Abstract
Hyaluronan is a simple repeating disaccharide polymer, synthesized at the cell surface by integral membrane synthases. The repeating sequence is perfectly homogeneous, and is the same in all vertebrate tissues and fluids. The polymer molecular mass is more variable. Most commonly, hyaluronan is synthesized as a high-molecular mass polymer, with an average molecular mass of approximately 1000–8000 kDa. There are a number of studies showing increased hyaluronan content, but reduced average molecular mass with a broader range of sizes present, in tissues or fluids when inflammatory or tissue-remodeling processes occur. In parallel studies, exogenous hyaluronan fragments of low-molecular mass (generally, <200 kDa) have been shown to affect cell behavior through binding to receptor proteins such as CD44 and RHAMM (gene name HMMR), and to signal either directly or indirectly through toll-like receptors. These data suggest that receptor sensitivity to hyaluronan size provides a biosensor of the state of the microenvironment surrounding the cell. Sensitive methods for isolation and characterization of hyaluronan and its fragments have been developed and continue to improve. This review provides an overview of the methods and our current state of knowledge of hyaluronan content and size distribution in biological fluids and tissues.
Highlights
Hyaluronan is found in vertebrate tissues, as a key component of the extracellular matrix
Quantification is accomplished by methods such as HPLC, capillary electrophoresis (CE), mass spectrometry, or fluorophore-assisted carbohydrate electrophoresis
To address the issues of limited sample amount, purification difficulty, and the importance of analyzing both high and low M HA simultaneously, we recently developed a method using size-dependent fractionation of HA by anion exchange on a spin column, and quantification of HA in the fractions using a competitive ELSA assay (IEX-ELSA) [37]
Summary
Hyaluronan (hyaluronic acid, HA) is found in vertebrate tissues, as a key component of the extracellular matrix. In inflammation and other specific tissue-remodeling processes, covalent transfer of the heavy chain domains of IαI to HA can be catalyzed by TSG-6 protein [11,12,13,14]. Unsulfated or undersulfated chondroitin, which is rare in normal tissues but may be significant in remodeling tissues, is not removed by this process At this point in the procedure, specific isolation of HA can be accomplished by affinity methods, such as use of a biotinylated HA-specific binding protein and streptavidin-coated magnetic beads, or other similar medium such as gel beads [36, 37]. Testing for degradation of HA during isolation can be accomplished by “spiking” the initial fluid or tissue with a pure HA sample of known M and low polydispersity in M, and testing its size in the final isolate. Spiking samples with known amounts of HA can be used to detect losses during isolation, including losses due to non-specific interactions with surfaces or other macromolecules that HA may not normally contact
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