Essentials of Glycobiology Lecture 36 Ajit Varki

1. Essentials of Glycobiology Lecture 36Ajit Varki Changes in Glycosylation in Systemic Diseases (other than Cancer)

2. CARDIOVASCULAR MEDICINE DERMATOLOGY ENDOCRINOLOGY AND METABOLISM GASTROENTEROLOGY HEMATOLOGY IMMUNOLOGY AND RHEUMATOLOGY INFECTIOUS DISEASE NEUROLOGY AND PSYCHIATRY PULMONARY MEDICINE ONCOLOGY (already covered in lecture 35) Changes in Glycosylation in Systemic Diseases

3. Role of Selectins in Reperfusion Injury Role of Selectins, Glycosaminoglycans and Sialic Acids in Atherosclerosis Cardiovascular Medicine

4. P-SELECTIN SELECTINS ACTIVATION EXIT OF WHITE BLOOD CELLS FROM THE CIRCULATION INTO TISSUES INVOLVES SELECTINS Platelets White cell Blood vessel wall INJURY INFLAMMATION EARLY PHASES OF BLOOD VESSEL INJURY RESPONSE ALSO INVOLVES SELECTINS

5. Many cardiovascular disorders (stroke, myocardial infarction, hypovolemic shock, etc.) characterized by period of decreased/absent blood flow, followed by re-perfusion (natural or therapeutic). Despite tissue rescue from anoxemic necrosis, entry of leukocytes into reperfused area results in tissue damage. P-selectin on activated endothelium in reperfused area and L-selectin on leukocytes play a key role in mediating initial steps of this cascade Substantial data in animal models indicates that blockade of initial selectin-based recognition can ameliorate subsequent tissue damage. Therapeutic potential in humans is likely, but remains unrealized Cardiovascular Medicine Role of Selectins in Reperfusion Injury

6. Pathology of Atherosclerosis

7. Earliest step in atherosclerosis involves entry of monocytes into subendothelium via endothelial P-selectin recognition of PSGL-1 ligand on circulating monocytes. Induction of endothelial P-selectin expression may result from oxidized lipids that are present in low-density lipoprotein (LDL) particles Atherosclerotic lesions in LDL Receptor-deficient mice show delayed progression in a P-selectin deficient background and even slower progression in combined P- and E-selectin deficiency. Therapeutic significance of the above facts are unexplored Glycobiology of Atherosclerosis (Selectins)

8. Subendothelial retention of LDLs in early atherosclerotic plaque thought occur at least partly via interactions with matrix proteoglycans. Interactions thought to cause irreversible structural alterations of LDL, potentiating oxidation and uptake by macrophages and smooth muscle cells. At molecular level, clusters of basic amino acids in Apo-B (protein moiety of LDL) thought to bind negatively charged GAG chains of proteoglycans. Some evidence for reduction of atherosclerosis in animals with heparin treatment. Practical therapeutic significance in humans unknown. Glycobiology of Atherosclerosis (Glycosaminoglycans)

9. Many reports of lowered sialylation of circulating LDL in patients with coronary artery disease. Pathophysiological significance of this finding in unclear Mechanism(s) involved remain unclear. Recent paper claims a serum LDL-associated trans-sialidase that transfers sialic acids from LDL to other serum proteins! Pathological hypothesis is that the desialylated LDL is more prone to be taken up and incorporated into atheromatous plaques. However, there are also some contradictory data in the literature concerning this issue Practical therapeutic significance in humans unknown. Glycobiology of Atherosclerosis (Sialic Acids)

10. Dermatology Role of Selectins in Inflammatory Skin Diseases

11. Some inflammatory skin diseases (e.g., atopic dermatitis, contact dermatitis) involve entry of T-helper (Th1) lymphocytes with a pathogenic role into dermis. Such skin lesions sometimes associated with persistent expression of E- and/or P-selectin on skin endothelial cells. E-selectin can recruit circulating Th1 lymphocytes carrying the Cutaneous Lymphocyte Antigen - a specific E-selectin ligand epitope carried on a subset of PSGL-1 molecules. P-selectin binding form of PSGL-1 is up-regulated in activated Th1 cells and also serves to mediate exit. Consistent observations made in experimental models, including selectin and selectin-ligand deficient animals Potential therapy in humans unexplored Dermatology Role of Selectins in Inflammatory Skin Diseases

12. Diabetes mellitus (DM) - dysregulated glucose metabolism resulting from absolute (Type I) or relative (Type II) lack of insulin action Characteristic long-term vascular and neurologic complications are the major causes of death High glucose in body fluids accelerates normal process in which open-chain (aldehyde) form of glucose reacts with lysine residues on proteins, giving reversible Schiff bases (non-enzymatic glucosylation). Adducts undergo irreversible Amadori rearrangement and "browning" (Maillard) reactions, giving Advanced Glycation End products (AGE). Resulting protein cross links can affect cell functions, and AGE can is recognized by some receptors (e.g., RAGE, macrophage scavenger receptor, perhaps participating in acceleration of atherogenesis). Is this a normal process of aging accelerated in the setting of the chronic persistent hyperglycemia of uncontrolled diabetes mellitus? Endocrinology and MetabolismNon-enzymatic Glycation and Complications of Diabetes Mellitus

13. Increased production of UDP-GlcNAc caused by the conversion of excess glucose via the Glucosamine:Fructose Aminotransferase (GFAT pathway). One hypothesis is that increased cytosolic UDP-GlcNAc gives rise to secondary increase of O-linked-GlcNAc levels on nuclear and cytosolic glycoproteins. Endocrinology and MetabolismAltered Glycosylation and Complications of Diabetes Mellitus GFAT

14. Nephropathy - diabetic complication with high mortality. Begins with microalbuminuria, progresses to nephrotic syndrome (macroalbuminuria) and then decreased glomerular filtration rate - finally end-stage renal disease. Proteinuria correlated with reduction in heparan sulfate proteoglycan of glomerular basement membrane. Underlying mechanism may involve reduction in HS synthesis by glomerular epithelial cells, somehow caused by the high glucose in the environment. Decrease in anionic charge thought to affect porosity of the glomerular basement membrane. N-deacetylase:N-sulfotransferase that plays a key role in heparan sulfate biosynthesis has reduced activity in poorly regulated diabetic animals. Endocrinology and MetabolismAltered Glycosylation and Complications of Diabetes Mellitus

15. Most gastrointestinal pathogens bind first to GI mucosal glycans. Changes in mucosal glycosylation associated with age, genetic background and/or diet may affect variation in susceptibility to certain infections. Ulcerative Colitis - non-infectious acquired disease with remitting and relapsing segmental ulcerations of the colon. Primary pathogenetic mechanisms remain unknown Sialic acids of normal colon are heavily O-acetylated Loss of sialic acid O-acetylation is seen in Ulcerative Colitis, but it is not known if this is cause or effect. The types of mucin molecules produced and their glycosylation can also be altered in Ulcerative Colitis. Again, it is not known if this is cause or effect. Some evidence for selectin involvement in leukocyte flux into colonic mucosa (beneficial effects of heparin reported!) GastroenterologyInfections and Ulcerative diseases of the GI tract

16. N-glycans in constant (CH2 or Fc) region of human IgG are buried between the folds of the two constant regions. Chains often immobilized by carbohydrate-protein interactions which can be seen in crystal structure (glycans typically invisible in crystals). Immunology and RheumatologyChanges in Immunoglobulin G Glycosylation in Rheumatoid Arthritis(RA).

17. Biantennary complex type N-glycans of IgG hardly ever completed into fully sialylated molecules. Most molecules remain with one or more terminal beta-linked Gal residues (so-called G1 and G2 molecules). In Rheumatoid Arthritis, major fraction of glycans on serum IgG molecules have decreased galactosylation, some carrying no galactose at all (so-called Go molecules). Severity of disease correlates with % Go, and spontaneous improvement during pregnancy correlates with increases in G1 and G2. Hypothesis: Fc N-glycans maintain conformation of Fc domains as well as the hinge regions - necessary for effector functions e.g., complement binding and antibody-dependent cytotoxicity. Immunology and RheumatologyChanges in Immunoglobulin G Glycosylation in Rheumatoid Arthritis(RA).

18. NMR studies indicate G0 glycans have increased mobility resulting from loss of interactions with Fc protein. Regions of protein surface normally covered by glycans may be exposed in RA. Some studies suggest that the more mobile G0 glycans are recognized by circulating mannose binding protein, activating complement directly. RA also characterized by immune complexes consisting of antibodies (rheumatoid factor) directed against Fc region of other IgG molecules. Immunology and RheumatologyChanges in Immunoglobulin G Glycosylation in Rheumatoid Arthritis(RA).

19. Lowered activities of beta-galactosyltransferase reported in lymphocytes from patients with RA. However, appearance of Go molecules is a general feature of other unrelated chronic granulomatous diseases as well as osteoarthritis (non-autoimmune arthritis of the elderly) Change in IgG glycosylation in RA remains an interesting phenomenon whose significance and pathogenic role have yet to be defined. Regardless, it may be of diagnostic and prognostic value. Immunology and RheumatologyChanges in Immunoglobulin G Glycosylation in Rheumatoid Arthritis(RA).

20. Inherited disease with skin eczema, altered cellular immune response and low platelet counts Early studies suggested absence of CD43 (leukosialin) the major O-glycosylated cell surface protein of leukocytes. Polypeptide is actually expressed normally, but has slower gel mobility because of increased branching of O-glycans. Recent data indicate that the primary defect is not in glycosylation, but in a transcription factor. However, glycan changes seen in resting T cells are same as those that are induced by activation of normal T cells. Remains possible that some aspects of the immune disorders are due to a secondary change in glycosylation Immunology and Rheumatology Changes in the O-glycans of CD43 in Wiskott-Aldrich Syndrome.

21. Immunology and Rheumatology Changes in the O-glycans of CD43 in Wiskott-Aldrich Syndrome.

22. Several microbes produce sialidases (neuraminidases) that are “virulence factors” in pathogenesis of diseases that they cause. In some severe infections, e.g. Clostridium perfringens gas gangrene, sialidase can appear in the plasma. Circulating blood cells and proteins can become desialylated, giving enhanced clearance and anemia. Detection of the circulating sialidase has been proposed to have diagnostic and prognostic significance. Whether inhibiting the sialidase with appropriate inhibitors will have a therapeutic value has not been investigated. Infectious DiseaseGlycan Recognition by Bacterial Adhesins, Toxins & Viral Hemagglutinins. Desialylation of Blood Cells by Microbial Sialidases During Infections.

23. Nephrotic syndrome - kidney glomerulus fails to retain serum proteins during filtration, allowing spill into urine. The mucin podocalyxin on foot processes (pedicles) of glomerular podocytes helps maintain pore integrity - excludes large proteins from filtrate. Sialic acid residues of podocalyxin critical in this process. Loss of glomerular sialic acid in "minimal change renal disease" and in nephrotic syndrome following infections. Proteinuria and renal failure after inoculation of sialidase in rats - loss of glomerular sialic acids and effacement of foot processes. Aminonucleoside nephrosis in rats by Puromycin injection. Defective sialylation of podocalyxin and glomerular glycosphingolipids detected. NephrologyLoss of Glomerular Sialic Acids in Nephrotic Syndromes

24. Non-immunologic glomerular accumulation of aggregated IgA1, with resulting damage and nephrotic syndrome. O-glycans on serum IgA are truncated. O-glycans may stabilize tertiary structure of IgA. Studies of heat-induced aggregation indicate that altered O-glycans on hinge region gives a loss of conformational stiffness. Removal of O-glycans from IgA1 gives noncovalent self-aggregation and a increased adhesion to ECM proteins. Underglycosylation of the IgAl molecule found in IgA nephropathy is likely to be involved in the pathogenesis. Primary mechanisms causing the underglycosylation remain unknown (Cosmc defect causing decreased Core 1 production?) NephrologyChanges in IgA O-glycans in IgA Nephropathy

25. Diseases associated with circulating antibodies against glycans enriched in nervous system - autoimmune damage. Antibodies arise via at least three distinct mechanisms. 1. Benign or malignant B cell neoplasms (benign monoclonal gammopathy of unknown significance - MGUS, Waldenstrom's Macroglobulinemia, plasma cell myeloma) can secrete monoclonal IgM or IgA specific for either ganglio-series gangliosides, or, sulfated glucuronosyl glycans (“HNK-1 epitope”). Resulting demyelinating peripheral neuropathy sometimes more damaging than primary disease. Therapy - try to treat primary disease with chemotherapy, or attempt removal of immunoglobulin by plasmapheresis. Both approaches are usually unsuccessful. Neurology and PsychiatryPathogenic Autoimmune Antibodies Directed Against Neuronal Glycans

26. GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN Neural Glycosphingolipids as Target Antigens of Paraproteins in Monoclonal Gammopathies GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P S N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein OUTSIDE P INSIDE O-LINKED GlcNAc O Ser

27. 2. Immune reaction to molecular mimicry of ganglioside structures by lipooligosaccharides (LOS) of bacteria like Campylobacter jejuni. Following intestinal infection, circulating antibodies against gangliosides appear. Associated with onset of demyelinating neuropathy involving peripheral or central nervous systems: Guillain-Barre syndrome or Miller-Fisher syndromes, respectively. Therapy -support patient until antibody production fades. Potential for active removal of antibodies to be explored. 3. “Iatrogenic” disease arising from recent attempts to treat stroke patients with intravenous bovine brain gangliosides. Some evidence that treatment is beneficial for primary disease. However, several cases of Guillain-Barre reported as a likely side-effect. Traces of Neu5Gc in bovine gangliosides may play a role in enhancing immunity Neurology and PsychiatryPathogenic Autoimmune Antibodies Directed Against Neuronal Glycans

28. Common primary degenerative dementia of humans with insidious onset and progressive course. Ultimate diagnosis made by histological examination of brain - characteristic amyloid plaques with neurofibrillary tangles that are associated with neuronal death. Paired helical filaments are major component of neurofibrillary tangles- composed of microtubule-associated protein tau. Tau is in a hyperphosphorylated state and no longer binds microtubules - self-assembles to form the paired helical filaments. Normal brain tau is multiply modified by Ser(Thr)-O-linked GlcNAc. Hypothesis: site-specific or stoichiometric changes in O-GlcNAc may allow excessive phosphorylation, causing paired helical filaments. Glycobiology of Alzheimer's Disease.

29. Hyperphosphorylated tau in Alzheimer's disease brain is found in association with heparan sulphate proteoglycans. Non-phosphorylated tau isoforms with three microtubule-binding repeats form paired helical-like filaments under physiological conditions in vitro, when incubated with heparan sulphate. Heparin also prevents tau from binding to microtubules and promotes microtubule disassembly. Heparin stimulates tau phosphorylation by protein kinases Thus, HS-GAGs may be a critical factor in forming neurofibrillary tangles. However, no significant difference between structure of HS obtained from control and Alzheimer's disease brains. Note: Topological separation of tau (in cytosol) from GAGs (extracellular) - thus, association can only occur after cell death. Heparan sulfate proteoglycans may also play an important role in regulating amyloid plaque deposition. Glycobiology of Alzheimer's Disease (continued).

30. Asthma - hyper-responsiveness of tracheobronchial tree, giving widespread airway narrowing - changes in severity, either spontaneously or from therapy. Two dominant pathological features are airway wall inflammation and luminal obstruction of airways by inflammatory exudate. Likely due to antigen-specific IgE antibodies, which become fixed to mast cells and basophils. Antigen cross-links IgE molecules, triggering an explosive release of vasoactive, bronchoactive, and chemotactic agents from mast cell granules into the extracellular milieu. Eosinophils may contribute to pathogenesis of asthma in several ways Evidence indicates that E and P-selectins are involved in the recruitment of eosinophils and basophils into the lung. Inhibitors of selectin should be explored. Pulmonary MedicineRole of Selectins in Bronchial Asthma.

31. Life-threatening final common pathway of lung injury arising from many events (e.g., shock, trauma and sepsis). Diffuse pulmonary endothelial injury, progressing to pulmonary edema due increase in capillary permeability. Selectins and integrins help circulating neutrophils to adhere to endothelium and release injurious oxidants, proteolytic enzymes, and arachidonic acid metabolites, resulting in endothelial cell dysfunction and destruction. Many neutrophils and products in bronchoalveolar lavage liquid emphasizing critical role of inflammatory response. Unfulfilled hope - selectin inhibitors used in early stages syndrome, before progression to lung damage and respiratory failure. Pulmonary MedicineRole of Selectins in the Acute Respiratory Distress Syndrome.

32. CF- common genetic disorder caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR). Causes defective chloride conduction across the apical membrane of epithelial cells. Associated with increased production of viscous mucins in the gut and lungs, which cause many of the symptoms. Reductions in sialylation and increases in the sulfation and fucosylation of mucus glycoproteins in CF. Higher Golgi pH in CF MAY cause abnormal glycosylation (controversial) Major cause of morbidity is colonization of respiratory epithelium by an alginate-producing form of Pseudomonas. Certain glycolipids are Pseudomonas receptors that help to maintain the colonization. Defects in Golgi sialylation as well as production of a sialidase by bacteria may help to enhance production of binding targets for colonization. Pulmonary MedicineAltered Glycosylation of Epithelial Glycoproteins in Cystic Fibrosis (CF)

33. GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN Paroxysmal Nocturnal Hemoglobinuria: Somatic Loss of Glycophospholipid Anchors in Hematopoietic Stem Cells GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

34. Mutation in PNH • The first step in biosynthesis of the GPI anchor requires at least four genes • One of them, PIG-A is an X-linked gene Biosynthesis of GPI anchors

35. Acquired clonal hematopoietic stem cell disorder - intravascular hemolytic anemia. Abnormal blood cells lack GPI-anchored proteins due to mutation in the PIG-A gene. Lack of GPI-anchored complement regulatory proteins, such as decay-accelerating factor (DAF) and CD59, results in complement-mediated hemolysis and hemoglobinuria. Factors that determine why mutant clones expand have not been determined. Do existing PNH clones have a conditional growth advantage depending on some factor present in the marrow environment of PNH patients? However, cells with PNH phenotype found at frequency of 22 per million in normal adults. These rare cells collected by flow sorting had PIG-A mutations. Thus, PIG-A gene mutations are not sufficient for the development of clinically evident PNH. Paroxysmal Nocturnal Hemoglobinuria

36. Biosynthesis of O-linked Glycans GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN O-Glycans as Antigens in the Polyagglutinin Syndromes GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

37. “Mixed Field Polyagglutination” - acquired condition - red cells become agglutinable with great majority of adult sera. Causes increased turnover of red cells and platelets In many instances, red cell membrane has exposed one (or more) latent O-glycan based antigens, i.e. T, Tn or sialylTn. Adult sera contain IgM antibodies directed against these antigens. When targets are exposed, cells are polyagglutinable upon testing with these sera. Transient Mixed Field Polyagglutination can be seen after various bacterial infections, probably due to entry of bacterial enzymes into the bloodstream In chronic cases, Core 1 Gal-T activity that synthesizes T antigen is low in some affected cells. Gene appears to be repressed instead of mutated. Is the primary defect in Cosmc on the X chromosome? The chronic form can be precursor of acute leukemia Polyagglutination Syndromes

38. I Antigen i Antigen GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN Polylactosamines as Antigens in Cold Agglutinin Syndromes GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS O-LINKED CHAIN P GLYCOPHOSPHO- LIPID ANCHOR Etn S P N N-LINKED CHAIN N O Asn NH Asn Ser/Thr 2 INOSITOL Glycoprotein OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

39. Acquired Hemolytic Anemias caused by cold-reactive IgM antibodies that agglutinate normal RBCs in the cold, and fix complement, resulting in lysis in warmer regions. Many of these antibodies react with the i / I antigens and are actually encoded by germline sequences. Mycoplasma pneumoniae infections associated with cold agglutinins that may cause a mild to moderate auto-immune hemolytic anemia. Major host cell receptor for Mycoplasma is sialylated i/I Hypothesis: Mycoplasma Anti-Id Antibody 2 Mycoplasma I Antigen I Antigen Antibody 1 Cold Agglutinin Syndromes

40. Congenital dyserythropoietic anemia type II or HEMPAS is an inherited anemia caused by a glycosylation deficiency. Red cell membrane glycoproteins (e.g.,band 3 & 4.5, which are normally glycosylated with polylactosamines lack these glycans, which accumulate on red glycolipids instead. HEMPAS red cells carry incompletely processed N-glycans, suggesting defects in the N-acetylglucosaminyltransferase II (GnT-II) or alpha-mannosidase II (M-II) steps Family studies identified two cases in which the M-II gene is defective. HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test)

41. N-LINKED CHAIN (INCOMPLETELY PROCESSED) GLYCOSPHINGOLIPID (LARGER) CHONDROITIN SULFATE HYALURONAN Altered expression of N-Glycans and Polylactosamines in HEMPAS(Hereditary Erythroblastic Multinuclearity with Positive Acidified Serum lysis test) GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S -O-Ser NS Proteoglycan N-LINKED CHAINS O-LINKED CHAIN P GLYCOPHOSPHO- LIPID ANCHOR Etn S P N N O Asn NH Asn Ser/Thr 2 INOSITOL Glycoprotein OUTSIDE P INSIDE O-LINKED GlcNAc O Ser

42. Mutant mice in which the MII gene was inactivated by homologous recombination resulted in a HEMPAS-like phenotype. Linkage analysis of another kindred excluded M-II and GnT-II as primary defects Regardless of which gene is defective, HEMPAS is characterized by incomplete processing of N-glycans. Further studies of HEMPAS may identify hitherto unknown factors affecting N-glycan synthesis Some patients who present late in life may have an acquired form of the disease? HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test)

43. CARDIOVASCULAR MEDICINE DERMATOLOGY ENDOCRINOLOGY AND METABOLISM GASTROENTEROLOGY HEMATOLOGY IMMUNOLOGY AND RHEUMATOLOGY INFECTIOUS DISEASE NEUROLOGY AND PSYCHIATRY PULMONARY MEDICINE ONCOLOGY (already covered in lecture 35) Acquired Glycosylation Changes in Relation to Human Disease