary CFTR defect allows a higher Golgi pH, resulting in abnormalities in glycosylation: however, there is controversy about this conclusion. Curiously, the CFTR is mainly expressed within nonciliated epithelial cells, duct cells, and serous cells of the tubular glands, but not highly expressed in the goblet cells and mucous glands of the acinar cells, which are the cells that synthesize respiratory mucins. Thus, the CFTR mutation may indirectly affect mucin glycosylation through the generation of inflammatory responses and/or changes in pH or chloride secretion. Another major cause of morbidity in the disease is the colonization of respiratory epithelium by an alginate-producing form of Pseudomonas aeruginosa. Certain glycolipids and mucin glycans have been suggested to be the Pseudomonas receptors that help to maintain the colonization. The changes in glycolipid and mucin glycosylation could enhance production of potential binding targets for organ colonization by the bacteria. The presence of bacterial products is also a proinflammatory condition, because the bacterial capsular polysaccharides may activate Toll-like receptors and lead eventually to neutrophil accumulation and organ damage.
Pulmonary vascular diseases include pulmonary embolism, pulmonary arterial hypertension (PAH), and arteriovenous malformations. These diseases increase pulmonary vascular resistance and pulmonary arterial pressure, ultimately leading to right ventricular hypertrophy and heart failure. PAH is the best studied—a progressive disease that shows nitric oxide deficiency, vasoconstriction, thrombosis, and enhanced vascular remodeling. Among other factors, dysregulated glucose metabolism may drive an increased flux into the hexosamine biosynthetic pathway. This results in PAH patients having increased hyaluronan in lung tissue, plasma, and pulmonary arterial smooth muscle cells. It also increases O-GlcNAc-modified proteins, a process shown to regulate pulmonary arterial smooth muscle cell proliferation associated with PAH progression, suggesting a potential therapeutic target.
The authors appreciate helpful comments and suggestions from Morten Thaysen-Andersen and Priya Umapathi.
Because of the wide range of topics covered in this chapter, it is not feasible to provide literature citations for all of them. Some examples are provided but the reader should consult references at the end of the other cited chapters.
The content of this book is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view the terms and conditions of this license, visit https://creativecommons.org/licenses/by-nc-nd/4.0/
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