Alzheimer disease (AD) is the most common neurodegenerative disease, affecting ~ 30 million people. Age is the most important risk factor, and as the life span of the population increases, there will be a steep increase in the number of AD cases in the coming years. Presently, only symptomatic treatment is available, but there is an ongoing massive research effort aimed at finding disease-modifying drugs. Neuropathologically, the disease is characterized by extracellular deposits composed of fibrils formed by amyloid β-peptide (Aβ), and intraneuronal tangles composed of hyperphosphorylated forms of the microtubule-associated protein tau. The tangles contain paired helical filament (PHF) structures. Since the discovery of Aβ 30 years ago, there has been intense research on this ~ 40-residue peptide, and several lines of evidence suggest that the polymerization of Aβ into neurotoxic aggregates is a key event in the pathological cascade that results in AD. Aβ is derived from its type 1 transmembrane (TM) precursor, amyloid precursor protein (APP), by the proteolytic action of β-secretase and γ-secretase. β-Secretase, or BACE-1 (β-site APP-cleaving enzyme 1), mediates the initial cleavage that generates soluble APPβ and a C-terminal membrane-bound fragment, C99, which is the immediate substrate for γ-secretase. Whereas BACE-1 consists of a single protein, γ-secretase is an assembly of at least four different proteins: presenilin (PS)1 or PS2, nicastrin, anterior pharynx-defective 1, and PS enhancer 2. γ-Secretase cleaves its substrate within the TM region, thereby releasing the APP intracellular domain, which could possibly be involved in transcription. The 48-residue or 49-residue C-terminal stub left in the membrane is thereafter further processed into different Aβ variants. The 40-residue variant, Aβ40, is the most common product, but it is the two residue longer Aβ42 that mediates most of the toxicity . Interestingly, the mutations that lead to familial forms of AD with an early onset (< 65 years of age) are found in APP or PS, and lead to increased Aβ production or an increased Aβ42/Aβ40 ratio .
Many of the drugs tested in clinical trials are aimed at lowering the Aβ levels in the brain, and the most popular strategies have been to reduce Aβ production by inhibiting β-secretase or γ-secretase, or to increase Aβ clearance with immunological approaches. Most of the trials have shown poor results, and it has been suggested that Aβ could be the wrong target, that the drug concentration in the central nervous system is too low, or that the clinical trials should start before the patients show clinical symptoms. In support of the last of these, recent data from a passive vaccination trial showed significant improvement in mild, but not in moderate, AD patients . Thus, there is an immediate need for an early biomarker of AD that could be used for selecting presymptomatic cases for clinical trials, and, when there is a drug on the market, to enable treatment to be started at an early stage of the disease. Furthermore, we need to expand our knowledge of the disease process in order to find complementary or alternative treatment strategies. One line of research that could be more thoroughly investigated is the role of post-translational protein modifications, in particular protein glycosylation. It is estimated that > 50% of all proteins are glycosylated, and glycoproteins usually exist in many glycosylation variants, giving rise to multiple gene products from one gene. Thus, glycans contribute significantly to proteome expansion in higher organisms and are vital for brain functions, including memory and learning . As several studies have suggested that protein glycosylation is altered in AD, this relatively unexplored topic deserves more attention, and could potentially be highly important for the development of improved biomarkers and treatment methods for AD. In the present review, we will summarize the current knowledge on the role of glycosylation in AD.
We will give a brief summary of protein glycosylation, but, for a comprehensive description of the topic, we refer to textbooks, e.g. Essentials of Glycobiology .
Glycans are referred to here as monosaccharides, oligosaccharides, or polysaccharides, either free or bound to glycoproteins. The smallest building blocks of glycans are cyclic forms of monosaccharides. Two stereoisomeric foms, denoted anomeric forms, can result upon cyclization of monosaccharides, generating the isomers α or β (Fig. 1A). In oligosaccharides and polysaccharides, monosaccharide units are held together by glycosidic linkages, i.e. covalent bonds formed between the anomeric carbon of one monosaccharide and an OH-group of another monosaccharide. Glycans have a nonreducing end and a reducing end, which has an anomeric carbon that is not involved in a glycosidic bond (Fig. 1B). The glycosidic bonds can exist in two stereoisomeric forms. These are named according to the position of the anomeric carbon in the nonreducing end monosaccharide followed by the isomeric form and the position of the OH-group to which the anomeric carbon is attached in the reducing end monosaccharide (Fig. 1B). Many monosaccharide units in glycans are five-carbon or six-carbon units, but more complicated monosaccharide units, such as sialic acid, also exist (Fig. 1C).
Figure 1. Examples of sugar structures. (A) Anomeric forms of Glc. In the α-isomer, the OH-group of the anomeric carbon and the CH2OH group at C5 point towards different sides of the plane of the ring, whereas in the β-form, they point to the same side. (B) Maltose, which is a disaccharide containing two α-d-Glc residues joined by an α-1,4-glycosidic bond. The reducing end is the side that is attached to proteins. (C) The structures of other monosaccharides that are important building blocks in glycans, including α-d-Man, β-d-Gal, α-l-Fuc, α-d-GlcNAc, and α-d-NeuAc, the sialic acid form present in humans. The image was drawn with accelrysdraw 4.1.
Download figure to PowerPoint
The functional properties of glycans in glycoproteins can be broadly divided into two categories: (a) structural and modulatory functions; and (b) recognition of glycans by other molecules. Glycans thus modulate cell–cell, cell–matrix and cell–molecule interactions, and are involved in many important processes in complex organisms, including the assembly and development of multicellular organs. They act as signals that determine how the glycoproteins are processed within the cell and which compartments they are targeted to. Glycans are also important in immunology, as the blood group antigens are determined by glycan epitopes. In infections, glycans mediate pathogen–host cell interactions. Specialized forms of intracellular glycans, which are rapidly added and removed, function as molecular switches. However, owing to the complexity of glycans and the resulting challenges in studying them, it is likely that many glycan functions remain to be revealed. Glycans are typically covalently linked to either an asparagine (N-glycans) or serine/threonine (O-glycans) residue on the glycoproteins.
N-glycosylation begins in the endoplasmic reticulum (ER) with the addition of a precursor oligosaccharide, Glc3Man9GlcNAc2 (Fig. 2), which is transferred from the lipid dolichyl-pyrophosphate (dolichyl-PP) to the luminal side of a polypeptide chain. This usually occurs on a growing polypeptide during protein translation. The sugar is typically attached to the asparagine within the sequence Asn-X-Thr/Ser, where X can be any amino acid except proline. A TM enzyme complex in the rough ER called the oligosaccharyl transferase complex (OST) catalyzes the transfer of the precursor oligosaccharide to the protein . Human OST is composed of the seven protein subunits ribophorin I, ribophorin II, defender against apoptotic cell death 1, N33/IAP, OST4, STT3, and OST48. STT3 is the catalytic subunit, and exists in two isoforms, A and B, which show somewhat different substrate selectivities. The sugar chain is then processed in the ER lumen by the sequential removal of glucose residues by α-glucosidases I and II. Many glycoproteins are further processed by ER α-mannosidase I, which removes the terminal mannose (Man) from Man9GlcNAc2 to yield Man8GlcNAc2. Thus, most glycoproteins enter the cis-Golgi carrying eight or nine Man residues. These structures are termed high-Man structures (Fig. 3A). In the cis-Golgi, Man5GlcNAc2 is formed by the action of α1,2-mannosidases IA, IB, and 1C. This is the intermediate for the generation of complex (Fig. 3B) and hybrid (Fig. 3C) types of N-glycan, which is initiated in the intermediate Golgi by an N-acetylglucosaminyl transferase; this is followed by processing by Golgi-resistent mannosidases and GlcNAc transferases. The Golgi complex also harbors enzymes to generate N-glycans with more than two branches (Fig. 3D,E) and bisecting GlcNAc, i.e. a GlcNAc attached to the first Man from the core (Fig. 3F). Further elongation of glycans during processing into mature N-glycans occurs mainly in the trans-Golgi network by the action of galactosidases and GlcNAc transferases to add GlcNAc and Gal residues. Some glycans are also processed by fucosidases, which add a fucose (Fuc) residue in an α1,6-linkage to the GlcNAc adjacent to asparagine in the core (Fig. 3G). The most important ‘capping’ or ‘decorating’ reactions, i.e. alteration of the terminal ends of glycans, involve the addition of sialic acid, Fuc, Gal, and GalNAc.
Figure 2. Dolichyl-PP–oligosaccharide: the structure of the dolichyl-PP-linked oligosaccharide, the precursor in the biosynthyesis of N-glycans. After the glycan has been attached to a polypeptide, it is processed while being transported through the ER–Golgi system to generate many different N-glycan structures. Blue square: GlcNAc. Green circle: Man. Blue circle: Glc. The image was drawn with glycoworkbench.
Download figure to PowerPoint
Figure 3. Nomenclature of some N-linked glycans. Glycans are broadly categorized into three major groups: (A) high-Man glycans, carrying only Man in addition to the core GlcNAc units; (B) complex glycans, which have more than one type of additional monosaccharide unit; and (C) hybrid glycans, carrying a mixture of high-Man and hybrid antenna. The glycans shown in (A)–(C) are called bi-antennary, as they have two branches. (D, E) Examples of tri-antennary and tetra-antennary glycans. (F) A glycan with a bisecting GlcNAc. (G) A core glycosylated glycan, with a Fuc attached to the innermost GlcNAc. Blue square: GlcNAc. Green circle: Man. Yellow circle: Gal. Purple diamond: NeuAc. Red triangle: Fuc. The image was drawn with glycoworkbench.
Download figure to PowerPoint
Some molecular tools for studying glycoproteins are as follows.
Inhibitors of glycosidases/glycosyltransferases: the functions of protein-linked glycans can be studied by treatment of cells or animal models with inhibitors of glycosidases or glycosyltransferases. One commonly used inhibitor is tunicamycin, which blocks the addition of N-linked glycans to glycoproteins by inhibiting the first step in the generation of dolichyl-PP–oligosaccharide. Deoxynojirimycin is a glucosidase I inhibitor that prevents the removal of Glc residues, and thereby increases the ER retention time. Mannosidase type 1 inhibitor (MNJ) inhibits the trimming of high-Man oligosaccharides, further preventing processing into complex oligosaccharides.
Enzymatic removal of glycans: peptide N-glycosidase F is a commonly used enzymatic tool that cleaves N-linked glycans between the innermost GlcNAc and the asparagines of high-Man, hybrid and complex oligosaccharides, leaving the entire released glycan intact. Endoglycosidase H (Endo H) cleaves between the two innermost GlcNAc residues, leaving one GlcNAc residue attached to the asparagine for oligomannose and most hybrid types of N-linked glycans, whereas complex glycans are not released. Neuraminidases (also called sialidases) catalyze the hydrolysis of terminal sialic acid residues, leaving the remaining glycan chain without this negatively charged monosaccharide.
Lectins: lectins are carbohydrate-binding proteins with high specificity for various sugar structures. They can thus be used for purification, ELISA, fluorescence microscopy, and many other methods. For example: concanavalin A (Con A) binds to α-d-Man and α-d-Glc residues; wheat germ agglutinin (WGA) binds to GlcNAc and sialic acid; Ricinus cummis agglutinin binds to Gal; and Lens culinaris agglutinin binds to α-d-Man.
O-glycosylation is more difficult to predict than N-glycosylation, although databases exist that determine the probability of a serine or threonine being O-glycosylated (e.g. NetOGlyc). In contrast to N-glycosylation, O-glycosylation occurs only after protein translation. Many O-linked glycans are linked via a GalNAc moiety to the protein. The first step in this type of O-glycosylation is the transfer of GalNAc from UDP-GalNAc to the amino acid, which is catalyzed by a polypeptide-N-acetyl-galactosaminyltransferase. Many different forms of polypeptide-N-acetyl-galactosaminyltransferase exist. The addition of the next glycan determines the core structure of the O-linked glycan. There are eight core structures (Fig. 4), which may be further substituted by other sugars. Thus, O-glycans differ from N-glycans in not having a common core structure. Many additional enzymes are involved in generating the final O-glycan structures, which are highly variable in size and composition. Some of the enzymes involved are common for N-glycans and O-glycans, and even glycolipids.
Figure 4. Core structures in O-glycans. The eight different core structures shown are attached to the OH-groups of Ser/Thr of glycoproteins (and in rare cases to Tyr). These core structures are further substituted and processed to form a huge variety of glycan structures. Yellow square: GalNAc. Blue square: GlcNAc. Yellow circle: Gal. The image was drawn with glycoworkbench.
Download figure to PowerPoint
Another, specialized, form of O-glycosylation is O-GlcNAcylation, which differs from other types of glycosylation by the product not being further processed after the addition of a single GlcNAc to serine or threonine. It also differs from other types of glycosylation by occurring on nuclear and cytoplasmic proteins, and by being more dynamic. O-GlcNAcylation is of importance in signal transduction, and addition of O-GlcNAc residues to a specific site on a protein and their release are regulated by the concerted action of O-GlcNAc transferase and O-GlcNAcase. O-GlcNAc and protein phosphorylation often compete for binding to either the same or proximal attachment sites, and thus interact in their signaling. Apart from the classes described above, there are additional types of O-glycans, e.g. in proteoglycans, that contain glycosaminoglycan chains with up to 200 monosaccharides, but those glycans will not be covered in this review.
Sialic acids comprise a group of monosaccharide units that are often found in the terminal position of the oligosaccharides of proteins. They are typically negatively charged at physiological pH, and are important recognition molecules for many cellular functions. The most common sialic acid found in mammalian cells is N-acetylneuraminic acid (NeuAc) (Fig. 1C). The enzymes that catalyze the transfer of sialic acids to glycans, STs, are membrane-bound proteins in the Golgi apparatus. Several forms with different specificities exist, and cleaved forms have been found in body fluids, e.g. serum and milk. Human STs typically add sialic acid to the nonreducing terminal position (Fig. 1B) of glycans in α-2,3-linkage or α-2,6-linkage to a Gal residue, or α-2,6-linkage to GalNAc or GlcNAc. Sialic acids are also found α-2,8-linked to sialic acid residues in gangliosides and in polysialic acid, which is expressed on, for instance, neural cell adhesion molecule (NCAM). ST6GalI is a member of the human ST family, and transfers sialic acid with an α-2,6-linkage to a terminal Gal residue of Gal-1-4GlcNAc disaccharide found as a free disaccharide or as a terminal disaccharide of N-linked or O-linked oligosaccharides.