Abstract
The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis (Calabro et al., 2000).
Keywords: Hyaluronan, Size distribution, Mammary gland, Extracellular matrix, Ion exchange fractionation
Background
The glycosaminoglycan HA consists of N-acetyl glucosamine and beta glucuronic acid disaccharides and is a ubiquitous component of the extracellular matrix. High molecular weight HA is fragmented by enzymatic degradation and oxidation by reactive oxygen and nitrogen species. In healthy tissues, the majority of HA is of high molecular weight. Accumulation of fragmented HA acts as danger signal during pathological processes (Tolg et al., 2012 and 2017; Yuan et al., 2015). For example, HA fragments stimulate inflammation whereas high molecular weight HA suppresses inflammation. HA influences cell behavior such as cell migration and proliferation by interaction with cell membrane receptors, leading to activation of signaling pathways. Since receptor-HA interactions are influenced by HA size, the effect of HA on tissue biology depends not only on HA amount but rather on HA size distribution and HA receptor expression by individual cells.
Materials and Reagents
Equipment
Procedure
Note: The dialysis and precipitation steps do not cause losses of HA fragments as small as 4 kDa.
Data analysis
The purified HA fractions can be quantified using an ELISA kit (for example kit K-1200, ECHELON) or by agarose gel electrophoresis and image analysis.
Recipes
Acknowledgments
This work was supported by a CBCF grant to ET and The Endre A. Balazs Foundation to MC. This protocol was adapted from Tolg et al. (2012 and 2017) and Yuan et al. (2015).
Competing interests
Authors have no conflict of interest.
References
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