Hyaluronic acid binding to CD44S is indiscriminate of molecular weight

The ubiquitous presence of hyaluronic acid (HA) in the extracellular matrix (ECM) of both healthy and diseased tissues underscores its importance in human physiology. Previous studies suggest that HA can be used as a probe to qualitatively monitor cell surface levels of CD44 and other important HA receptors; however, these studies use mixtures of HA at various molecular weights. Using fluorescently labeled HA, we evaluated the apparent differences of low (25 kilodalton) and high (700 kilodalton) molecular weight HA interacting with breast cancer cell lines of varying levels of CD44. Our results confirm that CD44 expression and the apparent level of HA interaction correlates with molecular weight. Importantly, we show that HA only binds a small fraction of the major CD44 isoform, CD44S, on cell surfaces and that CD44S interactions account for <50% of the total HA bound to cell surfaces. Although increased fluorescence level correlates with higher molecular weight of HA, this appears to be an artifact of chain length and not a result of multivalent binding between HA and CD44S. Accordingly, we verify that HA binding characteristics of cell surfaces is similar to previous artificial membrane models which proposed that HA anchors to CD44S and forms a non-binding corona of HA that extends beyond the surface.