Role of Selectins in Reperfusion Injury

Cardiovascular disorders such as stroke, myocardial infarction, and hypovolemic shock involve temporary interruption of blood flow that need to be fixed quickly. Natural or medical interventions restore blood flow, but suddenly reintroducing leukocytes to the traumatized tissue also causes tissue damage, called reperfusion injury. P-Selectin on the activated endothelium in the reperfused area and/or L-selectin on leukocytes initiate this cascade and E-selectin participates later (Chapter 34). Animal models show that blocking the initial selectin-based recognition ameliorates subsequent tissue damage. Pharmaceutical and biotechnology companies have tried to make small-molecule inhibitors to block these interactions in patients (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.

Roles of Selectins, Glycosaminoglycans, and Sialic Acids in Atherosclerosis

Heart attacks, strokes, and other serious diseases are associated with high levels of low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol. They increase the risk of atherosclerotic lesions in the large arteries. The earliest phase of the development of atherosclerotic lesions (the fatty streak) involves entry of monocytes into the subendothelial regions of the blood vessels. This process involves expression of P- and/or E-selectin on the endothelium, which recognizes correctly glycosylated and sulfated P-selectin glycoprotein ligand-1 (PSGL-1) or sialyl Lewis x on 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 may induce endothelial P-selectin in the early atheromatous plaque. Early intervention may be possible because these lesions develop very slowly and relatively early in life. Retention of LDLs in the early plaque probably involves their binding to proteoglycans. This interaction is thought to cause irreversible structural alterations of LDL, potentiating oxidation and uptake by macrophages and smooth-muscle cells. At the molecular level, clusters of basic amino acids in apolipoprotein B (the protein moiety of LDL) appear to bind the negatively charged glycosaminoglycans of proteoglycans. This binding also has a physiological function. Heparan sulfate (HS) found in the liver sinusoids clearly 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.

Role of Selectins in Inflammatory Skin Diseases

A common feature of atopic dermatitis and contact dermatitis is the entry of leukocytes into the dermis, where they recruit other types of cells that mediate tissue damage. Sometimes this results in the chronic expression of E-selectin on endothelial cells. E-selectin can recruit circulating lymphocytes carrying the cutaneous lymphocyte antigen (detected by the antibody HECA452), which appears to be a specific E-selectin ligand epitope carried on a subset of PSGL-1 molecules (see Chapter 34). Some T helper 1 (Th1) lymphocytes enter into the skin via expression of the PSGL-1 ligand for P-selectin. These results were derived from model systems and suggest potential selectin-based therapeutic targets for future development.

Pathogenesis and Complications of Diabetes Mellitus

Diabetes mellitus causes long-term vascular complications. One mechanism to account for this is that high glucose increases nonenzymatic glycation, wherein the open-chain (aldehyde) form of the glucose reacts with lysine residues on various proteins, generating reversible Schiff bases. Irreversible Amadori rearrangement then gradually generates adducts leading to the “browning” (Maillard) reactions, which eventually progress to advanced glycation end products (AGEs). The resulting protein cross-links can damage cellular functions. The glycation adducts bind to receptors, such as the receptor for advanced glycation end products (RAGE) and the macrophage scavenger receptor that may also participate in atherogenesis. Nonenzymatic glycation should not be confused with enzymatic glycosylation, which requires glycosyltransferases and nucleotide sugar donors.

The excess glucose in diabetes mellitus also increases production of UDP-GlcNAc through the glucosamine:fructose aminotransferase (GFAT) pathway. This leads to increased hyaluronan production (Chapter 16) as well as increased O-GlcNAc levels on nuclear and cytoplasmic glycoproteins, which in turn alters the phosphorylation of the same proteins and their functions (Chapter 19). Specific molecular mechanisms involving such altered O-GlcNAcylation have been defined in animal models for 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.

A very serious diabetic complication with high mortality is kidney dysfunction. Initial slight urinary albumin excretion (microalbuminuria) progresses to frank macroalbuminuria, to nephrotic syndrome, and to end-stage renal disease. The proteinuria has been correlated with a reduction in the HS proteoglycan content of the glomerular basement membrane. The underlying mechanism may involve a reduction in HS synthesis by glomerular epithelial cells that may, in turn, be caused by the high glucose in the environment. One theory is that the resulting decrease in anionic charge and loss of HS proteoglycan are thought to affect the porosity of the glomerular basement membrane, although recent genetic evidence in mice questions this hypothesis. Interestingly, high glucose also mediates increased plasminogen activator inhibitor-1 (PAI-1) gene expression in renal glomerular mesangial cells, via O-GlcNAc-mediated alterations in Sp1 transcriptional activity.

Role of Gut Epithelial Glycans in Gastrointestinal Infections

Numerous gastrointestinal pathogens interact with the gut mucosa via recognition of glycan structures (Chapter 37). Important examples are cholera toxin, which binds GM1 ganglioside, and Helicobacter pylori, the cause of peptic ulcers and gastritis, which binds Lewis type glycans in the stomach mucosa. A possible therapy is consuming soluble glycan inhibitors to block attachment of these gut pathogens. In this regard, it is interesting that a time-honored treatment for peptic ulcer disease (before the indentification of H. pylori) was a combination of antacids and milk (which contains large amounts of free sialyloligosaccharides). Indeed, the variety and high concentrations of free glycans found in human milk (especially in the early days after birth of the baby) do impede the ability of various gut pathogens to bind to the infant mucosa.

Heparan Sulfate Proteoglycans in the Pathogenesis of Protein-Losing Enteropathy

Protein-losing enteropathy (PLE) is the enteric loss of plasma proteins; however, 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, tumor necrosis factor-α (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 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.

Changes in Sialic Acid O-Acetylation in Ulcerative Colitis

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 lost in ulcerative colitis. Whether this contributes to the pathology is unknown, but these modified sialic acids are more resistant to bacterial sialidases. Some studies suggest heparin treatments improve the symptoms, but results are still inconclusive.

Clinical Use of Heparin as an Anticoagulant

Heparin (a highly sulfated form of HS; Chapter 17) is primarily derived from porcine intestines or bovine lungs. It is a fast-acting and potent anticoagulant that is often used to avoid thrombosis, in procedures such as 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 Factor IIa require a long chain that interacts both with the antithrombin and with the IIa 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 IIa levels. A synthetic pentasaccharide that binds and facilitates antithrombin inactivation of Factor Xa has thus been introduced as an alternative to heparin. Although these improvements have been valuable, it must be kept in mind that the original 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 being reduced or even eliminated by the switch to the low-molecular-weight heparins and the synthetic pentasaccharide.

A rare but feared complication of heparin treatment is heparin-induced thrombocytopenia. The pathogenesis appears to involve the formation of complexes between heparin and platelet factor-4 and the generation of antibodies against the complexes. These antibodies in turn then deposit on platelets, causing their aggregation and loss from circulation. Somewhat paradoxically, this process results in exaggerated thromboses, rather than bleeding. The incidence of this complication appears to be lower with the use of low-molecular-weight heparins, and may be absent with the use of the pure pentasaccharide.

Selectin Inhibition to Obviate Sickle Cell Crises

Sickle cell anemia is an inherited disorder of hemoglobin that leads to various acute and chronic painful complications. Although it was the first defined human genetic disorder, 40 years later there is still no effective therapy. 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 sialyl Lewis x or heparin can restore blood flow in a mouse model of sickle cell disease. The pan-selectin inhibitor GMI-1070 (now rivipansel) recently showed a dramatic benefit for patients with acute pain of sickle cell crisis, as it greatly reduced (>80%) their need for intravenous opioid analgesics.

Hemolytic Transfusion Reactions

Blood transfusion medicine first identified the ABO blood group system, defined by different alleles of an α-Gal(NAc) transferase (Chapter 14). These and other less prominent glycan antigens cause hemolytic transfusion and rejection reactions, mostly because of errors in blood typing. 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.

Acquired Anticoagulation Due to Circulating Heparan Sulfate

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.

Abnormal Glycosylation of Plasma Fibrinogen in Liver Disorders

Plasma fibrinogen is heavily sialylated and the sialic acids are involved in binding calcium. Certain genetic disorders of fibrinogen are known to be 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 can present clinically as a bleeding disorder associated with a prolonged thrombin time. Patients with congenital genetic disorders 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

Paroxysmal nocturnal hemoglobinuria (PNH) is an unusual form of acquired hemolytic anemia (excessive destruction of red blood cells) that usually appears in adults. The defect arises through a somatic mutation in bone marrow stem cells that causes the production of 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 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 these patients later also develop acute leukemia. It is now known that 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 did not become prominent contributors to the total pool of circulating red blood cells. In this scenario, the independent occurrence of a process damaging other stem cells allows the “unmasking” of the PNH defect.

Paroxysmal Cold Hemoglobinuria

Patients with the rare disorder paroxysmal cold hemoglobinuria have a cold-induced intravascular destruction of red cells (hemolysis), which appears to be caused by a circulating IgG antibody directed against the red cell P blood group antigens (Chapter 14). Biosynthesis of the P blood group antigens is dependent on B3GALNT1 (β1-3-N-acetylgalactosaminyltransferase 1) and A4GALT (α1-4-galactosyltransferase) genes. The pathogenesis of this disorder is unknown, but tends to occur in the setting of some viral infections and in syphilis. The IgG antibody is shown 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. Hemolysis occurs after warming back to 37°C.

Cold Agglutinin Disease

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 typically directed against the so-called “i” antigen (poly-N-acetyllactosamine), is 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 erythrocytes. Some 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.

Tn Polyagglutinability Syndrome

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 (Siaα2-6GalNAcα-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, leukemic clones that arise later may not have the same defect.

Changes in IgG Glycosylation in Rheumatoid Arthritis

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 with the level of galactosylation. Spontaneous clinical improvement occurs during pregnancy and correlates with restored galactosylation. 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 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 possibility is that the altered glycosylation changes interactions with Fc receptors. The basis of this decreased galactosylation in RA is unknown. Some evidence points to less β-galactosyltransferase activity in patients’ lymphocytes, but it is not known if the altered glycosylation of IgG has a specific pathogenic role in rheumatoid arthritis, because G0 molecules are seen in other unrelated diseases including chronic granulomatous diseases, Crohn's disease, and tuberculosis. The glycan changes are also seen to a lesser extent in osteoarthritis, a form of chronic degenerative arthritis with a different pathogenesis. Overall, this observation is an interesting phenomenon, but its significance in pathogenesis is unresolved.

Urinary Tract Infections

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, possible nonantibiotic treatment for this very common infection is D-mannose, which competes with the bacterial glycan binding to the urinary tract when it is excreted in the urine. Another alternative is optimized synthetic α-mannosides, which are promising candidates to treat and prevent UTIs.

Recognition of Glycans by Bacterial Adhesins, Toxins, and Viral Hemagglutinins

Many pathogens initiate infection by binding to 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, 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.

Desialylation of Blood Cells by Circulating Microbial Sialidases during Infections

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 enhanced clearance and anemia. Measuring plasma sialidase may aid diagnosis and prognosis. Sialidase-producing Streptococcus pneumoniae can cause hemolytic-uremic syndrome infections and selectively inhibiting their sialidase could have therapeutic value. On the other hand it appears that the rapid clearance of desialylated platelets by the Ashwell–Morell receptor in the liver may serve to protect againt unwanted disseminated intravascular coagulation.

Loss of Glomerular Sialic Acids in Nephrotic Syndrome

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 Vibrio 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.

Changes in O-Glycans in IgA Nephropathy

In humans, only IgA1 and IgD immunoglobulins contain O-glycans in the hinge regions, whereas all other immunoglobulin classes contain N-glycans in the Fc domain. Aggregation of the IgA1 molecule causes glomerulonephritis called IgA nephropathy. Studies of the O-glycans on serum IgA1 showed glycan truncations in the IgA nephropathy group compared with a negative control group. The IgA1 O-glycan chains are thought to stabilize the three-dimensional structure of the molecule. Studies of heat-induced aggregation support the notion that the altered glycosylation results in a loss of conformational stiffness, perhaps explaining the aggregation phenomenon. It appears that underglycosylation of the IgA1 molecule in IgA nephropathy is involved in the nonimmunologic glomerular accumulation of IgA1. The primary mechanism of underglycosylation remains unknown. One scenario is a defect in the C1GALT1C1 gene encoding 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 IgA.

Heparan Sulfate Changes in Systemic Lupus Erythematosus

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 situation 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).

Pathogenic Autoimmune Antibodies Directed against Neuronal Glycans

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 lipooligosaccharides 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.

Role of Glycans in the Histopathology of Alzheimer's Disease

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 multiply 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 are needed. Recently, genome-wide association studies (GWASs) showed a strong correlation between Alzheimer's disease and the higher expression of a form of CD33 (Siglec-3) that is expressed in brain microglia and may be suppressing clearance of amyloid.

Role of Selectins, Siglecs, and Mucins in Bronchial Asthma

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.

Role of Selectins in Acute Respiratory Distress Syndrome

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.

Altered Glycosylation of Epithelial Glycoproteins in Cystic Fibrosis

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.

Altered Glycosylation in Pulmonary Vascular Disease

Pulmonary vascular diseases (PVDs) include pulmonary embolism, pulmonary arterial hypertension (PAH), and arteriovenous malformations (AVMs). These diseases increase pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP), ultimately leading to right ventricular hypertrophy and heart failure. PAH is the best studied—a progressive disease that shows nitric oxide deficiency, vasoconstriction, in situ 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.