Heparan sulfate
Heparan sulfate (HS) is a linear
Proteoglycans
The major cell membrane HSPGs are the transmembrane
In the extracellular matrix, especially basement membranes and fractones,[13] the multi-domain perlecan,[14] agrin[15] and collagen XVIII[16] core proteins are the main HS-bearing species.
Structure and differences from heparin
Heparan sulfate is a member of the glycosaminoglycan (GAG) 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. Otherwise, it should be classified as HS.[17]
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.[18] It is in this form that HS is thought to exist at the cell surface.
-
GlcA-GlcNAc
-
GlcA-GlcNS
-
IdoA-GlcNS
-
IdoA(2S)-GlcNS
-
IdoA-GlcNS(6S)
-
IdoA(2S)-GlcNS(6S)
Abbreviations
- GAG = glycosaminoglycan
- GlcA = β-D-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
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, producing structural diversity encompassed by the term "heparanome" - which defines the full range of primary structures produced by a particular cell, tissue or organism.
In the 1980s, Jeffrey Esko was the first to isolate and characterize animal cell mutants altered in the assembly of heparan sulfate.[20] 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.[21]
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 tetrasaccharide primer O-linked to a serine of the core-protein:[22]
βGlcUA-(1→3)-βGal-(1→3)-βGal-(1→4)-βXyl-O-Ser.
The pathways for HS/heparin or chondroitin sulfate (CS) and dermatan sulfate (DS) biosynthesis diverge after the formation of this common tetrasaccharide linkage structure. The next enzyme to act, GlcNAcT-I or GalNAcT-I, directs synthesis, either to HS/heparin or CS/DS, respectively.[23]
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.[22][23]
Chain elongation
After attachment of the first
Mutations at the EXT1-3 gene loci in humans lead to an inability of cells to produce HS and to the development of the disease
Chain modification
As an HS 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
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.[28] 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.
There are four isoforms of NDST (NDST1–4). Both N-deacetylase and N-sulfotransferase activities are present in all NDST-isoforms but they differ significantly in their enzymatic activities.[29]
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-acetylation. GlcNH2 residues resulting from apparent uncoupling of the two activities have been found in heparin and some species of HS.[30]
Epimerisation and 2-O-sulfation
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
The 3OSTs are divided into two functional subcategories, those that generate an
Ligand binding
Heparan sulfate binds with a large number of extracellular proteins. These are often collectively called the “heparin interactome” or "heparin-binding proteins", because they are isolated by affinity chromatography on the related polysaccharide heparin, though the term “heparan sulfate interactome” is more correct. The functions of heparan sulfate binding proteins ranges from extracellular matrix components, to enzymes and coagulation factors, and most growth factors, cytokines, chemokines and morphogens [45] The laboratory of Mitchell Ho at the NCI isolated the HS20 human monoclonal antibody with high affinity for heparan sulfate by phage display.[46] The antibody binds heparan sulfate, not chondroitin sulfate.[5] The binding of HS20 to heparan sulfate requires sulfation at both the C2 position and C6 position. HS20 blocks the Wnt binding on heparan sulfate[5] and also inhibits infectious entry of pathogenic JC polyomavirus.[47]
Interferon-γ
The cell surface receptor binding region of
Wnt
Glypican-3 (GPC3) interacts with both Wnt and Frizzled to form a complex and triggers downstream signaling.[4][10] It has been experimentally established that Wnt recognizes a heparan sulfate motif on GPC3, which contains IdoA2S and GlcNS6S, and that the 3-O-sulfation in GlcNS6S3S enhances the binding of Wnt to the glypican.[5]
The HS-binding properties of a number of other proteins are also being studied:
- Antithrombin III
- Fibroblast growth factor
- Hepatocyte growth factor
- Interleukin-8
- Vascular endothelial growth factor
- Wnt/Wingless
- Endostatin
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.[49][50] Because heparan sulfate is broken down in chronic wounds by heparanase, the analogues only bind sites where natural heparan sulfate is absent and is thus resistant to enzyme degration. [51] 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.
Associated conditions
Hereditary multiple exostoses (also known as multiple hereditary exostoses or multiple osteochondromas is a hereditary disease with mutations on the EXT1 and EXT2 genes that affect biosynthesis of heparan sulfate.[52] [53]
References
- PMID 10913828.
- ^ Gallagher JT, Lyon M (2000). "Molecular structure of Heparan Sulfate and interactions with growth factors and morphogens". In Iozzo MV (ed.). Proteoglycans: structure, biology and molecular interactions. New York, New York: Marcel Dekker Inc. pp. 27–59.
- S2CID 14638091.
- ^ PMID 24492943.
- ^ PMID 27185050.
- PMID 15843372.
- PMID 11044095.
- PMID 32970989.
- PMID 21112773.
- ^ PMID 30352677.
- PMID 1556106.
- PMID 7532175.
- S2CID 28119663.
- S2CID 22934192.
- PMID 10595940.
- PMID 12556525.
- PMID 2933029.
- PMID 2932448.
- PMID 11166215.
- PMID 3858816.
- PMID 9737951.
- ^ PMID 23042481.
- ^ ISBN 9780080453828.
- ISSN 0945-053X.
- PMID 27920806.
- PMID 4228675.
- PMID 18487608.
- PMID 807579.
- PMID 11087757.
- PMID 9065769.
- S2CID 38249813.
- PMID 15303968.
- ^ S2CID 14139940.
- ^ PMID 12138164.
- ^ PMID 15303968.
- ^ PMID 17482450.
- S2CID 21853086.
- PMID 8900198.
- ^ PMID 9346953.
- PMID 9988767.
- PMID 12907690.
- PMID 15026143.
- PMID 16099108.
- PMID 16107334.
- PMID 18508513.
- PMID 30091851.
- PMID 29091757.
- PMID 9556569.
- S2CID 17262546.
- S2CID 7380997.
- S2CID 17262546.
- ISSN 0304-4165.
- PMID 26289657.