BIOCHEMISTRY: A TEXTBOOK FOR HIGHER EDUCATION - E. S. Severin - 2004

SECTION 15. BIOCHEMISTRY OF THE EXTRACELLULAR MATRIX

III. Glycosaminoglycans and Proteoglycans

Glycosaminoglycans — are linear, negatively charged Heteropolysaccharides. Previously, they were referred to as mucopolysaccharides because they were originally isolated from mucous secretions (mucus) and imparted viscous, lubricating properties to these fluids. These properties stem from the ability of glycosaminoglycans to bind large amounts of Water, giving the intercellular substance a gel-like consistency.

Proteoglycans — are high-molecular-weight compounds composed of protein (5 – 10%) and glycosaminoglycans (90 – 95%). They form the ground substance of the Extracellular matrix in Connective Tissue and can account for up to 30% of the dry weight of the tissue.

The protein component in proteoglycans is represented by a single polypeptide chain of varying molecular weight. The polysaccharide components differ among various proteoglycans. Proteoglycans are distinct from a large group of Proteins known as Glycoproteins. These proteins also contain oligosaccharide chains of varying lengths, covalently

attached to a polypeptide backbone. The carbohydrate component of glycoproteins is substantially smaller in mass than that of proteoglycans, making up no more than 40% of the total mass. Glycoproteins perform diverse functions in The Human Body and are found in all protein classes — Enzymes, Hormones, transport proteins, structural proteins, and others. Examples of glycoproteins include Collagen and Elastin, IMMUNOGLOBULINS, angiotensinogen, transferrin, ceruloplasmin, Castle's intrinsic factor, and thyrotropic hormone.

As essential Components of the extracellular matrix, Glycosaminoglycans and Proteoglycans play a vital role in intercellular interactions, the formation and maintenance of Cell and organ Morphology, and the creation of a structural framework during tissue development.

Due to their unique structural features and physicochemical properties, Proteoglycans and Glycosaminoglycans can perform the following functions in the human body:

✵ they serve as Structural components of the extracellular matrix;

✵ proteoglycans and glycosaminoglycans specifically interact with collagen, elastin, Fibronectin, Laminin, and other extracellular matrix proteins;

✵ as polyanions, all proteoglycans and glycosaminoglycans are capable of binding large amounts of cations (Nа⁺, К⁺, Са²⁺) In addition to water, thereby participating in The regulation of tissue turgor;

✵ proteoglycans and glycosaminoglycans act as a molecular sieve within the extracellular matrix, hindering the spread of pathogenic microorganisms;

✵ hyaluronic acid and proteoglycans provide a Shock-absorbing function in articular Cartilage;

✵ heparan sulfate-containing proteoglycans contribute to The formation of the filtration barrier in the Kidneys;

✵ keratan sulfates and dermatan sulfates ensure the transparency of the cornea;

✵ heparin acts as an anticoagulant;

✵ heparan sulfates are components of Plasma Membranes, where they can function as receptors and participate in Cell Adhesion and intercellular interactions. They are also constituents of synaptic and other vesicles.

A. Structure and Classes of Glycosaminoglycans

Glycosaminoglycans are long, unbranched heteropolysaccharide chains composed of repeating disaccharide units. One monomer of this disaccharide is a hexuronic acid (D-glucuronic acid or L-iduronic acid), while the second monomer is an amino sugar derivative (glucosamine or galactosamine). The NН₂ group of amino sugars is typically acetylated, which abolishes their intrinsic positive charge. With the exception of hyaluronic acid, all glycosaminoglycans contain sulfate groups in the form of O-esters or N-sulfate.

Currently, the structures of six Major Classes of glycosaminoglycans are known, as summarized in Table 15-4.

Class="center">Table 15-4. Structure of various classes of glycosaminoglycans

¹ The disaccharide unit may also contain D-glucuronic acid.

² May contain an N-sulfo derivative of glucosamine instead of N-acetylglucosamine, along with varying amounts of iduronic and glucuronic acids.

Hyaluronic acid is found in many Organs and Tissues. In cartilage, it is bound to protein and participates in the formation of proteoglycan aggregates, while in certain organs (such as the vitreous body of the eye, umbilical cord, and synovial fluid) it also occurs in a free state. It is believed that in synovial fluid, hyaluronic acid acts as a lubricant, reducing friction between articular surfaces.

The repeating disaccharide unit in hyaluronic acid has the following structure:

Hyaluronic acid contains several thousand disaccharide units, with a molecular weight reaching 10⁵ — 10⁷ Da.

Chondroitin sulfates are the most abundant glycosaminoglycans in the human body; they are found in cartilage, Skin, tendons, ligaments, Arteries, and the Cytology/practical/76.html">Cornea of the eye. Chondroitin sulfates are an essential component of aggrecan, the primary proteoglycan of the cartilage matrix. In the human body, Two Types of chondroitin sulfates occur: chondroitin-4-sulfate and chondroitin-6-sulfate. They have an identical structure, differing only in THE POSITION OF the sulfate group on the N-acetylgalactosamine molecule (see Scheme A).

A single chondroitin sulfate polysaccharide chain contains about 40 repeating disaccharide units and has a Molecular Weight of 10⁴ — 10⁶ Da.

Keratan sulfates are the most heterogeneous glycosaminoglycans, differing from one another in total carbohydrate content and distribution across various tissues. Keratan sulfate I is located in the cornea of THE EYE AND, in addition to the repeating disaccharide unit, contains L-fucose, D-mannose, and sialic acid. Keratan sulfate II has been found in cartilage tissue, bones, and intervertebral discs. Besides the sugars of the disaccharide unit, its composition includes N-acetylgalactosamine, L-fucose, D-mannose, and sialic acid. Keratan sulfate II is a component of aggrecan and certain small proteoglycans of the cartilage matrix. Unlike other glycosaminoglycans, keratan sulfates contain a galactose residue instead of hexuronic acid (see Scheme B).

The molecular weight of a single keratan sulfate chain ranges from 4 x 10³ to 20 x 10³ Da.

Dermatan sulfate is widely distributed in animal tissues, being particularly characteristic of the skin, Blood Vessels, and Heart Valves.

As part of small proteoglycans (biglycan and decorin), dermatan sulfate is found in the extracellular matrix of cartilage, intervertebral discs, and menisci. The repeating disaccharide unit of dermatan sulfate has the following structure (see Scheme A).

The molecular weight of a single dermatan sulfate chain ranges from 15 x 10³ to 40 x 10³ Da.

Heparin is a vital component of the BLOOD ANTICOAGULANT SYSTEM (it is used as an anticoagulant in the Treatment of thrombosis). It is synthesized by mast Cells and resides within granules inside these cells. The highest amounts of heparin are found in the Lungs, Liver, and skin. The disaccharide unit of heparin is similar to that of heparan sulfate. The difference between these glycosaminoglycans is that heparin contains more N-sulfate groups, whereas heparan sulfate contains more N-acetyl groups. The molecular weight of heparin ranges from 6 x 10³ to 25 x 10³ Da (see Scheme B).

Heparan sulfate is found in many organs and tissues. It is a constituent of basement membrane proteoglycans and serves as a permanent component of The Cell surface. The structure of the heparan sulfate disaccharide unit is identical to that of heparin. The molecular weight of a heparan sulfate chain ranges from 5 x 10³ to 12 x 10³ Da.

B. Synthesis and degradation of Glycosaminoglycans

Glycosaminoglycan METABOLISM depends on the balance between their rates of Synthesis and Breakdown.

Synthesis of Glycosaminoglycans

Glycosaminoglycan polysaccharide chains are almost always bound to a protein known as the core protein. The attachment of the polysaccharide to the protein occurs via a linkage region, which most frequently consists of the trisaccharide galactose-galactose-xylose (Fig. 15-14).

Fig. 15-14. The linkage region of glycosaminoglycans.

The Oligosaccharides of the linkage region are attached to the core protein via 3 types of covalent bonds:

1. An O-glycosidic bond between Serine and xylose;

2. An O-glycosidic bond between serine or Threonine and N-acetylgalactosamine;

3. An N-glycosylamide bond between the amide nitrogen of asparagine and N-acetylglucosamine.

The polysaccharide chains of glycosaminoglycans are synthesized via the sequential addition of Monosaccharides. The corresponding nucleotide sugars generally serve as monosaccharide Donors. The synthesis reactions of glycosaminoglycans are catalyzed by Enzymes of the transferase family, which exhibit absolute substrate Specificity. These transferases are localized on the membranes of the Golgi apparatus. Here, the core protein synthesized on polyribosomes is delivered via ER channels, after which the monosaccharides of the linkage region are attached, followed by the elongation of the entire polysaccharide chain. Sulfation of the carbohydrate moiety occurs here with the aid of sulfotransferase, with PAPS acting as the sulfate group donor (see Section 12).

Aminosugars are synthesized from glucose; in connective tissue, ~20% of glucose is utilized in this manner. Fructose-6-phosphate is the direct precursor of N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. Glutamine serves as the source of the NH₂ group in these sugars. The aminosugar is subsequently acetylated using acetyl-CoA. The activated forms of these aminosugars are their UDP derivatives (scheme, Fig. 15-15).

Scheme

Fig. 15-15. Scheme of aminosugar synthesis.

Sources of glucuronic acid in the human body can include diet, intracellular lysosomal degradation of glycosaminoglycans, and the de novo synthesis of glucuronic acid. The activated form of glucuronic acid (UDP-glucuronate) is formed via The oxidation of UDP-glucose (see scheme).

L-iduronic acid is formed after the incorporation of D-glucuronic acid into the carbohydrate chain As a result of an epimerization reaction.

Glucocorticoids influence the synthesis of glycosaminoglycans by inhibiting the synthesis of hyaluronic acid and sulfated glycosaminoglycans. The inhibitory effect of Sex Hormones on the synthesis of sulfated glycosaminoglycans in target organs has also been demonstrated.

Degradation of glycosaminoglycans

Glycosaminoglycans are characterized by a high turnover rate: the half-life (T_1/2) of many of them ranges from 3 to 10 days (only keratan sulfate has a T_1/2 of ~120 days). The degradation of polysaccharide chains is carried out by exo- and Endoglycosidases, as well as sulfatases, which include hyaluronidase, glucuronidase, galactosidase, iduronidase, etc. Glycosaminoglycans enter the cell from the extracellular space via endocytosis and are enclosed in endocytic vesicles, which subsequently fuse with Lysosomes. Lysosomal Hydrolases ensure the gradual and complete Cleavage of glycosaminoglycans down to monomers.

Mucopolysaccharidoses — severe hereditary disorders manifested by significant impairments in children's mental development, vascular lesions, corneal clouding, skeletal deformities, and reduced life expectancy. Mucopolysaccharidoses are caused by hereditary defects in any of the hydrolases involved in the Catabolism of glycosaminoglycans. These diseases are characterized by the excessive accumulation of glycosaminoglycans in tissues, leading to skeletal deformation and enlargement of organs containing large amounts of extracellular matrix. Tissues that normally synthesize the highest amounts of glycosaminoglycans are typically affected. In this process, incompletely degraded glycosaminoglycans accumulate in lysosomes, while their oligosaccharide fragments are excreted in the urine. Several types of mucopolysaccharidoses are known, caused by defects in various glycosaminoglycan-hydrolyzing enzymes. The MAIN TYPES OF mucopolysaccharidoses are listed in Table 15-5.

Table 15-5. Types of mucopolysaccharidoses

To diagnose a specific disorder, The activity of lysosomal hydrolases is typically measured. Since these diseases are currently untreatable, prenatal Diagnosis is necessary when carrier status for defective genes is suspected.

B. STRUCTURE AND TYPES of proteoglycans

Various proteoglycans are present in the extracellular matrix. Among them are very large ones, such as aggrecan and versican. In addition, the extracellular matrix contains a whole set of so-called small proteoglycans, which are widely distributed across various types of connective tissue and perform A wide variety of functions there.

The principal proteoglycan of the cartilage matrix is called aggrecan, accounting for 10% of the wet weight of the original tissue and 25% of the dry weight of the cartilage matrix. It is a very large molecule in which up to 100 chondroitin sulfate chains and about 30 keratan sulfate chains are attached to a single polypeptide chain. In shape, the aggrecan molecule resembles a bottle brush (Fig. 15-16).

Fig. 15-16. Structure of aggrecan. HA — hyaluronic acid; 1 — chondroitin sulfate; 2 — keratan sulfate; 3 — core protein. At the center of the molecule is the core protein (molecular mass ~220 kDa), which has three globular domains: G₁, G₂, and G₃, performing different functions. The N-terminal domain G₁ ensures the binding of aggrecan to hyaluronic acid and the low-molecular-weight link protein; the function of the G₂ domain is currently unknown; the C-terminal domain G₃ ensures the attachment of aggrecan to other extracellular matrix molecules and, possibly, participates in cell-cell interactions. Between the G₂ and G₃ domains are regions where keratan sulfate and chondroitin sulfate chains attach to the protein. In these regions, the core protein contains peptide segments consisting of 6 and 19 amino acid residues, which repeat 10 to 20 times.

In cartilage tissue, aggrecan molecules assemble into aggregates with hyaluronic acid and a small link protein. Both components attach to aggrecan via non-covalent bonds in the region of the G₁ domain. The G₁ domain interacts with approximately five disaccharide units of hyaluronic acid; this complex is subsequently stabilized by the link protein. The G₁ domain and the link protein together occupy 25 disaccharide units of hyaluronic acid. The final aggregate, with a molecular mass exceeding 200 × 10⁶ Da, consists of a single hyaluronic acid molecule and ~100 aggrecan molecules (and an equal number of link protein molecules). Coordinating the assembly of these aggregates is a central function of chondrocytes. Aggrecan and link protein are produced by these cells in the required quantities. These components can interact with each other intracellularly, but the aggregation process is fully completed in the extracellular matrix. It has been shown that hyaluronic acid is synthesized On the surface of chondrocytes by a specific synthetase and "pushed out" into the extracellular space to bind with aggrecan and the link protein. The maturation of the functionally active ternary complex takes about 24 hours.

The catabolism of aggrecan is currently insufficiently understood. There is Evidence for the presence of an aggrecanase enzyme in the cartilage extracellular matrix. The cleavage site of this enzyme is the interglobular region between the G₁ and G₂ domains. Additionally, in the region where chondroitin sulfate chains attach to the core protein, there are 3 more proteolytic cleavage sites for aggrecan. The final product of aggrecan degradation is a complex comprising the G₁ domain, link protein, and hyaluronic acid. It enters the chondrocyte via endocytosis and undergoes degradation by lysosomal hydrolases.

Small proteoglycans

Small proteoglycans are low-molecular-weight proteoglycans found in cartilage, tendons, ligaments, menisci, skin, and Other types of connective tissue.

These proteoglycans feature a small core protein attached to one or two glycosaminoglycan chains. The most extensively studied among them are decorin, biglycan, fibromodulin, lumican, and perlecan.

The core proteins of biglycan and decorin are similar in Size and Structure (molecular weights of 36,000 and 38,000 Da, respectively). They contain multiple leucine-rich tandem repeats that form α-helices or β-structures. The N- and C-termini of these proteins contain domains stabilized by disulfide (S-S) bonds. The primary structures of the core proteins differ significantly in their N-terminal regions, which accounts for variations in glycosaminoglycan attachment. Biglycan contains serine residues at positions 5 and 11, allowing for the attachment of two polysaccharide chains. Decorin contains a single serine at position 4, resulting in the attachment of only one polysaccharide chain. In these proteoglycans, the polysaccharide chains are represented by dermatan sulfate with a molecular weight of ~30,000 Da (Fig. 15-17).

Fig. 15-17. Small cartilage proteoglycans. 1 — regions of repetitive Amino acid sequences rich in leucine, forming α-helices or β-structures; 2 — domains in the N- and C-terminal regions of core proteins containing S-S bonds; 3 — dermatan sulfate chains attached to the serine (S) OH-group in the N-terminal region; 4 — short chains attached to the protein within the leucine-rich repeat region; this attachment is mediated by oligosaccharides ∇ linked to the protein via the NH₂ group of asparagine.

The core protein of fibromodulin (molecular weight ~40,000 Da) also contains leucine-rich tandem repeat regions; however, its N-terminal region lacks serine residues and instead features several sulfated Tyrosine residues. Consequently, one or two keratan sulfate chains are attached to the fibromodulin core protein not in the N-terminal region, but within the leucine-rich region via the NH₂ group of asparagine.

Small proteoglycans are multifunctional macromolecules. They can interact with other connective tissue components and influence their Structure and function. For example, decorin and fibromodulin bind to type II collagen fibrils and limit their diameter (i.e., preventing the formation of thick fibrils). By binding to fibronectin, decorin and biglycan inhibit cell adhesion, whereas their binding to transforming growth factor-β reduces its mitogenic activity. Furthermore, substantial evidence indicates that small proteoglycans play a crucial regulatory role in connective tissue development and repair processes.

Basement membrane proteoglycans

Basement membrane proteoglycans exhibit considerable heterogeneity. They are predominantly heparan sulfate proteoglycans (HSPGs), represented by two main variants: high-density and low-density forms (Fig. 15-18).

Fig. 15-18. Low-density (A) and high-density (B) heparan sulfate proteoglycans. High-density heparan sulfate proteoglycans have a star-like shape and consist of four short heparan sulfate chains linked to a small protein core. Low-density heparan sulfate proteoglycans feature a large multidomain protein core composed of a single polypeptide chain. Three long heparan sulfate chains are attached to one of the poles of the core.