10 hyaluronan and proteoglycans

What is a glycosaminoglycan GAG?

A linear unbranched polysaccharide made of repeating disaccharides

What is a proteoglycan?

A core protein with one or more GAG chains attached

What are the four major GAG families?

Hyaluronan chondroitin sulfate dermatan sulfate heparan sulfate heparin and keratan sulfate

Which GAG is not synthesised attached to a core protein?

Hyaluronan

Where is hyaluronan synthesised?

At the plasma membrane and extruded extracellularly

What disaccharide repeats make up hyaluronan?

Glucuronic acid and N acetylglucosamine

What linkages connect the HA repeat units?

Alternating beta 1-3 and beta 1-4 linkages

Is hyaluronan sulfated?

No it has no post polymer modifications

How long is a typical HA chain in disaccharides?

About 10000 disaccharides

What is meant by HA polydispersity?

HA exists as a mixture of sizes in tissues

What is the structural description of HA in solution?

A stiffened random coil

Why does HA occupy a large domain in solution?

Charge repulsion and hydrogen bond mediated stiffening

What is the critical overlap concentration of HA?

The concentration above which HA chains entangle

At roughly what concentration does large HA begin to entangle?

Above about 0.5 mg per mL

Does HA form strong direct chain to chain associations?

No its properties arise mainly from entanglement

How many water molecules associate with one HA disaccharide?

About 15

What happens to HA size in high salt like 0.5 M NaCl?

It collapses because charges are shielded

What does 0.5 M NaOH do to HA conformation?

It disrupts hydrogen bonds making HA more flexible and smaller

What property makes HA important in synovial fluid?

Viscoelasticity and molecular sieving

Name two medical uses of HA?

Viscosupplementation for joints and eye surgery

Where is HA found as a free polymer besides synovial fluid?

Vitreous gel of the eye and Whartons jelly in umbilical cord

How much HA is in an average human?

About 15 grams

What fraction of HA turns over each day?

About one third

Where is excess HA cleared from blood?

By uptake in the liver

When does serum HA increase?

Inflammation and liver disease and severe viral infection

Why do some bacteria make HA capsules?

To mimic host HA and evade immune detection

What are the three mammalian hyaluronan synthases?

HAS1 HAS2 and HAS3

What is unique about HAS enzymes?

A single membrane enzyme polymerises HA and exports it outside the cell

Which HAS knockout is embryonic lethal?

HAS2 knockout

Why is HAS2 knockout lethal?

Failure to form cardiac cushions during development

Are HAS1 and HAS3 knockouts lethal?

No they are viable and fertile

Which end does mammalian HAS extend HA from?

The non reducing end

What protein family contains many HA binding proteins?

The link module superfamily

What is the major cell surface receptor for HA?

CD44

Which HA receptor is mainly on lymphatic endothelium?

LYVE1

What is TSG6?

A secreted HA binding protein and catalyst in heavy chain transfer

How is CD44 regulated to bind HA during inflammation?

By conformational change influenced by glycosylation such as removal of sialic acid

What assay visualises pericellular HA coats?

Particle exclusion assay

What is inter alpha inhibitor IAI?

A plasma chondroitin sulfate proteoglycan with bikunin core protein and heavy chains

How are heavy chains linked in IAI?

Covalently attached to the chondroitin sulfate chain

What modification can heavy chains undergo during inflammation or ovulation?

Transfer from CS to hyaluronan forming HC-HA

What enzyme catalyses heavy chain transfer to HA?

TSG6

How does TSG6 transfer heavy chains to HA?

Via a covalent intermediate and two sequential transesterification reactions

Where are HC-HA complexes commonly formed?

In inflammation and during ovulation

What is the cumulus oocyte complex COC?

An oocyte surrounded by about 1000 to 1500 cumulus cells

What drives COC expansion?

Hyaluronan synthesis and crosslinking by heavy chain transfer

How much does COC volume increase during expansion in mouse?

About 40 fold

Where does IAI come from during ovulation?

From plasma after breakdown of the blood follicle barrier

What happens in bikunin knockout mice during ovulation?

COC fails to expand and females are infertile

What happens in TSG6 knockout mice?

COC fails to expand and females are infertile

What is PTX3 and what does it do in the COC matrix?

Pentraxin 3 acts as a crosslinking node linking HC-HA complexes

Why is the cumulus matrix important for fertilisation?

It captures sperm and allows regulated penetration

What enzyme helps sperm move through the HA rich cumulus matrix?

Hyaluronidase

What is the Youngs modulus of the cumulus matrix?

About 1 Pascal making it extremely soft

What is the main take home message about HA function?

Most HA biology is mediated by HA binding proteins and supramolecular assemblies