Heparan sulfate
Overview
 
Heparan sulfate is a linear polysaccharide
Polysaccharide
Polysaccharides are long carbohydrate molecules, of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure,...

 found in all animal tissues. It occurs as a proteoglycan
Proteoglycan
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan chain. The point of attachment is a Ser residue to which the glycosaminoglycan is joined through a tetrasaccharide bridge...

 (HSPG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. It is in this form that HS binds to a variety of protein ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...

s and regulates a wide variety of biological activities, including developmental processes, angiogenesis
Angiogenesis
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over terminology, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for the formation of new blood...

, blood coagulation and tumour metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...

. HS has been shown to serve as cellular receptor for a number of viruses including the respiratory syncytial virus (Hallak et al.
Unanswered Questions
Encyclopedia
Heparan sulfate is a linear polysaccharide
Polysaccharide
Polysaccharides are long carbohydrate molecules, of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure,...

 found in all animal tissues. It occurs as a proteoglycan
Proteoglycan
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan chain. The point of attachment is a Ser residue to which the glycosaminoglycan is joined through a tetrasaccharide bridge...

 (HSPG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. It is in this form that HS binds to a variety of protein ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...

s and regulates a wide variety of biological activities, including developmental processes, angiogenesis
Angiogenesis
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over terminology, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for the formation of new blood...

, blood coagulation and tumour metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...

. HS has been shown to serve as cellular receptor for a number of viruses including the respiratory syncytial virus (Hallak et al. 2000)

Proteoglycans

The major cell membrane HSPGs are the transmembrane syndecans and the glycosylphosphatidylinositol (GPI) anchored glypicans. Other minor forms of membrane HSPG include betaglycan and the V-3 isoform of CD44
CD44
The CD44 antigen is a cell-surface glycoprotein involved in cell–cell interactions, cell adhesion and migration. In humans, the CD44 antigen is encoded by the CD44 gene.- Tissue distribution and isoforms :...

 present on keratinocytes and activated monocytes.

In the extracellular matrix, especially basement membrane
Basement membrane
The basement membrane is a thin sheet of fibers that underlies the epithelium, which lines the cavities and surfaces of organs including skin, or the endothelium, which lines the interior surface of blood vessels.- Composition :...

s, the multi-domain perlecan
Perlecan
Perlecan also known as basement membrane-specific heparan sulfate proteoglycan core protein or heparan sulfate proteoglycan 2 , is a protein that in humans is encoded by the HSPG2 gene....

, agrin
Agrin
Agrin is a large proteoglycan whose best characterised role is in the development of the neuromuscular junction during embryogenesis. Agrin is named based on its involvement in the aggregation of acetylcholine receptors during synaptogenesis. In humans, this protein is encoded by the AGRN...

 and collagen XVIII
Type XVIII collagen
Type XVIII collagen is a type of collagen which can be cleaved to form endostatin....

 core proteins are the main HS-bearing species.

HS structure and differences from heparin

Heparan sulfate is a member of the glycosaminoglycan
Glycosaminoglycan
Glycosaminoglycans or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose or a hexuronic acid, linked to a hexosamine .-Production:Protein cores made in the rough endoplasmic reticulum are posttranslationally...

 family of carbohydrates and is very closely related in structure to heparin
Heparin
Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...

. Both consist of a variably sulfated repeating disaccharide
Disaccharide
A disaccharide or biose is the carbohydrate formed when two monosaccharides undergo a condensation reaction which involves the elimination of a small molecule, such as water, from the functional groups only. Like monosaccharides, disaccharides form an aqueous solution when dissolved in water...

 unit. The main disaccharide units that occur in heparan sulfate and heparin are shown below.

The most common disaccharide unit within heparan sulfate is composed of a glucuronic acid (GlcA) linked to N-acetylglucosamine
N-Acetylglucosamine
N-Acetylglucosamine is a monosaccharide derivative of glucose. It is an amide between glucosamine and acetic acid...

 (GlcNAc) typically making up around 50% of the total disaccharide units. Compare this to heparin where IdoA(2S)-GlcNS(6S) makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa.
Problems arise when defining hybrid GAGs that contain both 'heparin-like' and 'HS-like' structures. It has been suggested that a GAG should qualify as heparin only if its content of N-sulfate groups largely exceeds that of N-acetyl groups and the concentration of O-sulfate groups exceeds those of N-sulfate.

Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S) or a free amine
Amine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...

 group (GlcNH3+). Under physiological conditions the ester
Ester
Esters are chemical compounds derived by reacting an oxoacid with a hydroxyl compound such as an alcohol or phenol. Esters are usually derived from an inorganic acid or organic acid in which at least one -OH group is replaced by an -O-alkyl group, and most commonly from carboxylic acids and...

 and amide
Amide
In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...

 sulfate groups are deprotonated and attract positively charged counterions to form a salt. It is in this form that HS is thought to exist at the cell surface.



Abbreviations

  • GlcA = β-L-glucuronic acid
    Glucuronic acid
    Glucuronic acid is a carboxylic acid. Its structure is similar to that of glucose. However, glucuronic acid's sixth carbon is oxidized to a carboxylic acid...

  • IdoA = α-L-iduronic acid
    Iduronic acid
    L-Iduronic acid is the major uronic acid component of the glycosaminoglycans dermatan sulfate, and heparin. It is also present in heparan sulfate although here in a minor amount relative to its carbon-5 epimer glucuronic acid....

  • IdoA(2S) = 2-O-sulfo-α-L-iduronic acid
  • GlcNAc = 2-deoxy-2-acetamido-α-D-glucopyranosyl
  • GlcNS = 2-deoxy-2-sulfamido-α-D-glucopyranosyl
  • GlcNS(6S) = 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate

HS biosynthesis

Many different cell types produce HS chains with many different primary structures. Therefore, there is a great deal of variability in the way HS chains are synthesised. However, essential to the formation of HS regardless of primary sequence is a range of biosynthetic enzymes. These enzymes consist of multiple glycosyltransferases, sulfotransferase
Sulfotransferase
Sulfotransferases are transferase enzymes that catalyze the transfer of a sulfate group from a donor molecule to an acceptor alcohol or amine. The most common sulfate donor is 3'-phosphoadenosine-5'-phosphosulfate...

s and an epimerase. These same enzymes also synthesise heparin
Heparin
Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...

.

Many of these enzymes have now been purified, molecularly cloned and their expression patterns studied. From this and early work on the fundamental stages of HS/heparin biosynthesis using a mouse mastocytoma cell free system a lot is known about the order of enzyme reactions and specificity.

Chain initiation

HS synthesis initiates with the transfer of xylose
Xylose
Xylose is a sugar first isolated from wood, and named for it. Xylose is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms and includes an aldehyde functional group. It is the precursor to hemicellulose, one of the main constituents of biomass...

 from UDP-xylose by xylosyltransferase
Xylosyltransferase
Xylosyltransferase are transferase enzymes which act upon xylose and are classified under EC 2.4.2.More specifically, they can refer to:* Dolichyl-phosphate D-xylosyltransferase* Dolichyl-xylosyl-phosphate-protein xylosyltransferase...

 (XT) to specific serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

 residues within the protein core. Attachment of two galactose
Galactose
Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....

 (Gal) residues by galactosyltransferases I and II (GalTI and GalTII) and glucuronic acid
Glucuronic acid
Glucuronic acid is a carboxylic acid. Its structure is similar to that of glucose. However, glucuronic acid's sixth carbon is oxidized to a carboxylic acid...

 (GlcA) by glucuronosyltransferase I (GlcATI) completes the formation of a core protein linkage tetrasaccharide

βGlcA-1,3-βGal-1,3-βGal-1,4-βXyl.

Xylose
Xylose
Xylose is a sugar first isolated from wood, and named for it. Xylose is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms and includes an aldehyde functional group. It is the precursor to hemicellulose, one of the main constituents of biomass...

 attachment to the core protein is thought to occur in the endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...

 (ER) with further assembly of the linkage region and the remainder of the chain occurring in the golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....

.

The pathways for HS/heparin or chondroitin sulfate
Chondroitin sulfate
Chondroitin sulfate is a sulfated glycosaminoglycan composed of a chain of alternating sugars . It is usually found attached to proteins as part of a proteoglycan. A chondroitin chain can have over 100 individual sugars, each of which can be sulfated in variable positions and quantities...

 (CS) and dermatan sulfate
Dermatan sulfate
Dermatan sulfate is a glycosaminoglycan found mostly in skin, but also in blood vessels, heart valves, tendons, and lungs....

 (DS) biosynthesis diverge after the formation of this common linkage structure. The next enzyme to act, GlcNAcT-I or GalNAcT-I, directs synthesis, either to HS/heparin or CS/DS, respectively.

Chain elongation

After attachment of the first N-acetylglucosamine
N-Acetylglucosamine
N-Acetylglucosamine is a monosaccharide derivative of glucose. It is an amide between glucosamine and acetic acid...

 (GlcNAc) residue elongation of the tetrasacchride linker is continued by the stepwise addition of GlcA and GlcNAc residues. These are transferred from their respective UDP-sugar nucleotides. This is carried out by one or more related enzymes whose genes are members of the exostoses (EXT) gene family of tumour suppressors.

Mutations at the EXT1-3 gene loci in humans leads to an inability of cells to produce HS and to the development of the disease Multiple Hereditary Exostoses (MHE).

MHE is characterized by cartilage-capped tumours, known as osteochondromas or exostoses, which develop primarily on the long bones of affected individuals from early childhood until puberty. Although exostoses are in themselves benign, surgery may be required to alleviate secondary complications such as joint pain and restricted movement.

For further information on this disease see the dedicated web site here

Chain modification

As the chain polymerises, it undergoes a series of modification reactions carried out by four classes of sulfotransferases and an epimerase. The availability of the sulfate donor PAPS is crucial to the activity of the sulfotransferases.

N-deacetylation/N-sulfation

The first polymer modification is the N-deacetylation/N-sulfation of GlcNAc residues into GlcNS. This is a prerequisite for all subsequent modification reactions, and is carried out by one or more members of a family of four GlcNAc N-deacetylase/N-sulfotransferase enzymes (NDSTs). In early studies, it was shown that modifying enzymes could recognize and act on any N-acetylated residue in the forming polymer. Therefore the modification of GlcNAc residues should occur randomly throughout the chain. However, in HS, N-sulfated residues are mainly grouped together and separated by regions of N-acetylation where GlcNAc remains unmodified.

Generation of GlcNH2

Due to the N-deacetylase and N-sulfotransferase being carried out by the same enzyme N-sulfation is normally tightly coupled to N-desulfation. GlcNH2 residues resulting from apparent uncoupling of the two activities have been found in heparin and some species of HS.

Epimerisation and 2-O-sulfation

Epimerisation is catalysed by one enzyme, the GlcA C5 epimerase or heparosan-N-sulfate-glucuronate 5-epimerase . This enzyme epimerises GlcA to iduronic acid
Iduronic acid
L-Iduronic acid is the major uronic acid component of the glycosaminoglycans dermatan sulfate, and heparin. It is also present in heparan sulfate although here in a minor amount relative to its carbon-5 epimer glucuronic acid....

 (IdoA). Substrate recognition requires that the GlcN residue linked to the non-reducing side of a potential GlcA target be N-sulfated. Uronosyl-2-O-sulfotransferase (2OST) sulfates the resulting IdoA residues.

6-O-sulfation

Three glucosaminyl 6-O-transferases (6OSTs) have been identified that result in the formation of GlcNS(6S) adjacent to sulfated or non-sulfated IdoA. GlcNAc(6S) is also found in mature HS chains.

3-O-sulfation

Currently seven glucosaminyl 3-O-sulfotransferases (3OSTs) are known to exist in mammals (eight in zebrafish) . The 3OST enzymes create a number of possible 3-O-sulfated disaccharides, including GlcA-GlcNS(3S±6S) (modified by 3OST1 and 3OST5), IdoA(2S)-GlcNH2(3S±6S)(modified by 3OST3a, 3OST3b, 3OST5 and 3OST6) and GlcA/IdoA(2S)-GlcNS(3S) (modified by 3OST2 and 3OST4) . As with all other HS sulfotransferases, the 3OSTs use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfate donor. Despite being the largest family of HS modification enzymes, the 3OSTs produce the rarest HS modification, the 3-O-sulfation of specific glucosamine residues at the C3-OH moiety .

The 3OSTs are divided into two functional subcategories, those which generate an antithrombin III binding site (3OST1 and 3OST5) and those which generate a herpes simplex virus 1 glycoprotein D (HSV-1 gD) binding site (3OST2, 3OST3a, 3OST3b, 3OST4, 3OST5 and 3OST6) . As the 3OSTs are the largest family of HS modification enzymes and their actions are rate-limiting, substrate specific and produce rare modifications, it has been hypothesized that 3OST modified HS plays an important regulatory role in biological processes .

Interferon-γ

The cell surface receptor binding region of Interferon-γ overlaps with the HS binding region, near the protein's C-terminal. Binding of HS blocks the receptor binding site and as a result, protein-HS complexes are inactive.

The HS-binding properties of a number of other proteins are also being studied:
  • Antithrombin III
  • Fibroblast Growth Factors
  • Hepatocyte Growth Factor
    Hepatocyte growth factor
    Hepatocyte growth factor/scatter factor is a paracrine cellular growth, motility and morphogenic factor. It is secreted by mesenchymal cells and targets and acts primarily upon epithelial cells and endothelial cells, but also acts on haemopoietic progenitor cells...

  • Interleukin-8
  • Vascular Endothelial Growth Factor
    Vascular endothelial growth factor
    Vascular endothelial growth factor is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate....

  • Wnt/Wingless
  • Endostatin
    Endostatin
    ]Endostatin is a naturally-occurring 20-kDa C-terminal fragment derived from type XVIII collagen. It is reported to serve as an anti-angiogenic agent, similar to angiostatin and thrombospondin....


Heparan sulfate analogue

Heparan sulfate analogues are thought to display identical properties as heparan sulfate with exception of being stable in a proteolytic environment like a wound. Because heparan sulfate is broken down in chronic wounds by heparanase, the analogues only bind sites where natural heparan sulfate is absent and cannot be broken down by any known heparanases and glycanases. Also the function of the heparan sulfate analogues is the same as heparan sulfate, protecting a variety of protein ligands such as growth factors and cytokines. By holding them in place, the tissue can then use the different protein ligands for proliferation.

For more information see:Heparan sulfate analogue
Heparan sulfate analogue
Heparan sulfate analogues are polymers engineered to mimic several properties of heparan sulfates. They can be constituted with a backbone of polysaccharides, such as poly glucose or glucuronates or a polyester such as co polymers of lactic or malic acid to which sulfates, sulfonate or...

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