How to measure Glycosaminoglycans and Proteoglycans?

Glycosaminoglycans (or mucopolysaccharides) are long un-branched polysaccherides which consist of disaccharide repeats. The repeats usually (with the exception of Keratan sulfate) consist of on amino sugar (N-acetylgalactosamine of N-acetylglucosamine) and a uronic sugar (iduronic acid or glucuronic acid) or galactose.

Glycosaminoglycans are highly polar and function as water attractants, thus they are useful as shock absorbers or lubricants.

Four glycosaminoglycan classes can be distinguished:

Structure chondroitin sulfate

Chrondroitin sulfate

  • Heparin/heparan sulfate
  • Chondroitin sulfate/dermatan sulfate
  • Keratan sulfate
  • Hyaluronic acid

Dermatan sulfate

 

 

 

 

 

These structures differ in their core disaccaride structure and their synthesis pathways. While Heparin/heparan sulfate, Chondroitin sulfate/dermatan sulfate and Keratan sulfate are produced by the Golgi apparatus, Hyaluronic acid is synthezised by integral membrane synthases which immediately secrete the elongated disaccharide chain.

Proteoglycans – core proteins attached to glycosaminoglycans

Proteoglycans consist of core proteins which are covalently linked to glycosaminoglycans – either mono- or poly-glycosylated. The glycosaminoglycan is coupled though a tetrasaccharide bridge to a Serin residue of the protein. Proteoglycans represent the major component of the animal extracellular matrix where they form complex networks with other proteoglycans and fibrous matrix proteins such as collagen. These networks are involved in binding water and diverse cations.

Mucopolysaccharidoses are genetic discorders which lead to the inability to degrade proteoglycans. As a consequence an accumulation of proteoglycans occurs which subsequently leads to a number of pathological symptoms (dependent on the type of proteoglycan which cannot be degraded).

One assay to measure almost all glycosaminoglycans and peptidoglycans

Flowchart Blyscan

Fig. 1: Blyscan Assay Flowchart

The Blyscan Assay (see flowchart Fig. 1) is a quantitative dye-binding method for the analysis of sulfated proteoglycans and glycosaminoglycans. Test material can be assayed directly when present in a soluble form, or following papain extraction from biological materials. The assay can be used to measure the total glycosaminoglycans content and can also be adopted to determine the O- and N-sulfated glycosaminoglycan ratio within test samples.
The dye label used in the assay is 1, 9-dimethylmethylene blue and the dye is employed under conditions that provide a specific label for the sulfated polysaccharide component of proteoglycans or the protein free sulfated glycosaminoglycan chains.

Note: the assay is not suitable for small sulfated disaccharide fragments or for samples containing alginates, as these contain uronic acid.

Results Blyscan

Fig. 2: Recovery of soluble glucosaminoglycans from mouse tissues, with 3 and 18 hours of hot papain.

For analysing which soluble extracts?

  • fibrous and hyaline cartilages
  • arteries, heart, lung, skin and other material containing extracellular matrix, (connective tissue) and solid tumours (results see Fig. 2)
  • extracellular matrix components that may be released by live cells into the culture medium, some of which can be attached to cell culture plasticware
  • soluble glycosaminoglycans from synovial fluid, urine and gel chromatography fraction aliquots

Would the Blyscan assay be of use in your research? Share your comments or questions below!

 

 

 

Hyaluronic acid – size matters!

HA or Hyaluronan measurement | tebu-bio

Hyaluronan (HA) Competitive ELISA standard curve (cat. nr 117K-1200-1ea)

Hyaluronic acid (HA), or hyaluronan, is an ubiquitous, very high molecular mass polysaccharide that has applications in a variety of fields, including cosmetics, some types of surgery (e.g. opthalmic) and regenerative medicine. It can even be present as a contaminant in some bio-production processes. HA has also been suggested as a possible biomarker for Alzheimer’s disease (AD).

HA acts as a molecular shock-absorber and stabilizer for cells. Its visco-elastic properties, biologically speaking, are valuable for separating tissue and maintaining shape. It is a key in tissue lubrication, and it may play a role in wound repair. It is the ideal choice for some implants, as it does not usually cause an immune response (contrary to what may happen with some biomaterials). Size of the HA used in therapy has an impact on its success. Usually, higher weight forms usually render longer benefits. For bio-production, however, smaller HA forms are usually the main concern.

So, depending on the reason why you are studying this marker, keep size in mind in order to choose the best assay to measure the HA levels in your experimental model. It is of key importance that whatever product you use, analysis of the HA sizes detected with it are clearly mentioned in the technical documentation.

HA or Hyaluronan measurement | tebu-bio

Hyaluronic acid (HA) sandwich ELISA standard curve (direct assay cat. nr 117K-4800-1kit)

A post by my colleague Dr. Philippe Fixe is of great help for choosing the right assay!

It’s not that these assays will allow you to discriminate between high- and low-weight HA. What is intended here is that these assays will allow you to detect all or part of the HA forms depending on its weight. And that may be crucial, as you may be using the assay that does not detect the HA sizes relevant for your experiment.

It is also possible now to outsource HA measurements to an external lab performing regularly validated Hyaluronic acid-specific immuno-assays. As an example, Echelon’s Competitive or Sandwich HA ELISAs outsourcing by tebu-bio’s lab in cosmetology or drug discovery.

Any comments? Feel free to share them below!

 

How to select Hyaluronic Acid (HA) quantitative tests?

Hyaluronic acid (also called HA or Hyaluronan) is a glycosaminoglycan with unique characteristics. HA possesses elevated viscosity, enabling tissues (eye, skin, joint and synovial fluid…) to resist to physical and mechanical constraints (torsion, flexion…). Over time, when HA production is declining, tissues progressively loss these tensile properties, leading to wrinkles and fold, weak reepithelization and age-related troubles. [Read more…]