Complex Carbohydrates Glycosaminoglycans(GAGs) Glycoproteins and proteoglycans

LECTURE OUTLINE Differences between glycoproteins and proteoglycans Structures of glycoproteins and proteoglycans Functions of glycoproteins and proteoglycans Synthesis and degradation of glycoproteins and proteoglycans Pathology related to glycoproteins and proteoglycans

Differences Between Glycoproteins and Proteoglycans Proteins conjugated to saccharides lacking a serial repeat unit Glycoproteins Protein>>carbohydrate Carbohydrate>>protein Proteoglycans Proteins conjugated to polysaccharides with serial repeat units Glycosaminoglycans Mucopolysaccharides

Glycoprotein Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide side-chains. The process of attaching the glycans is known as glycosalation. The sugar groups attached to glycoprotein can assist in protein folding or improve a proteins’ stability. 4

The disaccharide units contain either of two modified sugars, called amino sugars N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and an acidic sugar uronic acid such as glucuronic acid or iduronic acid. The amino group is usually acetylated.

In some glycosaminoglycans, one or more of the hydroxyls of the amino sugar is esterified with sulfate. The combination of these sulfate groups and the carboxylate groups of the uronic acid residues gives the glycosaminoglycans a very high density of negative charge. Keratan sulfate is an exception in which galactose is present, instead of an acidic sugar. Hyaluronic acid does not contain sulfate.

Structure of Glycosaminoglycans GAGs in the body are linked to core proteins ( except hyaluronic acid), forming proteoglycans (also called mucopolysaccharides). The GAGs extend perpendicularly from the core in a brush-like structure. e.g. in cartilage proteoglycan the GAGs present are chondriotin sulfate and keratan sulfate

Proteoglycan Aggregates Proteoglycan monomers associate with a molecule of hylauronic acid to form proteoglycan aggregates in association with linker proteins in a “bottle brush” arrangement. Association is not covalent but ionic between hyaluronic acid and the core protein. Stabilized by link proteins

Proteoglycan aggregate of the extracellular matrix One very long molecule of hyaluronan is associated noncovalently with about 100 molecules of the core protein aggrecan

Interactions between cells and the extracellular matrix with binding sites for both integrin and the proteoglycan

Linkage The linkage of GAGs to the protein core involves a specific trisaccharide composed of two galactose residues and a xylose residue (Gal-Gal-Xyl-O-CH2-protein).

The trisaccharide linker is coupled to the protein core through an O-glycosidic bond to a Serine residue in the protein. Some forms of keratan sulfates are linked to the protein core through an N-glycosidic bond. The protein cores of proteoglycans are rich in Serine and Threonine residues, which allows multiple GAG attachments.

STRUCTURE OF GLYCOPROTEINS One or more carbohydrate chains--covalently linked to a protein. The chains may be neutral or negatively charged. They are frequently branched. There are two types of glycosidic links: 1. O-glycosidic link O-glycosidic link between galactose or glucose and the hydroxyl group of hydroxylysine (i.e. collagen). Other O-linked glycoproteins have a glycosidic link between N-acetyl galactosamine and either serine or threonine (i.e. blood group substances and salivary mucins). 2. N-glycosidic link N-glycosidic links exist between N-acetylglucosamine and asparagine. There are two types: A. High mannose B. Complex. For example, in addition to mannose they may contain N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid (sialic acid)

Lippincott

Their core pentasaccharide is the same. In the complex form additional sugar residues are present: N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NANA). Fucose

Eight Sugars in Glycoproteins Abbreviation β-D-Glucose Glc β-D-Galactose Gal β-D-Mannose Man α-L-Fucose Fuc N-Acetylgalactosamine GalNAc N-Acetylglucosamine GlcNAc N-Acetylneuraminic acid NeuNAc Xylose Xyl

Glycoproteins Glycoproteins are proteins that contain oligosaccharide (glycan) chains covalently attached to their polypeptide backbones. Glycoproteins occur in most organisms, from bacteria to humans. Their carbohydrate content ranges from 1% to over 85% by weight.

They differ from proteoglycans: Length of the chain is relatively short (usually 2-10 sugar residues) very long in GAGs. Do not have repeating disaccharide units. They are branched. May or may not be negatively charged.

The distinction between proteoglycans and glycoproteins resides in the level and types of carbohydrate modification.

Proteoglycans also contain the sugar glucuronic acid (GlcA). The carbohydrate modifications found in glycoproteins are rarely as complex as that of proteoglycans. Most proteins that are secreted, or bound to the plasma membrane, are modified by carbohydrate attachment. The part that is modified, in plasma membrane-bound proteins, is the extracellular portion of the protein.

Intracellular proteins are less frequently modified by carbohydrate attachment. However, the attachment of carbohydrate to intracellular proteins confers unique functional activities on these proteins

Structure of Glycoprotein The oligosaccharide components of glycoproteins  is branched heteropolymers  composed of D-hexoses, with the addition in some cases of neuraminic acid, and of L-fucose (6-deoxyhexose)

FUNCTIONS

Function Glycoprotein 1. Structural molecule Collagens/Arthritis Some Functions of Glycoproteins _________________________________________________ Function Glycoprotein 1. Structural molecule Collagens/Arthritis 2. Lubricant Mucins/ Peptic ulcer 3. Transport molecule e.g. Transferrin, Ceruloplasmin 4. Immune system Immunoglobulins, Histocompatibility antigens, Blood group determinants 5. Hormone e.g. HCG, TSH 6. Enzymes e.g. Alkaline phosphatase 7. Blood clotting e.g. Fibrinogen 8. Blood groups 9. Cell surface recognition Lectins

Glycoproteins antibodies are glycoproteins Glycoproteins contain carbohydrate units covalently bonded to a polypeptide chain antibodies are glycoproteins carbohydrates play a role as antigenic determinants, the portions of the antigenic molecule that antibodies recognize and to which they bond.

Almost all the plasma proteins of humans—except albumin—are glycoproteins. For example, O-linked oligosaccharides on the surface of RBCs help provide the ABO blood group determinants Many proteins of cellular membranes contain substantial amounts of carbohydrate.

Blood Group Substances Membranes of animal plasma cells have large numbers of relatively small carbohydrates bound to them: these membrane-bound carbohydrates act as antigenic determinants among the first antigenic determinants discovered were the blood group substances in the ABO system, individuals are classified according to four blood types: A, B, AB, and O at the cellular level, the biochemical basis for this classification is a group of relatively small membrane-bound carbohydrates

ABO Blood Classification

ABO Blood Classification in type A, the nonreducing end is NAGal in type B it is Gal in type AB, both types are present in Type O, neither of these terminal residues is present

Recognition and adhesion at the cell surface

Stronger interaction near the site of inflammation lectin-ligand interactions in lymphocyte movement to the site of an infection Stronger interaction near the site of inflammation

Helicobacter pylori Interaction between a bacterial surface lectin and an oligosaccharide of the gastric epithelium

The extracellular space in animal tissues is filled with a gel-like material, the extracellular matrix, also called ground substance, which holds the cells of a tissue together and provides a porous pathway for the diffusion of nutrients and oxygen to individual cells.

extra- cellular matrix Epithelial cells extra- cellular matrix Underlying cells

The extracellular matrix is composed of an interlocking meshwork of heteropolysaccharides and fibrous proteins. Heteropolysaccharides in the body are the glycosaminoglycans (GAGs). These molecules are long unbranched polysaccharides containing a repeating disaccharide unit.

GAGs are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution. GAGs are located primarily on the surface of cells or in the extracellular matrix (ECM).

Along with the high viscosity of GAGs comes low compressibility, which makes these molecules ideal for a lubricating fluid in the joints. At the same time, their rigidity provides structural integrity to cells and provides passage ways between cells, allowing for cell migration.

Hyaluronic acid Hyaluronic acid is unique among the GAGs in that it does not contain any sulfate and is not found covalently attached to proteins as a proteoglycan. It is, however, a component of non-covalently formed complexes with proteoglycans in the ECM.

Only GAG present both in animals and bacteria. Found in synovial fluid, vitreous humor, ECM of loose connective tissue Umbilical cord Cartilage

Glycosaminoglycans b-1,3 b-1,4 GlcUA GlcNAc No protein link No sulfate 1/2/2019 Glycosaminoglycans b-1,3 b-1,4 GlcUA GlcNAc No protein link No sulfate b-1,3 glycosidic linkage Hyaluronate GlcUA: glucuronic acid GlcNAc: N-acetylglucosamine

Specific function: 1.Hyaluronic acid is especially high in concentration in embryonic tissues and is thought to play an important role in permitting cell migration during morphogenesis and wound repair. 2. Act as lubricators and shock absorbers.

Association with major diseases: Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM. Tumor cells can induce fibroblasts to synthesize greatly increased amounts of this GAG, thereby perhaps facilitating their own spread.

Glycosaminoglycans b-1,3 b-1,4 GlcUA GalNAc 1/2/2019 Glycosaminoglycans b-1,3 b-1,4 GlcUA GalNAc GlcUA-Gal-Gal-Xyl-O-Ser link Sulfate at 4 or 6 C of GalNAc b-1,3 glycosidic linkage Chondroitin sulfate GlcUA: glucuronic acid Gal: galactose Xyl: xylose Ser: serine GalNAc: N-acetylgalactosamine

Chondroitin sulfate Most abundant GAG Cartilage (bind collagen and hold the fibers strongly) Tendons Ligaments Heart valves

Glycosaminoglycans a-1,4 a-1,4 GlcUA GlcNAc GlcN and GlcUA or IdUA 1/2/2019 Glycosaminoglycans a-1,4 a-1,4 GlcUA GlcNAc GlcN and GlcUA or IdUA N and O sulfate (C2,3,6) a-1,4 glycosidic linkage Heparin GlcN: glucosamine GlcUA: glucuronic acid IdUA: iduronic acid NAc: N-acetyl > NAc < N and O sulfate Heparan sulfate

Heparan sulfate Extracellular GAG contains higher acetylated glucosamine than heparin And less sulphated groups found in the basement membrane of the kidney along with type IV collagen and laminin where it plays a major role in determining the charge selectiveness of glomerular filtration

are associated with the plasma membrane of cells, with their core proteins actually spanning that membrane. In it they may act as receptors and may also participate in the mediation of cell growth and cell-cell communication.

Association with the disease: Some tumor cells have less heparan sulfate at their surfaces, and this may play a role in the lack of adhesiveness that these cells display.

Heparin The repeating unit of heparin

Heparin It is an intracellular GAG. Component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin more sulfated than heparan sulfate

Heparin is an important anticoagulant Heparin is an important anticoagulant. It binds with factors IX and XI, but its most important interaction is with plasma antithrombin III. Heparin can also bind specifically to lipoprotein lipase present in capillary walls, causing a release of this enzyme into the circulation.

Specific function: Heparin and warfarin are widely used in the treatment of thrombotic and thromboembolic conditions, such as deep vein thrombosis and pulmonary embolus. Heparin is administered first, because of its prompt onset of action, whereas warfarin takes several days to reach full effect. Their effects are closely monitored by use of appropriate tests of coagulation because of the risk of producing hemorrhage.

Glycosaminoglycans b-1,3 b-1,4 IdUA GalNAc IdUA with some GlcUA 1/2/2019 Glycosaminoglycans b-1,3 b-1,4 IdUA GalNAc IdUA with some GlcUA Sulfate at 4 or 6 C of GalNAc b-1,3 glycosidic linkage Dermatan sulfate IdUA: iduronic acid GlcUA: glucuronic acid GalNAc: N-acetylgalactosamine

Dermatan sulfate Sclera- gives shape to the eye. Binds LDL –plays a role in the development of atherosclerosis. skin, blood vessels, heart valves

Glycosaminoglycans b-1,4 b-1,3 GlcUA GlcNAc GlcNAc and Gal (no UA) 1/2/2019 Glycosaminoglycans b-1,4 b-1,3 GlcUA GlcNAc GlcNAc and Gal (no UA) Sulfate on C6 of Gal or HexN b-1,4 glycosidic linkage Keratan sulfate I GlcNAc: N-acetylglucosamine Gal: galactose UA: uronic acid HexN: hexosamine GalNAc: N-acetylgalactosamine Ser: serine Thr: threonine GlcUA: glucuronic acid Keratan sulfate II GalNAc-O-Ser or Thr

Keratan sulfate cornea, bone, cartilage aggregated with chondroitin sulfates

Both keratan sulfate I and dermatan sulfate are present in the cornea Both keratan sulfate I and dermatan sulfate are present in the cornea. They lie between collagen fibrils and play a critical role in corneal transparency.

In various types of arthritis, proteoglycans may act as autoantigens, thus contributing to the pathologic features of these conditions. The amount of chondroitin sulfate in cartilage diminishes with age.

Whereas the amounts of keratan sulfate and hyaluronic acid increase. These changes may contribute to the development of osteoarthritis. Changes in the amounts of certain GAGs in the skin are also observed with aging.

glycosaminoglycans of extracellular matrix lubricants in the synovial fluid of joints cartilage, tendons, ligaments a variety of horny structures formed of dead cells: horn, hair, hoofs, nails

SYNTHESIS OF GLYCOPROTEINS

Synthesis of O-linked glycosides The protein to which the oligosaccharides are to be attached is synthesized on the RER, and extruded into its lumen. Glycosylation begins with the transfer of an N-acetylgalactosamine (from UDP-N-acetylgalactosamine) onto a specific seryl or threonyl R-group

The glycosyl-transferases responsible for the stepwise synthesis of the oligosaccharides are bound to the membranes of the Golgi apparatus.

Synthesis of the N-linked glycosides First, as with the O-linked glycosides, protein is synthesized on the RER and enters its lumen. The protein itself does not become glycosylated with individual sugars at this stage of glycoprotein synthesis, but rather a lipid-linked oligosaccharide is first constructed

This consists of dolichol (an ER membrane lipid 80 to 100 carbons long) attached through a pyrophosphate linkage to an oligosaccharide containing N-acetylglucosamine, mannose, and glucose.

The sugars to be added to the dolichol by the membrane-bound glycosyltransferases are first N-acetylglucosamine, followed by mannose and glucose . The oligosaccharide is transferred from the dolichol to an asparagine side group of the protein by a protein-oligosaccharide transferase present in the ER.

Final processing of N-linked oligosaccharides After incorporation into the protein, the N-linked oligosaccharide is processed by the removal of specific mannosyl and glucosyl residues as the glycoprotein moves through the ER.

Finally, the oligosaccharide chains are completed in the Golgi by addition of a variety of sugars (for example, N-acetylglucosamine, N-acetylgalactosamine, and additional mannoses, and then fucose or NANA as terminal groups

The ultimate fate of N-linked glycoproteins is the same as that of the O-linked, for example, they can be released by the cell, or become part of a cell membrane. In addition N-linked glycoproteins can be translocated to the lysosomes.

Enzymes destined for lysosomes N-linked glycoproteins being processed through the Golgi can be phosphorylated at one or more specific mannosyl residues. Mannose 6-P receptors, located in the Golgi apparatus, bind the mannose 6-P residues of these targeted enzymes, resulting in their translocation to the lysosomes.

Proteoglycans: cell surface or extracellular matrix A typical tetrasaccharide linker (blue) connects a glycosamino-glycan—in this case chondroitin 4-sulfate (orange)—to a Ser residue (pink) in the core protein. The xylose residue at the reducing end of the linker is joined by its anomeric carbon to the hydroxyl of the Ser residue.

linkages in glycoproteins Ser/thr

Glycopeptide bonds Asn Glc NAc Type I N-Glycosyl linkage to Asn Ser 1/2/2019 Glycopeptide bonds Asn Glc NAc Type I N-Glycosyl linkage to Asn Ser Glc Glc HOLys NAc Asn: asparagine Glc: glucose NAc: N-acetyl Ser: serine Thr: threonine HOLys: 5-hydroxylysine Type II O-Glycosyl linkage to Ser (Thr) Type III O-Glycosyl linkage to 5-HOLys

Examples Mucins – mucins are secreted in the mucus of the respiratory and digestive tracts. The sugars attached to the mucins give them considerable water-holding capacity and make them resistant to proteolysis by digestive enzymes. Immune System Glycoproteins – antibodies, major histocompatibility complex (MHC) (interacts with T-cells)

Hormones That Are Glycoproteins Follicle-stimulating hormone (FSH) Luteinizing hormone (LH) Thyroid Stimulating hormone (TSH) Human chorionic gonadotropin (HCG) Alpha-fetoprotein (α-FP) Erythropoietin

Glyconutrient Conversion A series of enzyme controlled steps converts one glyconutrient sugar to another. Enzyme conversions require energy. Toxins, stress, drugs, processed foods, lack of enzymes, age, etc. can all inhibit an enzymes ability to convert these glyconutrients. It is more efficient to obtain glyconutrients in the diet than to have to convert them.

Glyconutrient Effects Raise the level of natural killer cells and macrophages to fight against infectious organisms. Activate immune T-cell activity only when invaders are present. Decrease cell death in people suffering from chronic fatigue syndrome.

Glyconutrient Effects Elevate disease resistance in weakened individuals. Act as antioxidant compounds. Protect the body from toxin and pollution exposure. Slow premature aging.

Glyconutrient Effects Decrease inflammation in diseases like rheumatoid arthritis. Helps immune cells recognize invaders due to a mutual “sugar exchange” of information. Enable cellular components to stick to each other initiating the proper reactions.

Classification of Glycosaminoglycans The classification is based on: OR the GAGs differ from each other by: Monomeric (acidic & amino sugar) composition Degree & location of sulfation Type of glycosidic linkages Chain length of the disaccharides Nature of the core protein Their tissue distribution Their biologic functions

SYNTHESIS OF GLYCOPROTEINS/ PROTEOGLYCANS Synthesized on ribosomes attached to the RER then transported via vesicles to Golgi complex for sorting. The units in the saccharide chains are elongated in alternating acidic/amino sugars, donated from UDP derivatives. Last step is sulfation of some amino sugars. These additions are catalyzed by specific glycosyltransferases. For glycosaminoglycan synthesis and synthesis of O-linked glycoproteins, the addition is direct. For N-linked glycoproteins, the chain is formed on dolichol pyrophosphate and then transferred to the protein.

I-cell disease rare syndrome acid hydrolase enzymes normally found in lysosomes are absent, results in an accumulation of substrates normally degraded by lysosomal enzymes within these vesicles. Individuals with I-cell disease: are lacking the enzymic ability to phosphorylate the mannose residues of potential lysosomal enzymes, causing an incorrect targeting of these proteins to extracellular sites, rather than lysosomal vesicles (Note: I-cell disease is considered to be a glycoprotein storage disease ).

Defects can lead to a number of diseases/disorders DEGRADATION OF PROTEOGLYCANS Some proteoglycans must be phagocytosized first Degradation of the saccharide chains is achieved by hydrolytic enzymes present in lysosomes. The enzymes act on the ends of the chains on a last-on-first-off basis. Defects can lead to a number of diseases/disorders

Mucopolysaccharidosis Several genetically inherited diseases, for example the lysosomal storage diseases, result from defects in the lysosomal enzymes responsible for the metabolism of complex membrane-associated GAGs.

These specific diseases, termed mucopolysaccharidoses (MPS) lead to an accumulation of GAGs within lysosomes of affected cells. There are at least 14 known types of lysosomal storage diseases that affect GAG catabolism.

All are autosomal recessive disorders except hunters syndrome which is X- linked. Specific lab tests: Urine Enzymes assay Tissue biopsy DNA testing Prenatal diagnosis

MUCOPOLYSACCHARIDOSES (MPS) Rare inborn errors in the degradation of glycosaminoglycans result in a series of diseases called mucopolysaccharidoses; characterized by mental retardation and/or structural defects. MPS Type I Hurler’s syndrome results from a deficiency of alpha-L-iduronidase. Heparan sulfate and dermatan sulfate accumulate. There is growth and mental retardation with characteristic facial changes. MPS Type II Hunters syndrome is similar to Hurler’s syndrome but the enzyme deficiency is for iduronate sulfatase and the inheritance is X-linked. MPS Type III Sanfilipo’s syndrome is caused by a deficiency of one of four enzymes of which three are hydrolases and one is an N-acetyltransferase. There is severe mental retardation but only mild structural features. Other MPS Types are IV, VI and VII. There is no MPS Type V.

MPS I (Hurler Syndrome) A deficiency of L-iduronidase leads to mental retardation and structural changes due to accumulation of dermatan sulfate and heparan sulfate 96

MPS II (Hunter Syndrome) X-linked disease due to a deficiency of iduronate sulfatase 97

MPS III (Sanfilippo Syndrome) Deficiency in one of four degradative enzymes leads to severe mental retardation but little structural change 98

MPS IV (Morquio Syndrome) Deficiency of a galactose-6-sulfatase or a beta-galactosidase leads to accumulation of keratan sulfate with normal intelligence but severe deformity 99

Summary Glycoproteins and proteoglycans are distinct: --functions/structures --synthesis/degradation --associated pathologies