1.) What are glycosaminoglycans importance/significance.

and

proteoglycans?

Give

their

biologic

Glycosaminoglycans (GAGs) is a type of polysaccharide based on a repeating disaccharide in which one of the sugar is an amino acid sugar and at least one of them has a negative charge owing to the presence of a sulphate group or a carboxyl group. This is the most abundant heteropolysaccharide in the body. These polysaccharides are involved in a wide variety of cellular functions and tissues including blood plasma, joints and the mucosal (mucous membrane) lining of a variety of organs, including the GIT and the bladder. Characteristics of GAGs

GAG Hyaluronate Chondroitin sulfate Heparan sulfate

Localization synovial fluid, vitreous humor, ECM of loose connective tissue cartilage, bone, heart valves

Comments large polymers, shock absorbing most abundant GAG contains higher acetylated glucosamine than heparin more sulfated than heparan sulphates

basement membranes, components of cell surfaces component of intracellular granules of mast Heparin cells lining the arteries of the lungs, liver and skin Dermatan sulfate skin, blood vessels, heart valves cornea, bone, cartilage aggregated with Keratan sulfate chondroitin sulphates

GAG Hyaluronate

Biological Significance Chief components of the extracellular matrix , contributes to cell proliferation and migration, may also be involved in the progression of some malignant tumors. Dietary supplement for treatment of osteoarthritis, maintaining the structural integrity of the tissue, regulates the growth and development as well as the nervous system response to injury.

Chondroitin Sulfate

Heparan Sulfate

Regulates developmental processes, angiogenesis, blood coagulation and tumour metastasis. Act as an anticoagulant May have roles in coagulation, cardiovascular disease, carcinogenesis, infection, wound repair, and fibrosis Supporting functional roles in cellular recognition of protein ligands, axonal guidance, cell motility, and embryo implantation.

Heparin Dermatan Sulfate

Keratan Sulfate

Hyaluronates: composed of D-glucuronate + GlcNAc linkage is (1, 3)

Dermatan sulfates: composed of L-iduronate (many are sulfated) + GalNAc-4-sulfate linkages is (1, 3)

Chondroitin 4- and 6-sulfates : composed of D-glucuronate and GalNAc-4- or 6-sulfate linkage is (1, 3) (the figure contains GalNAc 4sulfate)

Heparin and Heparan sulfates: composed of iduronate-2-sulfate (D-glucuronate-2-sulfate) and N-sulfo-D-glucosamine-6sulfate linkage is (1, 4) (heparans have less sulfate than heparins)

Keratan sulfates: composed of galactose + GlcNAc6-sulfate linkage is (1, 4)

Proteoglycans are glycoproteins with an extremely high carbohydrate content approximately 85% to 95% by weight. The majority of GAGs in the body are linked to core proteins, forming proteoglycans (also known as mucopolysaccharides). Proteoglycans make up a major part of the extracellular matrix, the material between cells that provides structural support. Proteoglycan s are heavily glycosylated glycoprotein which means that they are protein with chains of polysaccharide. The specific type of polysaccharides attached to proteoglycans are

called glycosaminoglycans. The GAG chains of proteoglycan may be made of chondroitin sulphate, dermatan sulphate, heparin sulphate, heparan sulphate, or keratin sulphate. Structure of the GAG Linkage to Protein in Proteoglycans

2.) What are the different mucopolysaccharides? Identify the defect/deficient enzyme in each type. Give the clinical characteristics of each type Mucopolysaccharides are proteins covalently linked to glycosaminoglycans. Types of proteoglycans vary from each other in terms of tissue distribution, nature of protein core, attached glycosaminoglycans and functions. Linkages of glycosaminoglycans to the core protein involves specific trisaccharide structure composed of two galactose residues and a xylose residue (Galactose-GalactoseXylose). An O-glycosidic bond is formed between the xylose and serine residue of the protein. There are about six types of glycosaminoglycans (GAGs): hyaluronate, chondrotin sulfate, heparin sulfate, heparan, dermatan sulfate, keratin sulfate I and II. Hyaluronate is composed of N-acetylglucosamine and glucoronic acid. It is usually nonsulfonated and non-covalently attached to proteins. Hyaluronate is found in synovial fluid of joints, vitreous humor or eye, umbilical cord, loose connective tissue and cartilage. This compound serves as lubricant and shock absorber.

Chondroitin sulfate is composed of N-acetylgalactosamine with sulfate (on either carbon 4 or carbon 6) and glucoronic acid dissacharide units. It is said to be the most abundant GAG in the body. Also, it is usually found in cartilage, tendons, ligaments and aorta. Heparan sulfate is almost the same with heparin, however, it has less sulfate groups. The disaccharide unit is composed of N-glucosamine and mainly glucoronic acid. Dermatan sulfate is composed of N-acetylgalactosamine and L-iduronic acid. This is mainly found in skin, blood vessels and heart valves. Lastly, Keratan sulphates I and II are composed of the same disaccharide unit Nacetylglucosamine and galactose (no uronic acid). However, they differ in their protein linkages: GlcNAc-Asn for Keratan sulfate I and GlcNAc-Thr for Keratan sulfate II. The first one is mainly found in the cornea and the second one is found in loose connective tissue. Table 1 shows the summary of each glycosaminoglycans mentioned above. Table 1: Summary of GAGs (attachments, localization and special features) GAGs Sugar Sulfate Protein Localization attachment linkages Hyaluronate Glucoronic acid and Nacetlyglucosamin e Glucosamine None Synovial fluid, loose connective tissue, cartilage and vitreous body of eye Cartilage, bone, cornea, heart valve Skin fibroblast, aortic wall Special features -Present in bacteria -Shock absorbers

Chondroitin sulphate

Glucoronic acid and Nacetylgalactogluc -osamine Glucoronic acid and Nglucosamine

N-acetylgalactoglucosamine

Xyl-Ser Oglycosidic bond Xyl-Ser

Most abundant GAG Contains higher acetylated glucosamine than heparin More sulfated than heparin sulfates

Heparan sulphate

N-Glucosamine

Dermatan sulfate

N-Glucosamine and Iduronic acid

N-Glucosamine N-Glucosamine Iduronic acid

Ser

Mast cells, lining the arteries of the lungs, liver

and skin Keratan sulfate I Keratan sulfate II N-acetlygalactosamine and galactose N-acetlygalactosamine and galactose Nacetylgalactosamine Nacetylgalactosamine GlcNAc-Asn Cornea --

GlcNAc-Thr

Loose connective tissue

--

(A)

(B)

(C)

(D)

(E) Figure 1: Structures of different GAGs: (A) Hyaluronate, (B) Chondroitin sulfate, (C) Heparan sulfate, (D) Dermatan sulfate and (E) Keratan sulfate. Note that Sulfate groups (labelled as S=)

are shown in all possible positions. Also, structure of Keratan sulfate I differs from Keratan sulfate II via protein attachments. (Images taken from Champe et al., 2008) Mucopolysaccharidoses are hereditary disorders that are characterized by accumulation of GAGs on various tissues. Basically, it is caused by deficiency of any lysosomal hydrolases. Incomplete lysosomal degradation of GAGs results in the presence of oligosaccharides in urine. Few of the disorders are summarized in Table 2. Table 2: Few known mucopolysaccharidoses and their specific enzyme defects, urinary metabolites and symptoms Mucopolysaccharidoses Hurler (MPS1H) Enzyme defect -L-iduronudase Urinary metabolites Dermatan sulfate Heparan sulfate Schele (MPS1S) -L-iduronudase Dermatan sulfate Heparan sulfate Hurler-Schele (MPS1HS) -L-iduronudase Dermatan sulfate Heparan sulfate Hunter (MPS II) Iduronate sulfatase Dermatan sulfate, Heparan sulfate Physical deformity, mental retardation, dystosis multiplex, only X-linked MPS Severe nervous system disorders, profound mental retardation, hyperactivity, skin, brains, lungs Severe nervous system disorders, profound mental retardation, hyperactivity, skin, brains, lungs Severe nervous system Intermediate between H and S Symptoms Heart disease, dwarfism, corneal clouding, dystosis, mental retardation, early mortality

Corneal clouding, aortic valve disease, joint stiffening

Sanfilippo A (MPS IIIA) Heparan sulfate N- Heparan sulfatase (sulfamidase) sulfate

Sanfilippo B (MPS IIIB)

-NHeparan Acetylglucosaminidase sulfate

Sanfilippo C (MPS IIIC)

Acetyltransferase

Heparan

sulfate

disorders, profound mental retardation, hyperactivity, skin, brains, lungs Severe nervous system disorders, profound mental retardation, hyperactivity, skin, brains, lungs Corneal clouding, odontoid hypoplasia, aortic valve disease, distinctive skeletal abnormalities Corneal clouding, odontoid hypoplasia, aortic valve disease, distinctive skeletal abnormalities Aortic valve disease, dystosis multiplex, normal intelligence, corneal clouding, coarse facial features Hepatosplenomegaly, dystosis multiplex, hydrops fetalis

Sanfilippo D (MPS IIID) -NAcetylglucosamine-6sulfatase Morquio A (MPS IVA) Galactose-6-sulfatase

Heparan sulfate

Keratan sulfate Chondroitin-6 sulfate

Morquio B (MPS IVB)

-galactosidase

Keratan sulfate

Maroteaux-Lamy (MPS VI)

Arylsulfatase B (NAcetylgalactosamine4-sulfatase) -glucoronidase

Dermatan sulfate

Sly (MPS VII)

Heparan sulfate Dermatan sulfate Chondroitin4,6-sulfates

3.) What is the role of glycosaminoglycans in: a.) Cancer:

Hyaluronic acid localized in the synovial fluid, vitreous humor and extracellular matrix of loose connective tissue is significant since they are involved in the progression of cancer. Exposure of the said glycosaminoglycan with that of the cancer cells will activate series of activities which in turn will result in cell migration. Heparan sulphate glycosaminoglycans (HSGAGs), which are complex polysaccharides also regulate aspects of cancer life process (metastasis, tumor progressing, tumorgenesis). They are found on the cell surfaces and extracellular matrix. HSGAGs are part of the glycosaminoglycan family that are complex polysaccharides having repeating disaccharide units of uronic acid linked to a glucosamine. These can be found at the cell-tissue-organ interface of every eukaryotic cell and are shown play roles in physiological processes including tumor progression and onset. The structure of HSGAGs enables binding and interaction with different proteins (growth factors, chemokines, morphogens, and enzymes). The binding of these proteins to the HSGAGs can affect cancer cells. The growth factors involved in tumor development are the Fibroblast Growth Factors (FGF1 and FGF2), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), Transforming Growth factor , and Platelet-derived Growth Factor. Although the presence of HSGAGs contributes to the metastasis of cells, studies have indicated that heparin might interfere with tumor progression and metastasis by means of several mechanisms: 1. Anticoagulation Heparin interferes with fibrin clot formation that surrounds the tumor cells. 2. Immune modulation Heparin inhibit metastasis by making circulating cancer cells more vulnerable to immune response. It also regulated the activities of several cytokines. Heparin binds to granulocytes and macrophages to promote destruction of tumors. 3. Cell adhesion Heparin inhibit tumor metastasis by blocking P selectin mediated interaction between platelates and sialylated, fucosylated mucins that reside on the surface of the circulating cancer cells B.) Atherosclerosis The intima of the arterial wall contains hyaluronic acid and chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans. Immunohistochemistry reveals that versican, which is the principal chondroitin sulfate proteoglycans in the blood vessels, is prominent in the intima adventitia of most arteries and veins. The accumulation of versician in the normal arterial intima is mainly responsible for the proteoglycan-rich nature of this layer. Versician interacts with hyaluronan and link protein to form higher molecular weight stable aggregates that fill the ECM space not occupied by the other fibrous proteins such as collagens and elastic fibers. These complexes create a reversibly compressive compartment and provide a swelling pressure within the ECM that is offset by collagen and elastic fibers. Analysis of binding constants of Chondroitin Sulfate Proteoglycans from lesions reveals that multiple LDL particles can bind to a single CS chain. Thus, GAG chain length is a determining factor in lipid binding. Vascular injury produces elongated GAG chains on the large vascular CSPG promoting LDL binding. Conditions that promote CS chain elongation in Arterial

Smooth Muscle Cells such as cell proliferation, treatment of the cells with oxidized LDL and transforming growth factor (TGF) also cause increased binding of versican to LDL. In addition, exposure of human ASMCs to nonesterified free acids, as occurs in diabetes, increases the production of proteoglycans including versican, which then binds LDL more effectively Inflammation, infection, or physical damage to the intima of the arterial wall can lead to the release of soluble GAGs. This, in turn, promotes binding of LDL to the large vascular CSPG. Buildup of lipids, cholesterol, calcium, and cellular debris within the intima of the vessel wall results to Atherosclerosis. C.) Arthritis Arthritis is a form of joint disorder that involves inflammation of one or more joints. The most common form, osteoarthritis (degenerative joint disease) is a result of trauma to the joint, infection of the joint, or age. Other arthritis forms are rheumatoid arthritis, psoriatic arthritis, and related autoimmune diseases. Although many infectious agents can cause inflammatory arthritis, the actual antigen behind autoimmunity may be GAGs. According to a study conducted by Roehrl and Wang, circulating or locally released GAGs induce the clonal expansionof various GAG-binding cells, for example, T and B cells and macrophages. These cells, because of their enhanced or matured binding to GAGs, preferentially migrate and adhere to connective tissue where GAGs are abundant. GAGs expressed on endothelial and synovial lining cells facilitate the extravasations and adherence of GAG-binding cells from the bloodstream into GAG-rich environments, such as connective tissue and cartilage. Excessive and prolonged accumulation of these abnormal cells eventually leads to pathological symptoms, including damage of joint cartilage and bone erosion.

Development of arthritis, especially osteoarthritis, is due to the changes brought about by the amount of chondroitin sulfate in cartilage diminishing and amounts of keratan sulfate and hyaluronic acid increasing along with age.

Changes in the amounts of certain GAGs in the skin are also observed with aging and help to account for the characteristic changes noted in this organ in elderly.

Hands affected by Rheumatoid Arthritis, an autoimmune form of arthritis

Sources: Champe, et. al. (2008). Biochemistry: 4th ed. Lippincott Williams & Wilkins. Philadelphia, PA. Merrilees & Wright. (2004). Proteoglycans in Atherosclerosis and Restenosis : Key Roles for American Heart Association. Circulation Research 2004, 94:1158-1167

Roehrl & Wang. (2002). Glycosaminoglycans are a potential cause of rheumatoid arthritis. Channing Laboratory, Department of Medicine, Brigham and Womens Hospital, and Department of Biological Chemistry and Molecular Pharmacology. Harvard Medical School, Boston. Sasisekharan, et. al. (2002). Roles of Heparan-Sulphate Glycosaminoglycans in Cancer. Biological Engineering Division and Center for Biomedical Engineering, Massachusetts Institute of Technology, Division of Hematology and Oncology,New England Medical Center,Tufts University School of Medicine. Nature, July 2002: Vol 2

GLYCOSAMINOGLYCANS (GAGs)
1D-2 Members: PERALTA, JP QUE, Arbie QUESADA, Gab RABAGO, Gian RENDON, Katrina ROBERTO, Kathleen ROBLES, Kristine