CD44 is a multifunctional glycoprotein that binds to hyaluronan and fibrin(ogen). Alternative splicing is responsible for the generation of numerous different isoforms, the smallest of which is CD44s. Insertion of variant exons into the extracellular membrane proximal region generates the variant isoforms (CD44v). Here, we used force spectroscopy to delineate the biophysical and molecular requirements of CD44-HA and CD44-fibrin(ogen) interactions at the single-molecule level. CD44v-HA and CD44s-HA single bonds exhibit similar kinetic and micromechanical properties because the HA-binding motif on CD44 is common to all of the isoforms. Although this is the primary binding site, O- and N-linked glycans and sulfation also contribute to the tensile strength of the CD44-HA bond. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than CD44s-fibrinogen but is weaker than the CD44-HA bond. In contrast to CD44-HA binding, the molecular interaction between CD44 and fibrin(ogen) is predominantly mediated by the chondroitin sulfate and dermatan sulfate on CD44. Blocking sulfation on CD44s modestly decreases the tensile strength of CD44s-fibrin(ogen) binding, which is in stark contrast to CD44v-fibrin interaction. Collectively, the results obtained by force spectroscopy in conjunction with biochemical interventions enable us to delineate the biophysical parameters and molecular constituents of CD44 binding to hyaluronan and fibrin(ogen).
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