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, 181 (3), 264-73

Age-related Nanostructural and Nanomechanical Changes of Individual Human Cartilage Aggrecan Monomers and Their Glycosaminoglycan Side Chains

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Age-related Nanostructural and Nanomechanical Changes of Individual Human Cartilage Aggrecan Monomers and Their Glycosaminoglycan Side Chains

Hsu-Yi Lee et al. J Struct Biol.

Abstract

The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecan was significantly weaker in compression than newborn aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of aggrecan, with direct implications for the effects of aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.

Figures

Figure 1
Figure 1
Tapping mode AFM height images of (a, b, c) newborn and (d, e, f) 38-year old adult human aggrecan monomers. Globular domains are indicated by arrows (a and d). An example of core protein trace, LCP, end-to-end distance, Ree, and GAG chain trace, LGAG, are shown in (c).
Figure 2
Figure 2
Histograms and box-and-whisker plots of structural parameter distributions of newborn and 38-year-old adult human aggrecan monomer core proteins: (a) trace length, LCP, of all observed aggrecan, (b) trace length, LCP, of full length aggrecan, (c) end-to-end distance, Ree, of full length aggrecan, and (d) extension ratio, Ree/LCP, of full length aggrecan. *: p < 0.01 between the newborn and adult human aggrecan populations via Mann-Whitney U test.
Figure 3
Figure 3
Histograms and box-and-whisker plots of newborn and 38-year-old adult human aggrecan monomer GAG side chain trace length, LGAG, distributions: (a) individual aggrecan, (b) all observed aggrecan, (c) full length aggrecan. *: p < 0.0001 between the newborn and adult human aggrecan populations via Mann-Whitney U test.
Figure 4
Figure 4
Tapping mode AFM height images of (a, b) untreated, (c, d) keratanase II-treated and (e, f) chondroitinase ABC-treated 29 year old adult human aggrecan monomers. An example of core protein trace, LCP, and end-to-end distance, Ree, are shown in (b).
Figure 5
Figure 5
Tapping mode AFM height images of chondroitinase ABC-treated aggrecan monomers. The presence of short GAG side chains, presumably KS-GAGs, are visualized near one end of the core proteins (arrows), likely within the KS-domain along the core protein.
Figure 6
Figure 6
Histograms and box-and-whisker plots of the structural parameter distributions of 29-year-old full length adult human aggrecan monomers: (a) core protein trace length, LCP, (b) end-to-end distance, Ree, and (c) extension ratio, Ree/LCP. Only full length aggrecan monomers with indentifiable G1 and G3 globular domains are included. *: p < 0.05 for each population compared with the other two populations via Mann-Whitney U test.
Figure 7
Figure 7
(a–d) Three dimensional height images (at ~ 3 nN applied normal force) and compressed aggrecan layer height, H, versus applied normal force, F, curves of end-grafted (a,c) newborn and (b,d) adult human aggrecan layers, measured via contact mode AFM imaging on an aggrecan and hydroxyl-terminated self-assembled monolayer (OH-SAM) micro-patterned surface (top right schematic). Each data point represents the average of eight different scan locations under the same normal force, the SDs are smaller than the size of the data symbols. (e–f) Normal force, F, versus distance, D, curves of end-grafted (e) newborn and (f) adult human aggrecan layers, measured via AFM-based colloidal force spectroscopy. The probe tip approached the substrate perpendicular to the plane of the substrate (bottom right schematic). Each curve represents the average of 30 different locations on the aggrecan pattern, the SDs are smaller than the width of the data curves. (g) Comparison of the H versus F (open triangles, replotted from c and d) and F versus D (dashed lines, replotted from e and f) measurements at 0.01 M ionic strength. All the experiments were conducted in NaCl aqueous solutions at 0.001 – 1.0 M ionic strengths using a gold-coated spherical colloidal probe tip (R ~ 2.5 µm), functionalized with OH-SAM.
Figure 8
Figure 8
Stress versus sGAG concentration curves converted from force-distance curves in Fig. 7e and f (0.1 M NaCl). Each curve is an average of 30 approaches at different locations on the aggrecan pattern. The 95% confidence intervals of each averaged curve are smaller than the width of the data curves.

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