Biochemistry, Glycosaminoglycans

Cellular Level

The cellular organelles involved in the synthesis and modification of GAGs to their final, bioactive structures are numerous and differ depending on the GAG being synthesized. This section provides an overview of the cellular mechanisms involved in GAG biosynthesis. It is important to note that, unlike proteins and nucleic acids, GAG biosynthesis is a non-template-driven process carried out by the combined action of several tissue-specific enzymes.[2]

The process of GAG biosynthesis begins in the cellular cytoplasm with the synthesis of 5 uridine diphosphate (UDP)-activated sugars. These sugars include UDP-glucuronic acid, UDP-N-acetylglucosamine, UDP-xylose, UDP-galactose, and UDP-N-acetylgalactosamine.[4] These UDP-activated sugars are then transported from the cytoplasm to the Golgi apparatus through an antiporter transmembrane transporter for further modification. The noteworthy exception to the following steps in GAG biosynthesis is hyaluronic acid. Instead of undergoing modification and sulfation in the Golgi apparatus, the hyaluronic acid precursor sugars, UDP-glucuronic acid and UDP-N-acetylglucosamine, are transported from the cytoplasm to the plasma membrane for further processing without sulfation, leading to the production of hyaluronic acid.[4]

All other GAGs require additional modification steps that occur in and around the Golgi apparatus, including sulfation of functional groups by the sulfate donor compound 3`-phosphoadenosine-5`-phosphosulfate (PAPS). The availability of PAPS for the sulfation of GAGs significantly affects the biosynthetic rate of production of sulfated GAGs.[4] The sulfated GAGs synthesized in the Golgi apparatus undergo covalent linkage to proteins, forming proteoglycans. The tethering process for the GAGs heparin/heparan sulfate, chondroitin sulfate, and dermatan sulfate occurs via a serine residue on the protein core, which connects to a common tetrasaccharide linker between the GAG and proteoglycans. Keratan sulfate is the only sulfated GAG that is not linked to a proteoglycan protein core by this mechanism and is instead linked by various other compounds depending on the subtype of keratan sulfate, described in further detail below.[3] Modification of the resulting polysaccharide structures by enzymatic epimerization is responsible for the production of the various molecular structures of GAGs and their resulting properties. The molecular structures of individual GAGs are described in the following section.

Molecular Level

As the name suggests, the “glyco-” prefix refers to galactose or a uronic sugar (glucuronic acid or iduronic acid) attached to an aminoglycan, or amino sugar (N-acetylglucosamine or N-acetylgalactosamine). Variations in the types of monosaccharides and in the presence or absence of sulfation result in the major categories of GAGs, including hyaluronic acid, heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, and keratan sulfate. The molecular structures of the major categories are shown below.

Hyaluronic Acid

Hyaluronic acid has the simplest structure of all GAGs and does not require additional sulfation of functional groups in the Golgi apparatus, as do the other GAGs. Instead, the structure consists of sequentially bound glucuronic acid and N-acetylglucosamine residues.[4] These monosaccharide building blocks are synthesized in the cell cytoplasm and are recruited to the plasma membrane by diffusion for hyaluronic acid synthesis.[3] After synthesis within the plasma membrane, haluronic acid gets secreted from the cell into the extracellular space unmodified.

Heparan Sulfate/Heparin

Heparan sulfate and heparin (Hep) contain repeating disaccharide units of N-acetylglucosamine and hexuronic acid residues. The hexuronic acid residue, glucuronic acid, is seen in heparan sulfate, while iduronic acid is present in heparin. Sulfation of the various hydroxyl groups and the amino group on the glucosamine moiety of Heparan sulfate/Hep determines its ability to interact with various proteins, cytokines, and growth factors, and ultimately its bioactive function.[1] Heparan sulfate/Hep is tethered to a proteoglycan protein core via a serine residue connected to a tetrasaccharide linker consisting of 1 xylose, 2 galactose, and 1 glucuronic acid residue.[3]

Chondroitin Sulfate/Dermatan Sulfate

Chondroitin sulfate and dermatan sulfate are similar in structural composition to heparan sulfate. Their disaccharide repeat consists of N-acetylgalactosamine and hexuronic acids, iduronic acid in dermatan sulfate, and glucuronic acid in chondroitin sulfate. They are tethered to a proteoglycan protein core via the same serine residue and tetrasaccharide linker as heparan sulfate.[2] Similar to heparan sulfate/Hep, the sulfation pattern of chondroitin sulfate/dermatan sulfate that takes place in the Golgi apparatus determines the biological activity of the resulting compound. Chondroitin sulfate polysaccharide chains linked to carrier proteins range in their number of repeat units from 10 to 200 and are found both on cell surfaces and in the extracellular matrix.[5] 

Keratan Sulfate

Keratan sulfate contains the disaccharide repeat consisting of galactose and N-acetylglucosamine. Sulfation patterns may be present on either unit of the disaccharide repeat of keratan sulfate, with increased frequency on the N-acetylglucosamine residue. As previously mentioned, keratan sulfate is the only sulfated GAG that is not connected to the proteoglycan protein core by a tetrasaccharide linker compound. Instead, the subtypes of keratan sulfate, including KSI, KSII, and KSIII, each use a distinct mechanism for linking the proteoglycan protein core. Keratan sulfate type I GAG chains are tethered to a proteoglycan protein core by a complex glycan structure utilizing an asparagine amino acid link. Keratan sulfate type II chains are predominantly found in cartilage and utilize an N-acetylgalactosamine link via a serine or threonine residue. Keratan sulfate type III is most frequently noted in brain tissue and uses a mannose linker to the protein core via serine or threonine residues.[3]

Pathophysiology

Pathophysiological processes related to GAGs are very broad due to their ubiquitous nature in the body. This section describes how GAGs are involved in the pathophysiology of various infectious processes, as well as in a group of rare genetic diseases known as Mucopolysaccharidoses (MPS), which are related to GAG metabolism.

Infection

GAGs are very important to the infectious processes of various viral, bacterial, fungal, and parasitic pathogens. The mechanisms by which these pathogens utilize GAGs to promote virulence vary based on the unique GAGs expressed in each organ system.[6] Pathogens that invade through the skin provide many examples of how GAGs are targeted to promote dermal infection.

An intact skin epithelium is arguably the body’s most important defense against infection by providing a physical barrier composed of thick layers of dead keratinocytes. When this outer layer of skin is compromised, pathogens can invade and proliferate, causing infection, using GAGs. Merkel cell polyomavirus (MCV) is a double-stranded DNA virus that uses heparan sulfate and chondroitin sulfate on dermal cell surfaces to bind to and invade host cells, causing infection.[6]

Group A Streptococci (GAS, Streptococcus pyogenes) are Gram-positive bacteria that represent another mechanism by which pathogens use GAGs to promote virulence. GAS utilizes a hyaluronic acid GAG-containing capsule to evade host immune defenses through molecular mimicry. Due to the abundance of hyaluronic acid already present in the dermis and epidermis, the hyaluronic acid capsule of GAS prevents recognition and subsequent phagocytosis by host leukocytes.[7] Examples of other pathogens that use GAGs to promote dermal infection include Herpes Simplex Virus (HSV), Candida, Staphylococcus Aureus, and Leishmania.[6]

Mucopolysaccharidoses

Mucopolysaccharidoses comprise a group of rare genetic diseases characterized by a deficiency of lysosomal enzymes required for the metabolism of GAGs.[8] This deficit results in lysosomal accumulation of GAG intermediates that eventually leads to cellular dysfunction and death. Mucopolysaccharidoses manifest with variable symptoms depending on the dysfunctional enzyme and the associated effects on GAG metabolism in organ systems.

Initial diagnostic steps of mucopolysaccharidoses following clinical suspicion include urinary GAG and enzyme assays. Confirmatory testing for mucopolysaccharidosis is via molecular diagnosis. Previously, treatment for mucopolysaccharidoses was based on symptom management. However, both enzyme replacement therapy and hematopoietic stem cell transplantation have been successfully used to treat certain subgroups of mucopolysaccharidosis.[9]

Clinical Significance

As previously mentioned, GAGs play an essential role in many physiological processes present throughout the body. The clinical significance of each GAG class is summarized below. Note that the information provided is concise and is not intended to represent all physiological processes that involve GAGs.

Hyaluronic Acid

Hyaluronic acid is ubiquitous in body tissues and is best known for its capability of attracting water molecules. The highly polar structure of hyaluronic acid enables it to bind 10,000 times its own weight in water. Due to this characteristic, it plays a key role in lubricating synovial joints and in wound healing.[5] Hyaluronic acid is also used exogenously by clinicians to promote tissue regeneration and skin repair, and has demonstrated safety and efficacy for this purpose.[10] Hyaluronic acid is used in a variety of cosmetic products and shows promising efficacy in promoting skin tightness and elasticity, as well as improving aesthetic scores.[11] In addition to its water-binding capabilities, hyaluronic acid has been shown to promote and inhibit angiogenesis and, therefore, to be involved in the process of carcinogenesis.[5]

Heparan Sulfate/Heparin

Heparan sulfate is among the best-studied GAGs due to its many roles and potential as a pharmacological target for cancer treatment. Noteworthy functions of heparan sulfate include extracellular matrix (ECM) organization and modulation of cellular growth factor signaling by acting as a bridge between receptors and ligands. In the extracellular matrix, heparan sulfate interacts with many compounds, including collagen, laminin, and fibronectin, to promote cell-to-cell and cell-to-extracellular matrix adhesion. In the setting of malignancy, such as melanoma, degradation of heparan sulfate in the extracellular matrix by the enzyme heparanase promotes the migration of malignant cells and metastasis. This mechanism makes heparanase and heparan sulfate viable pharmacological targets for preventing cancer metastasis.[1]

Heparan sulfate also plays a key role in cellular growth factor signaling. One example of this role involves the interaction between heparan sulfate and fibroblast growth factor (FGF) and its receptor (FGFR). Heparan sulfate facilitates the formation of FGF-FGFR complexes, resulting in a signaling cascade that leads to cellular proliferation. The degree of sulfation of heparan sulfate influences the formation of these complexes. For example, the proliferation of melanoma cells is stimulated by the action of highly sulfated heparan sulfate on FGF.[1]

Heparin represents the earliest recognized biological role of GAGs, as an anticoagulant. The mechanism for this role involves its interaction with the protein antithrombin III (ATIII). The interaction of heparin with ATIII induces a conformational change that enhances ATIII's ability to function as a serine protease inhibitor of coagulation factors. Differences in heparin molecular weight have been shown to result in varying clinical anticoagulation intensities.[5]

Chondroitin Sulfate

Chondroitin sulfate is historically known for its clinical use as a disease-modifying osteoarthritis drug (DMOAD). Clinical trials have documented its potential for symptomatic pain relief and a structure-modifying effect in osteoarthritis, as evidenced by radiographic joint findings.[12] There are multiple mechanisms by which chondroitin sulfate produces these clinical effects. The pain-relieving properties of chondroitin sulfate in osteoarthritis relate to its anti-inflammatory effects, which attenuate the nuclear factor-kappa-B (NF-kappa-B) pathway, which is overactive in osteoarthritis.[13]

One of the leading pathophysiological causes of osteoarthritis is the loss of chondroitin sulfate from articular cartilage, leading to inflammation and cartilage and subchondral bone catabolism. The structure-modifying role of chondroitin sulfate in osteoarthritis is due to its role in stimulating type II collagen and proteoglycan production in both articular cartilage and the synovial membrane. This anabolic effect of chondroitin sulfate prevents further tissue damage and synovial tissue remodeling.[13]

Keratan Sulfate

Keratan sulfate has been well-studied for its functional role in both the cornea and the nervous system. The cornea comprises the richest known source of keratan sulfate in the body, followed by brain tissue.[14] The role of keratan sulfate in the cornea includes regulation of collagen fibril spacing that is essential for optical clarity, as well as optimization of corneal hydration during development based on its interaction with water molecules. As with other GAGs, the degree of sulfation of keratan sulfate determines its functional status. Abnormal sulfation patterns of keratan sulfate due to specific genetic mutations increase corneal opacity and cause visual disturbances.[14]

Keratan sulfate has also been shown to play an important regulatory role in neural tissue development. Various subgroups of keratan sulfate in the brain play key roles in stimulating microglial cell growth and promoting axonal repair following injury. Abakan is an example of a type of keratan sulfate found in brain tissue that blocks neural attachment and marks the boundaries of neural growth in the developing brain.[14] In conclusion, GAGs have widespread functions within the body. They play a crucial role in cell signaling, including regulating cell growth and proliferation, promoting cell adhesion, anticoagulation, and wound repair.