Abstract
Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.
Highlights
HA is a negatively charged linear polysaccharide consisting of repeating disaccharide units of glucuronic acid and N-acetylglucosamine at the reducing end
The saturation of the gold quartz-crystal microbalance with dissipation monitoring (QCM-D) sensor surface with small hyaluronan oligosaccharides (sHA)-eSH was almost complete within 10 minutes (Fig. 1b) and the following washing steps with buffer did not induce any change in frequency, indicating strong binding between the Synthesis of End-Thiolated sHA (sHA-eSH) and the gold sensor, Introducing unmodified sHA on the gold QCM-D sensor did not show a significant frequency change, which confirms formation of a dense adlayer by sHA-eSH, but not by unmodified sHA (Supporting Information, Figure S1)
When BSA is washed over sHA-eSH modified surfaces, BSA doesn’t induce any significant change in frequency apart from its typical buffer effect[22], which is related to the viscosity difference of BSA solution
Summary
Hyaluronan-Protein Interactions received: 09 September 2015 accepted: 27 January 2016. The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA They are an ideal tool to study the interactions of hyaluronanbinding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end. HA can be chemically modified to facilitate covalent attachments[2], an essential property to generate hydrogels[3] and immobilize it onto surfaces to control cell adhesion and protein adsorption[4,5]. The unique chemical properties of the reducing ends have been implicated in the specific recognition of hyaladherins[20]
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