Distinct kinetic and molecular requirements govern CD44 binding to hyaluronan versus fibrin(ogen)

Abstract

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).

Figure 1

Single-molecule force spectroscopy as used to probe the biomechanical properties of CD44s-fibrin(ogen) bonds. (A) Schematic diagram of the standard and variant isoforms of CD44. Orange hexagons represent putative sites of N-linked glycosylation; green circles represent putative sites for O-linked glycosylation (SS, disulfide bond; P, serine phosphatases). (B) Schematic diagram of the MFP used to investigate the kinetic and micromechanical properties of receptor-ligand interactions at single-molecule resolution. (C) CD44s was immunoprecipitated from HL60 human myeloid leukemia cells, and CD44v was immunoprecipitated from LS174T colon carcinoma cells and subjected to SDS-PAGE under reducing conditions, followed by Western blotting using the anti-CD44 mAb 2C5. (D) Typical force-distance traces acquired from force spectroscopy experiments in which fibrin or fibrinogen immobilized on a cantilever tip was brought into contact hundreds of times with immunopurified CD44s incorporated into lipid vesicles and layered onto a PEI-cushioned glass slide. The red arrows in the first two curves indicate two distinct CD44s-fibrin rupture events. The rupture event in the red box is enlarged on the right to show the drop in the force. The slope before rupture multiplied by the retraction velocity gives the loading rate. The third curve is typically obtained when there is no binding. The scale bar for the three curves is also indicated.

Figure 2

Kinetic and micromechanical properties of CD44-HA binding at the single-molecule level. (A) Average rolling velocities (μm/s) of microspheres (2 × 10⁶/ml) decorated with either CD44v immunopurified from LS174T cells or CD44s immunopurified from HL60 cells on 50 μg/ml HA at prescribed wall shear stresses. Data represent the mean ± SE from n = 3 independent experiments. (B) Frequency of binding events between HA and CD44v or CD44s in the absence and presence of the function-blocking CD44 antibody Hermes-1. DMPC-CD44v or DMPC-CD44s lipid solutions were premixed with 20 μg/ml of the Hermes-1 mAb just before bilayers were formed on PEI-coated glass slides. Data represent the mean ± SE of n = 3 independent experiments; ^∗p < 0.05 with respect to control. (C) Rupture force (pN) as a function of loading rate (pN/s) for CD44v-HA and CD44s-HA interactions. Data were acquired for a range of retraction velocities from 5 to 25 μm/s, using a dwell time of 20 ms. Data represent the mean ± SE of n = 4–6 experiments, with each experiment having at least 1200 approach/reproach cycles.

Figure 3

Role of glycosylation and sulfation of CD44 in CD44-HA binding. (A and B) Average rupture force of (A) CD44v-HA and (B) CD44s-HA as a function of contact duration. CD44v was immunoprecipitated from LS174T cells cultured in medium containing either 2 mM benzyl-GalNAc to inhibit O-linked glycosylation or DPBS. CD44s was immunoprecipitated from HL60 cells containing either 2 mM benzyl-GalNAc or DPBS. CD44v and CD44s were treated with PNGaseF according to the manufacturer's instructions to cleave N-linked glycans. (C and D) Average rupture force of (C) CD44v-HA and (D) CD44s-HA bonds as a function of contact duration. CD44v was immunoprecipitated from LS174T cells cultured in medium containing either sodium chlorate (60 mM) to block sulfation or DPBS. CD44s was immunoprecipitated from HL60 cells cultured in medium containing either sodium chlorate (20 mM) or DPBS. Data represent the mean ± SE of n = 3 independent experiments performed at a retraction velocity of 20 μm/s; ^∗p < 0.05 with respect to untreated control.

Figure 4

Kinetic and micromechanical properties of CD44s-fibrin and CD44s-fibrinogen interactions at the single-molecule level. (A) Rupture force (pN) as a function of loading rate (pN/s) for CD44s-fibrin and CD44s-fibrinogen interactions. Data were acquired for a range of retraction velocities from 5 to 25 μm/s and using a dwell time of 20 ms. Data represent mean ± SE of n = 4–6 experiments, where each experiment had at least 1200 approach/reproach cycles. (B) Distribution of adhesion forces obtained experimentally (bars) or computed using a Monte Carlo simulation (line) based on Bell model kinetic parameters for CD44s-fibrin interactions. The retraction velocity was maintained for 20 μm/s.

Figure 5

Effect of Hermes-1 on CD44s-fibrin(ogen) binding. (A and B) The frequency of binding events (A) and average rupture force (B) of CD44s binding to fibrin were evaluated in the absence and presence of the function-blocking CD44 antibody Hermes-1. (C and D) The frequency of binding events (C) and average rupture force (D) of CD44s binding to fibrinogen were evaluated in the absence and presence of Hermes-1. DMPC-CD44s lipid solutions were premixed with 20 μg/ml of the Hermes-1 mAb just before bilayers were formed on PEI-coated glass slides. Data represent the mean ± SE of n = 3 independent experiments; ^∗p < 0.05 with respect to untreated control, and ^∗∗p < 0.05 with respect to CD44s binding to fibrinogen in the presence of Hermes-1 at 2 ms.

Figure 6

Role of glycosylation and sulfation of CD44 in CD44-fibrin(ogen) binding. (A and B) Frequency of binding between CD44s treated with chondroitinase ABC (1 U/ml) or PNGaseF according to the manufacturer's instructions, and (A) fibrin or (B) fibrinogen as a function of contact duration compared with untreated control. (C and D) Average rupture force of (C) CD44s-fibrin and (D) CD44s-fibrinogen bonds as a function of contact duration. CD44s was immunoprecipitated from HL60 cells cultured in medium containing sodium chlorate (20 mM) to block sulfation or DPBS. Data represent the mean ± SE of n = 3 independent experiments performed at a retraction velocity of 20 μm/s; ^∗p < 0.05 with respect to untreated control.