Heparan sulfate

Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (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 ligands and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation and tumour metastasis. 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 present on keratinocytes and activated monocytes.

In the extracellular matrix, especially basement membranes, the multi-domain perlecan, agrin and collagen XVIII core proteins are the main HS-bearing species.

HS structure and differences from heparin
Heparan sulfate is a member of the glycosaminoglycan family of carbohydrates and is very closely related in structure to heparin. Both consist of a variably sulfated repeating disaccharide 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 (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 group (GlcNH3+). Under physiological conditions the ester and amide 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
 * IdoA = α-L-iduronic 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, sulfotransferases and an epimerase. These same enzymes also synthesise heparin.

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 from UDP-xylose by xylosyltransferase (XT) to specific serine residues within the protein core. Attachment of two galactose (Gal) residues by galactosyltransferases I and II (GalTI and GalTII) and glucuronic 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 attachment to the core protein is thought to occur in the endoplasmic reticulum (ER) with further assembly of the linkage region and the remainder of the chain occurring in the golgi apparatus.

The pathways for HS/heparin or chondroitin sulfate (CS) and dermatan sulfate (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 (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 (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 subcatagories, 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
 * Interleukin-8
 * Vascular Endothelial Growth Factor
 * Wnt/Wingless
 * Endostatin