Hyaluronic acid

The glycosaminoglycan hyaluronic acid contains alternating residues of D-glucuronic acid and
N-acetylglucosamine. They form clear, highly viscous solutions that serve as lubricants in the
synovial fluid of joints and give the vitreous humor of the vertebrate eye its jellylike consistency.
Hyaluronate is also an essential component of the extracellular matrix of cartilage and tendons,
to which it contributes tensile strength and elasticity as a result of its strong interactions with
other components of the matrix. Hyaluronidase can hydrolyze the glycosidic linkages of
hyaluronate, rendering tissues more susceptible to bacterial invasion. In many organisms, a
similar enzyme
in sperm hydrolyzes an outer glycosaminoglycan coat around the ovum, allowing sperm
penetration.
Other glycosaminoglycans differ from hyaluronate in two respects: they are generally much
shorter polymers and they are covalently linked to specific proteins.

Biosynthesis of Hyaluronic acid

Biosynthesis of HA begins with the phosphorylation of glucose by hexokinase to produce the

main precursor, glucose-6-phosphate. From here, HA synthesis pathway can then be divided into
two distinct pathways that synthesise the two building blocks of HA, glucuronic acid and N-
acetylglucosamine. In the first set of reactions, phosphoglucomutase converts glucose-6-
phosphate to glucose-1-phosphate before a phosphate group from UTP is transferred to glucose-
1-phosphate by UDP-glucose pyrophosphorylase (hasC) to produce UDP-glucose. Finally, UDP-
glucose is oxidised by UDP-glucose dehydrogenase (hasB) to yield the first HA precursor, UDP-
glucuronic acid (UDP-GlcUA). In the second set, glucose-6-phosphate is converted to fructose-
6-phosphate by phosphoglucoisomerase (hasE). Once converted, fructose-6-phosphate is tagged
with an amido group transferred from a glutamine residue via amidotransferase (glmS) to
produce glucosamine-6-phosphate and then is modified by mutase (glmM) to yield glucosamine-
1-phosphate. This intermediate is then sequentially acetylated and phosphorylated by
acetyltransferase and pyrophosphorylase, respectively (hasD) to yield the second HA precursor,
UDP-N-acetylglucosamine (UDP-GlcNAc). Once the two precursors are synthesised, hyaluronic
acid/hyaluronan synthase (hasA) polymerises the two components in an alternate manner to
produce the HA polymer. It is important to note that HA biosynthesis is an energy-consuming
process for the bacteria as several intermediates are also used in cell wall biosynthesis, biomass
formation and lactate formation via glycolysis.

Chondroitin sulfates
Chondroitin 4-sulfate is a major constituent of various mammalian tissues (bone, cartilage,
tendons,heart valves,skin, cornea etc.).Structurally it is comparable with hyaluronic acid.
Chondroitin 4-sulfatec consists of repeating disaccharide nits composed of D-glucuronic acid
and N-acetyl D-galactosamine4 -sulfate.

Dermatan sulfate

The name dermatan sulfate is derived from the fact that this compound mostly occurs in the skin. lt is
structurally related to chondroitin 4-sulfate.The only difference is that there is an inversion in the
configuration around C5 of D-glucuronic acid to form L-iduronic acid

Dermatan sulfate

CS/DS Biosynthesis

The biosynthesis of CS/DS is a complex, multistep process that occurs in endoplasmic

reticulum/Golgi compartments and is initiated by the synthesis of GAG-protein linkage region
covalently linked to specific serine residues embedded in different core proteins. The linkage
region is a specific tetrasaccharide structure GlcAβ1-3Galβ1-3Galβ1-4Xylβ1, in which Gal and
Xyl represent galactose and xylose residues, respectively. This structure is catalyzed by the
corresponding glycosyltransferase in the tetrasaccharide sequence. Firstly, a Xyl residue is
connected to a specific Ser residue of core protein through the catalyzation of xylosyltransferase;
then, β1,4-galactosyltransferase I and β1,3-galactosyltransferase II catalyze the connection of
two Gal residues in turn; and, finally, β1,3-glucuronyltransferase I catalyzes the addition of GlcA
residue to form the tetrasaccharide linkage region.

Once the synthesis of the linkage tetrasaccharide is completed, the extension of CS/DS chain will
be triggered by the transfer of a GalNAc residue to the nonreducing terminal GlcA residue of the
tetrasaccharide linkage region by GalNAc transferase I, and then GlcA and GalNAc residues will
be added in turn to form the chondroitin (Chn) skeleton composed of repeating disaccharide
GlcA-GlaNAc through alternating catalysis of GalNAc transferase II and GlcA transferase II.
During the process of polymerization, some GlcA residues in the Chn skeleton can be
transformed into IdoA under the control of two GlcA C-5 epimerases, thereby transforming the
corresponding Chn domains into its stereoisomer dermatan domains . Furthermore, some
hydroxyl groups of GalNAc or GlcA/IdoA residues in the chains can be site-specifically
modified by a variety of sulfotransferases by using 3’-phosphoadenosine 5’-phosphosulfate as a
donor substrate. Under the control of chondroitin 4-O-sulfotransferase-1, 2 and , and dermatan 4-
O-sulfotransferase the sulfate group is transferred to the hydroxyl group at the C-4 location of
GlcA to generate an A unit and an iA unit, respectively. The 6-O-sulfation of the C unit is
catalyzed by chondroitin 6-O-sulfotransferase-1. The GalNAc 4-sulfate 6-O-sulfotransferase
transfers sulfate to the C-6 position of the A/iA unit to generate an E/iE unit, and uronyl 2-O-
sulfotransferase sulfates GlcA in the C-2 position of the C/iA unit to generate a D/iB unit . The
space-time-dependent expression and combined action of these enzymes make the structure of
CS/DS chains extremely diverse and complex, which presents significant challenges for the
structural and functional studies of CS/DS.

The structure of CS/DS. The CS/DS chain consisting of D-glucuronic acid (GlcA) or L-iduronic
(IdoA) acid glycosidically linked to N-acetylgalacyosamine (GalNAc) [(-4GlcAβ1-3GalNAcβ1-)
or (-4IdoAα1-3GalNAcβ1-). CS/DS chains are covalently attached to the core protein by GAG-
protein linkage region tetrasaccharide.

Heparin

Heparin is a natural anticoagulant made in mast cells (a type of leukocyte) and released into the
blood, where it inhibits blood coagulation by binding to the protein antithrombin. Heparin
binding causes antithrombin to bind to and inhibit thrombin, a protease essential to blood
clotting. The interaction is strongly electrostatic; heparin has the highest negative charge density
of any known biological macromolecule. Purified heparin is routinely added to blood samples
obtained for clinical analysis, and to blood donated for transfusion, to prevent clotting.

Heparin biosynthesis

Heparin and heparan sulfate share the same polysaccharide backbone structure but differ in sulfation
degree and expression pattern. Heparan sulfate is found in virtually all cells of the human body, where as
heparin expression is restricted to mast cells. Heparin and heparan sulfate consisting of N-acetyl-D-
glucosamine (GlcNAc) and glucuronic acid (GlcA) units that are partly modified by epimerization of
GlcA to iduronic acid (IdoA) and by sulfation at different positions of mainly GlcNAc and IdoA residues.

Initiation

The biosynthesis of heparin and HS begins with the formation of a tetrasaccharide linkage region (-GlcA-
Gal-Gal-Xyl-). This process is catalyzed by four enzymes adding individual monosaccharides
sequentially to the growing glycosaminoglycan (GAG) chain. A xylose residue from UDP-xylose is first
transferred to the hydroxyl group of a serine residue on the core protein by xylosyltransferase. The
serglycin protein contains a stretch of repetitive Ser–Gly residues that can be decorated with heparin
chains, making the protein densely glycosylated

The attachment of xylose is followed by a stepwise transfer of two galactose and one glucuronic acid
residues. The enzymes responsible for the subsequent steps of the linkage region formation,
galactosyltransferase-I (GalT1), galactosyltransferase-II (GalT2), and glucuronyltransferase (GlcAT1).
These units may be modified by phosphorylation (xylose) and/or sulfation (galactose units).
Phosphorylation/sulfation of the tetrasaccharide linker may thus be a way of regulating GAG synthesis.
Addition of GlcNAc results in HS/heparin formation.

Elongation

Addition of the first GlcNAc residue to the GlcA of the heparin/HS linkage region is performed by
enzymes with GlcNAcT-I activity. The heparin/HS chain is elongated by addition of alternating
glucuronate and N-acetyl-glucosamine residues from their respective UDP-sugars are catalysed by
enzymes with GlcNAcT-II activity.

Modification
As the HS/heparin chain grows, it is modified by a set of various enzymes. Mammalian HS/heparin
modification enzymes include C5-epimerase, 2-O-sulfotransferase, 6-O-sulfotransferases and 3-O-
sulfotransferases. HS chains are only partly modified, with the modifications occurring in clusters,
resulting in polysaccharide chains having regions that are highly sulfated interspersed with unmodified
regions. Heparin is more heavily sulfated, containing 80–90% N-sulfated glucosamine, whereas about
30–60% of the glucosamine residues in HS are N-sulfated. D-Glucuronic acid residues adjacent to N-
sulfated glucosamine can be epimerized to L-iduronic acid followed by 6-O-sulfation of GlcNAc and 2-
O-sulfation of IdoA and, more rarely, of GlcA. Sulfate groups can also be found at the C3-position of
GlcNAc, although this modification is not very common.

Keratan sulfate

Keratan sulfate is the only GAG that does not contain uronic acid. They are present in cornea,
cartilage, bone, and a variety of horny structures formed of dead cells: horn, hair, hoofs, nails,
and claws. Keratan sulfate is a sulfated polylactosamine. It resembles the other GAGs by the backbone
structure of alternating β1,3 and β1,4 bonds. N-acetylglucosaminyltransferase and β-1,4-
galactosyltransferase transfer GlcNAc and Gal to a nonreducing terminus of a carbohydrate core structure
connected to carrier proteins, are involved in elongation of the poly-N-acetyllactosamine backbone of KS.
In KSPGs, the lactosaminoglycans are normally sulfated on the 6-position of either or both sugars.
Two sulfotransferases (GlcNAc 6-O-sulfotransferase and Gal 6-O-sulfotransferase), which transfer a
sulfo group to the 6-O-position of GlcNAc or Gal.
Two types of KSPGs exist depending upon their linkage to the core protein. KSI which is
prominent in the cornea is linked through N-glycosylamine bonds to asparagine residues in the
core protein in a mannose-containing linkage oligosaccharide. This is the main KS in the cornea
and is part of at least three KSPGs, fibromodulin, lumican, and keratocan. These are members of
the small leucine-rich proteoglycans (SLRPs).
Chain elongation involves the glycosyl transfer of galactose and GlcNA. UDP galactose is an
essential precursor. UDP-galactose is formed from UDP-glucose and requires UDP-glucose-4-
epimerase. UDP-glucose is used for either the formation of UDP-glucuronic acid for CS
synthesis or UDP-galactose for KS synthesis. The enzyme UDP-glucose dehydrogenase which
permits CS synthesis is inhibited by NADH so that NAD/NADH levels are also important
regulators of KS biosynthesis.
The Golgi membrane fractions of corneal cells contain a KS acetylglucosaminyl transferase and
galactosyltransferase activity and sulfation is catalyzed by at least two sulfotransferases, GlcNAc
6-O-sulfotransferase and Gal 6-O-sulfotransferase. The Gal 6-O-sulfotransferase from cornea is
specific for KS and does not act on chondroitin. In fact, the activity of these polymerizing
enzymes decreases with increasing molecular mass and sulfation degree, suggesting a feedback
in which degree of sulfation may dictate chain termination. Thus, these enzymes are key
regulators of KS biosynthesis.
The second type of KS is linked to core protein via a linkage to Ser/Thr as in the mucins and
follows rules outlined for the synthesis of mucins. This form, found in skeletal muscle is
frequently referred to as KS II.