Langmuir 2008, 24, 1495-1508
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Molecular Aspects of Boundary Lubrication by Human Lubricin: Effect of Disulfide Bonds and Enzymatic Digestion† Bruno Zappone,‡,§ George W. Greene,§ Emin Oroudjev,| Gregory D. Jay,⊥ and Jacob N. Israelachvili*,§ Centro di Eccellenza LiCryL, UniVersity of Calabria, Rende (CS), 87036 Italy, Materials Department and Materials Research Laboratory, UniVersity of California Santa Barbara, Santa Barbara, California 93106, Life Science, UniVersity of California Santa Barbara, California 93106, and Department of Emergency Medicine and DiVision of Engineering, Brown UniVersity, ProVidence, Rhode Island 02903 ReceiVed August 2, 2007. In Final Form: October 30, 2007 Lubricin (LUB) is a glycoprotein of the synovial cavity of human articular joints, where it serves as an antiadhesive, boundary lubricant, and regulating factor for the cartilage surface. It has been proposed that these properties are related to the presence of a long, extended, heavily glycosylated and highly hydrated mucinous domain in the central part of the LUB molecule. In this work, we show that LUB has a contour length of 220 ( 30 nm and a persistence length of e10 nm. LUB molecules aggregate in oligomers where the protein extremities are linked by disulfide bonds. We have studied the effect of proteolytic digestion by chymotrypsin and removal of the disulfide bonds, both of which mainly affect the N- and C- terminals of the protein, on the adsorption, normal forces, friction (lubrication) forces, and wear of LUB layers adsorbed on smooth, negatively charged mica surfaces, where the protein naturally forms lubricating polymer brush-like layers. After in situ digestion, the surface coverage was drastically reduced, the normal forces were altered, and both the coefficient of friction and the wear were dramatically increased (the COF increased to µ ) 1.1-1.9), indicating that the mucinous domain was removed from the surface. Removal of disulfide bonds did not change the surface coverage or the overall features of the normal forces; however, we find an increase in the friction coefficient from µ ) 0.02-0.04 to µ ) 0.13-1.17 in the pressure regime below 6 atm, which we attribute to a higher affinity of the protein terminals for the surface. The necessary condition for LUB to be a good lubricant is that the protein be adsorbed to the surface via its terminals, leaving the central mucin domain free to form a low-friction, surface-protecting layer. Our results suggest that this “end-anchoring” has to be strong enough to impart the layer a sufficient resistance to shear, but without excessively restricting the conformational freedom of the adsorbed proteins.
Introduction Lubricin (LUB) is a glycoprotein found in the synovial fluid that specifically binds to the surface of cartilage in human articular joints.1-3 In Vitro solutions of LUB in saline buffer lubricate two shearing cartilage surfaces,4,5 cartilage and glass,6 latex and glass,7 and two mica surfaces8 as effectively as the whole synovial fluid. Consequently, it has been proposed that LUB serves as the primary boundary lubricant in articular joint lubrication and possibly acts synergistically with hyaluronic acid (HA)3,7,9 and/or lipids10,11 †
Part of the Molecular and Surface Forces special issue. * To whom correspondence should be addressed. E-mail: jacob@ engineering.ucsb.edu. ‡ University of Calabria. § Materials Department and Materials Research Laboratory, University of California Santa Barbara. | Life Science, University of California Santa Barbara. ⊥ Brown University. (1) Jay, G. D. Curr. Opin. Orthop. 2004, 15, 355-359. (2) Jones, A. R. C.; Gleghorn, J. P.; Hughes, C. E.; Fitz, L. J.; Zollner, R.; Wainwright, S. D.; Caterson, B.; Morris, E. A.; Bonassar, L. J.; Flannery, C. R. J. Orthop. Res. 2007, 25 (3), 283-292. (3) Schmidt, T. A.; Gastelum, N. S.; Nguyen, Q. T.; Schumacher, B. L.; Sah, R. L. Arthritis Rheum. 2007, 56, 882-91. (4) Radin, E. L.; Swann, D. A.; Weisser, P. A. Nature 1970, 228 (5269), 377-378. (5) Swann, D. A.; Sotman, S.; Dixon, M.; Brooks, C. Biochem. J. 1977, 161 (3), 473-485. (6) Swann, D. A.; Hendren, R. B.; Radin, E. L.; Sotman, S. L.; Duda, E. A. Arthritis Rheum. 1981, 24 (1), 22-30. (7) Jay, G. D.; Lane, B. P.; Sokoloff, L. Connect. Tissue Res. 1992, 28, 245255. (8) Zappone, B.; Ruths, M.; Greene, G. W.; Jay, G. D.; Israelachvili, J. N. Biophys. J. 2007, 92 (5), 1693-1708. (9) Jay, G. D.; Torres, J. R.; Warman, M. L.; Laderer, M. C.; Breuer, K. S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6194-6199.
to protect the surfaces of articular cartilage against wear. LUB has also been shown to be an efficient antiadhesive agent and cytoprotective factor in the synovial cavity, preventing cartilagecartilage integration12 and the excessive accumulation of proteins, cells, and other components of the synovial fluid at the surfaces of joints13 (which can reduce lubrication efficiency and increase the risk of premature joint wear). The absence of LUB and the expression of mutated LUB gene products cause joint malformation, decreased joint lubrication, and increased cartilage wear that often leads to complete joint failure.14 These symptoms are typical of common debilitating arthritic diseases such as osteoarthritis which currently affects more than 13% of the world population.15 The unexplained origins and prevalence of this disease motivate our investigation of LUB as a regulatory factor and possible palliative supplement in joint diseases that affect the adhesion, lubrication, and wear of articular cartilage.16 Human LUB has a molecular weight of Mw ≈ 280 kDa and possesses a characteristic molecular structure composed of a (10) Ozturk, H. E.; Stoffel, K. K.; Jones, C. F.; Stachowiak, G. W. Tribol. Lett. 2004, 16, 283-289. (11) Schwarz, I. M.; Hills, B. A. Br. J. Rheumatol. 1998, 37, 21-26. (12) Schaefer, D. B.; Wendt, D.; Moretti, M.; Jakob, M.; Jay, G. D.; Heberer, M.; Martin, I. Biorheology 2004, 4 (3-4), 503-508. (13) Rhee, D. K.; Marcelino, J.; Baker, M.; Gong, Y.; Smits, P.; Lefebvre, V.; Jay, G. D.; Stewart, M.; Wang, H.; Warman, M. L.; Carpten, J. D. J. Clin. InVest. 2005, 115 (3), 622-631. (14) Marcelino, J.; Carpten, J. D.; Suwairi, W. M.; Gutierrez, O. M.; Schwartz, S.; Robbins, C.; Sood, R.; Makalowska, I.; Baxevanis, A.; Johnstone, B.; Laxer, R. M.; Zemel, L.; Kim, C. A.; Herd, J. K.; Ihle, J.; Williams, C.; Johnson, M.; Raman, V.; Alonso, L. G.; Brunoni, D.; Gerstein, A.; Papadopoulos, N.; Bahabri, S. A.; Trent, J. M.; Warman, M. L. Nat. Genet. 1999, 23 (3), 319-322. (15) World Health Organization, WHO Tech. Rep. Ser. 2003, 919 (i-x), 1-218.
10.1021/la702383n CCC: $40.75 © 2008 American Chemical Society Published on Web 12/08/2007
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Figure 1. Schematic representation of the lubricin structure. Exons are represented with different shades of gray and are numbered from the N-terminal. Exon 6, called the “mucin” domain, is densely coated with short lateral sugar chains containing two or three sugar units, about 2/3 of which are negatively charged. The mucin domain accounts for approximately 67% of the AA and 50% of the protein molecular weight, Mw ≈ 280 kDa. Exons 2, 4, and 5 are subject to alternative splicing, producing four lubricin isoforms. Exons 1-5 and 7-12 contain subdomains similar, respectively, to somatomedin-B (SMB) and homeopexin (HPX). The vertical bars indicate the location of the cysteine residues, which can form intra- and intermolecular disulfide bonds.
long, heavily glycosylated (sugar-coated) and highly hydrophilic central domain flanked by two nonglycosylated globular end domains (Figure 1). The central domain carries most of the negative charges of the protein, while positive charges and hydrophobic amino acids (AA) are mainly located in the end domains. The composition of LUB is similar to that of mucin proteins that lubricate and protect many surfaces in the human body, including teeth, eyelids, respiratory tract, gastrointestinal tract, urogenital tract, and other biolubricating surfaces.17 Mucins specifically bind to the epithelial surfaces, often in the form of a gel (“mucus”), where molecules are physically entangled or covalently linked in long chains via disulfide bonds between cysteine (Cys) residues. It is believed that the molecular basis of the lubricating properties of LUB and mucins is related to the presence of the central, heavily hydrophilic region which is commonly referred to as the “mucin domain” (Figure 1). This idea is supported by studies on synthetic polymers in good solvents18 and charged polyelectrolytes in aqueous solutions19 adsorbed on molecularly smooth surfaces in the form of “brushes”, that is, densely grafted to the substrate with only one of their ends. When brought into contact, the adsorbed brush layers repel each other primarily through steric-entropic forces that arise from the tendency of flexible molecules to coil randomly (entropically) and, therefore, resist the conformational restriction imposed by confinement.20 Two contacting brushes can show a very low friction coefficient, µ < 0.01 (“superlubricity”), as a result of two effects: (1) the low degree of interpenetration of the brushes leading to the formation of a sharp interface, that is, with few cross-border entanglements, and (2) the presence of hydrated counterions which bind to the charged polymer chain to form hydration sheaths. A strong hydration prevents adhesion and reduces the friction between the chains. Superlubricity is normally limited to pressures below a few atmospheres. For higher pressures, the friction rapidly increases due to increasing entanglement and decreasing level of hydration of the polymer layers. It thus appears that the presence of a long, flexible, and highly hydrated portion in the structure of a polymeric molecule plays a central role in determining its lubricating capability. However, efficient boundary lubrication cannot be attributed solely to the presence of this domain. The boundary lubricating properties of LUB, mucins, and polymeric molecules in general arise from a (16) Rhee, D. K.; Marcelino, J.; Al-Mayouf, S.; Schelling, D. K.; Bartels, C. F.; Cui, Y. J.; Laxer, R.; Goldbach-Mansky, R.; Warman, M. L. J. Biol. Chem. 2005, 280 (35), 31325-31332. (17) Bansil, R.; Stanley, E.; LaMont, J. T. Annu. ReV. Physiol. 1995, 57, 635-657. (18) Klein, J.; Kumacheva, E.; Mahalu, D.; Perahia, D.; Fetters, L. J. Nature 1994, 370 (6491), 634-636. (19) Raviv, U.; Giasson, S.; Kampf, N.; Gohy, J. F.; Jerome, R.; Klein, J. Nature 2003, 425 (6954), 163-165. (20) de Gennes, P. G. AdV. Colloid Interface Sci. 1987, 27 (3-4), 189-209.
complex interplay of molecular properties (chemistry and structure) and physical interactions. First, the binding of the molecule to the substrate must be strong enough to create a stable adsorption and to prevent the molecules from becoming detached and squeezed out from the contact junction by compression and shear forces. For example, HA is a flexible biopolymer that is highly hydrated due to the high density of negative charges along the main backbone. HA binds weakly to negatively charged mica surfaces and is quickly expelled from the contact junction under modest loads (pressures) and shear forces. As a result, HA fails to lubricate under the conditions where LUB acts as a good lubricant.21 Second, the adsorbed layer of lubricating molecules must expose the hydrated portions of the molecules to the shearing interface. Third, these domains must not bind to or interpenetrate with the opposing surface layer. Positively charged and highly hydrated chitosan, for example, binds strongly to mica and is a good lubricant at very low contact pressures,22 but it fails to lubricate at higher pressures. In fact, adsorbed chitosan molecules penetrate and become entangled with the opposing layer, where they also form adhesive “bridges” with the opposing surface, therefore increasing the friction. Cross-linking also plays an important role in lubrication and wear: HA becomes an excellent wear protector for mica after being chemically cross-linked to form a surface gel layer,23 even though the friction coefficient is little changed. This phenomenon also raises an important question of whether the role of a biological boundary lubricant is (i) to reduce the friction force and simultaneously protect the surfaces from wear or (ii) to act synergistically with other boundary lubricants, with each one serving a specific function which may also include antiadhesive, chemical inertness, biorecognition, and signaling. The case of LUB is representative of this complexity. LUB binds to negatively charged hydrophilic mica and to hydrophobized mica in the form of a dense and thick polymer brush-like layer.8 The adsorbed layers have similar composition and structure on both surfaces. This arises due to LUB’s ability to bind to the different substrates preferentially via the globular end-domains2 which contain most of the protein’s positive charges and hydrophobic residues (Figure 1). The central mucin domain, on the other hand, is strongly negatively charged and hydrophilic and has a low affinity for mica and hydrophobic surfaces. The preferential adsorption of the globular end-domains gives the brush-like layers a conformation that is either “tail-like” when only one end is adsorbed or “loop-like” when both ends are adsorbed. In each of these configurations, the outermost exposed (21) Tadmor, R.; Chen, N.; Israelachvili, J. N. J. Biomed. Mater. Res. 2002, 61, 514-23. (22) Kampf, N.; Raviv, U.; Klein, J. Macromolecules 2004, 37 (3), 11341142. (23) Benz, M.; Chen, N. H.; Israelachvili, J. J. Biomed. Mater. Res., Part A 2004, 71 (1), 6-15.
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glycosylated segments give rise to a pure brush-like repulsion when pressed against another, similar, surface. LUB acts a good boundary lubricant when adsorbed on mica, producing a coefficient of friction µ ) 0.02-0.04 for pressures up to 6 atm. For higher pressures, the adsorbed LUB layers undergo an irreversible shear-induced rearrangement which is accompanied by a sudden increase of the friction coefficient to µ ) 0.2-0.6. In contrast, LUB layers adsorbed on a hydrophobic surface show high friction at all pressures but no shear-induced rearrangement.8 The causes of such a difference in lubricating behavior lie in the molecular-level details of the interactions such as the strength of the binding to mica or hydrophobic surfaces and the resulting relative abundance and configuration of the loops and tails. In the present work, we have considered how changes in the molecular structure of LUB affect the protein adsorption and conformation on negatively charged surfaces such as mica and, ultimately, the ability of LUB to act as an efficient antiadhesive, lubricant, and wear-protecting agent. We aimed at selectively modifying LUB terminal domains, leaving the lubricating mucin domain intact. We used a proteolytic enzyme, chymotrypsin, to selectively cleave aromatic AA, which are abundant in the end domains but are rare in the mucin domain and located close to its extremities.24 Alternatively, we used reducing and alkylating agents to remove intra- and intermolecular disulfide bonds between Cys residues, most of which are located in the end domains. We found that the end domains regulate the selfassembly of LUB proteins into oligomers and determine the strength of the adsorption of the protein on the mica surface. These factors strongly influence the ability of the mucin domain to lubricate and protect the surfaces from wear. Materials and Methods Preparation of Lubricin (LUB) Samples. Human LUB protein was purified from synovial fluid according to the procedure described in ref 25 and diluted in phosphate filtered buffered saline (PBS from Sigma, 120 mM NaCl, 10 mM phosphate salt, 2.7 mM KCl, catalog #P3744). To remove disulfide bonds, we followed the procedure described in ref 26 to reduce and alkylate the Cys residues. A protein solution at a concentration of 0.7 mg/mL in PBS was reduced by an equal volume of PBS containing 4 µL of 0.5 M dithiothreitol and boiled at 100 °C for 3 min. The alkylating agent iodoacetamide (0.25 M) was subsequently added in a volume which was 5% of the total reaction volume and boiled at 100 °C for 1 min. Solutions of native and reduced protein samples were further reduced in PBS in aliquots of about 100 µL at a concentration of 0.25 mg/mL, near the value typically found in human synovial fluid.2 The pH was also kept in the physiological range of 7.2-7.6.27 The solutions were stored at -18 °C for less than 4 months before use, allowing a maximum of four short freeze/thaw cycles during the sample preparation. From our measurements, we could not detect any degradation of the protein following storage and freezing. In the following, we will refer to these solutions of native and reducedand-alkylated protein as LUB and R-LUB, respectively (Table 1). Chymotrypsin digestion was done before or after adsorption of LUB on the mica surfaces. In the former case, digestion in solution was done by adding TLCK-treated chymotrypsin to a 0.77 mg/mL LUB/PBS solution containing the protease inhibitors leupeptin (10 µg/mL) and aprotinin (5 µg/mL).28 The solution was diluted to (24) Genbank, Accession Code NM 005798. (25) Jay, G. D.; Harris, D. A.; Cha, C. J. Glycoconjugate J. 2001, 18, 807815. (26) Lane, L. C. Anal. Biochem. 1978, 86, 655-664. (27) Sokoloff, L. The joints and synoVial fluid; Academic Press: New York, 1980; Vol. 2. (28) Jay, G. D.; Tantravahi, U.; Britt, D. E.; Barrach, H. J.; Cha, C. J. J. Orthop. Res. 2001, 19 (4), 677-687.
Langmuir, Vol. 24, No. 4, 2008 1497 Table 1. Abbreviations Used for the Different Forms of LUB Protein Considered LUB R-LUB D-LUB SD-LUB
native form reduced and alkylated forma digested with chymotrypsin in solution before adsorptionb digested in situ with chymotrypsin after adsorption on micac
a The solution also contained reducing and alkylating agents. b The solution also contained chymotrypsin and protease inhibitors. c The surfaces were immersed in a solution containing chymotrypsin and Ca2+.
concentrations between 0.25 and 0.29 mg/mL before the measurements and stored as described before. In the following, we refer to this solution as to D-LUB (Table 1). Although digestion by chymotrypsin was expected to leave the longest portion of the mucin domain intact, we could not isolate the mucin domain from the D-LUB solution neither by electrophoresis and Western blotting nor by inhibition enzyme-linked immunoadsorbent assay (ELISA) using a specific antibody for the mucin domain. This indicated that chymotrypsin had an unexpected ability to digest nonaromatic AA in the mucin domain (see the Discussion section). For in situ digestion after adsorption, mica surfaces bearing adsorbed layers of LUB were first rinsed with pure PBS (no LUB) and then with PBS containing 0.05 mg/mL CaCl2, and finally immersed in PBS/CaCl2 solution containing 10 units of chymotrypsin/1 mL. In the following, this surface-digested form of the protein will be called SD-LUB (Table 1). All protein solutions were prepared in a dust-free laminar flow hood using filtered PBS. The samples were centrifuged for a few minutes at 1000-3000 rpm before any measurement. Atomic Force Microscopy (AFM). For AFM imaging of single molecules and aggregates, droplets of a few microliters of 0.5 µg/ mL LUB or R-LUB in PBS were deposited on freshly cleaved muscovite mica or highly ordered pyrolytic graphite (HOPG). Mica was either left bare or pretreated in a solution of NiCl2 to adsorb positively charged Ni2+ on the negatively charged lattice sites of the mica surface.29 In the latter case, mica was dipped in NiCl2 solution, rinsed in Millipore purified water, and thoroughly dried with a flow of N2. After adsorbing the protein from PBS, the surfaces were rinsed in purified water and dried with N2. AFM imaging was performed in dry air under a steady flow of N2 (relative humidity ≈ 5%) with a Multimode microscope (Veeco Digital Instruments) used in tapping mode. Dynamic Light Scattering (DLS). DLS samples from 1.2 mg/ mL solutions of LUB and 0.6 mg/mL solutions of R-LUB in PBS were studied with a Malvern Zetasizer Nano ZS instrument (Malvern Instruments, U.K.), using a He-Ne laser (λ ) 633 nm) in a backscattering geometry. Solutions were loaded in a 45 µL quartz cuvette that was thoroughly cleaned in organic solvents and acid solutions before each use. The time autocorrelation function of the scattered intensity, G1(q,t), was fitted using the method of cumulants:30 1 1 G1(q,t)/G1(q,0) ≈ exp -K1t + K2t2 - K3t3 + ... , 2 3!
[
]
for t f 0 (1)
where q is the scattering vector. K1/q2 ) C is the z-averaged diffusion coefficient given by C)
∑ p (q) M C ∑ p (q) M i
i
i
i i
i
(2)
i
where pi(q), Mi, and Ci are, respectively, the scattering form factor, molecular weight, and diffusion coefficient of the ith scattering species. From eq 2, one can calculate an apparent average hydrodynamic radius of the scattering objects, Rh, using the Stokes-Einstein (29) Hansma, H. G.; Laney, D. E. Biophys. J. 1996, 70 (4), 1933-1939. (30) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814.
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equation: Rh ) kBT/3πhC, where kBT and η are, respectively, the Boltzmann factor and viscosity of PBS. Another useful parameter is the so-called polydispersity index, PdI ) K2/2K12, which is zero for a monodisperse sample. The DLS setup was tested (calibrated) on a solution of monodisperse latex nanospheres of nominal diameter d ) 60 nm (Figure 4) (supplied by Malvern Instruments as a calibration sample). The test correctly gave a value of Rh ) 31 nm and a small polydispersity of PdI ) 0.036. We also tested the filtered PBS (no LUB) used to prepare the protein solutions, and we did not detect any contaminating particles. Surface Forces Apparatus (SFA). Normal and friction forces were measured using a surface forces apparatus (model SFA3). A detailed description of the SFA3 can be found in ref 31. Two backsilvered sheets of muscovite mica were glued with UV-curable glue (NOA61, by Norland) onto half-cylindrical glass lenses with a radius of R ) 2 cm. The lenses were assembled in the SFA chamber with their mica surfaces facing one another in a crossed geometry, approximating a sphere near a flat surface (see schematic inset in Figure 5a). A 50-100 µL droplet of LUB/PBS solution was introduced between the surfaces, drawn in by capillary forces. To prevent evaporation, the surfaces were sealed in the SFA chamber together with a reservoir of water that was not in direct contact with the surfaces but ensured saturation of the surrounding vapor. The protein solution was “incubated” between the surfaces for at least 3 h at 25.0 ( 0.1 °C before any measurements were made to allow adsorption to reach equilibrium and reduce thermal and mechanical drifts to a rate of 150 nm, where the forces between the adsorbed LUB layers were always negligible, and 500 atm), and even after a permanent rearrangement of the LUB layers occurred.8 Effect of Chymotrypsin Digestion and Ca2+Ions. Digestion of LUB with chymotrypsin had a much more profound effect on the adsorption and lubrication of LUB than reduction and alkylation. For both D-LUB and SD-LUB, where LUB was digested before and after adsorption (see Table 1), respectively, there was very little adsorption and the surface coverage was below the detection limit, Γ < 1 mg/m2. After adsorption of D-LUB on Ni2+-mica, AFM images did not show any of the
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Figure 6. Summary of the normalized forces, F/R, shown on a semilog scale as a function of the mica-mica distance, D, for 0.250.29 mg/mL solutions of LUB (gray data points, dotted and dashed lines) and R-LUB (black data points and solid line) in PBS. The data points for R-LUB are taken from Figure 5 (with the same meaning of the symbols), excluding the data obtained during and after shearing. The solid, dotted, and dashed lines are fits to the AlexanderDeGennes model, eq 3, with values of the brush height, L, average grafting distance, s, and offset distance, D0, given in the figure. For LUB, two values of L were observed. The gray data points correspond to the lower value of L (from ref 8). The noise level in the force measurements was about 0.1 mN/m.
Figure 7. Friction force, f, as a function of the normal force (load), F, or the pressure, P (from eq 4), between two mica surfaces in the presence of 0.29 mg/mL R-LUB in PBS solution. The sliding speed was V ≈ 1 µm/s. Straight lines are the best linear fits to the data, giving the friction coefficient µ ) df/dF. The data labeled as (b-d) correspond to the force curves shown in Figure 5b-d, respectively, with the same meaning of the symbols. The data labeled with the symbols b/O (approach/retraction) belong to another measurement, showing a sudden rearrangement of the R-LUB layer at high loads associated with a decrease of the friction. Error bars are only shown for the points of highest load of each measurement. The error bars on F are much smaller than the sizes of the data points.
features observed for nondigested LUB (Figure 2). The presence of the digested protein on bare mica was only revealed by the normal force curves, shown in Figures 8 and 9, respectively, for
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D-LUB and SD-LUB, which were different from those between untreated mica surfaces in pure PBS (LUB-free) solutions. Adsorption of D-LUB produced a purely repulsive force that was reversible during approach/retraction cycles (no hysteresis), as shown in Figure 8a and b. The range of the repulsion, about 40 nm, was much smaller than the range measured for undigested LUB. The adsorbed layers of D-LUB were thus less dense and much thinner than the adsorbed layers of the nondigested protein. The force curves also showed a certain variability in the range of the repulsion from one contact position to another (Figure 8b), indicating a nonuniformity in the adsorbed layer. We notice that the range of the repulsion was larger than but of similar magnitude to the forces measured in pure PBS (no LUB) between bare mica surfaces (Figure 8a). In this situation, therefore, we cannot separate the effect of the steric-entropic forces from the exponential electrostatic double-layer forces; the former are simply too weak to distinguish them from the latter. Also, since the coverage is now low, the Alexander-de Gennes model for brushes is not expected to be valid. Instead, the steric-entropic forces should be described by the equation for isolated surface-adsorbed molecules or “mushrooms”32 (which has a similar, roughly exponential, functional form to the Alexander-de Gennes equation). Thus, the F(D) curve measured for D-LUB was similar to the electrostatic repulsion between bare mica surfaces in pure PBS, except for a shift D0 of a few nanometers toward larger distances, suggesting that the thickness of a compacted D-LUB layer on each surface is ∼2 nm. After rinsing the digested protein-coated surfaces in pure PBS buffer and replacing one of them with a bare mica surface, hysteresis and adhesion were observed (Figure 8c and d). During approach, the force was repulsive and the range of the repulsion was about 40 nm, comparable to the value measured for two symmetrically coated surfaces (Figure 8a and b). This suggests that in the symmetrical geometry the protein layers were deeply interpenetrated or that the mica surface was not completely covered by the protein, resulting in a relatively rough surface layer. Additional forces appeared during retraction of the surfaces, increasing with the time the D-LUB-coated surface was kept in contact with the bare mica surface. These attractive forces are due to the “bridging” bonds formed when the surfaces are close enough to allow the molecules on the coated surface to form bonds with the other, initially uncoated, surface. “Bridging forces” are commonly observed between polymer-coated colloidal particles, and they have been observed before for LUB molecules under similar conditions.8 They require low coverage and weak to intermediate strength binding of the polymers to the surfaces, or else the time to form these bridges, that is, attain the equilibrium bridging configuration, is too long compared to typical experimental time scales.39,40 The maximum surface separation at which an attractive bridging force is detected gives an estimate of the fully extended “contour” length of the bridging molecule which, in these experiments, was ∼70 nm for D-LUB (Figure 8d), which may be compared to ∼200 nm for LUB.8 When the surfaces are separated beyond this distance, all bridges are broken and the surfaces “jump out” from this “adhesive contact” position. The bridging distance as well as the adhesive bridging force measured during retraction depend on the “waiting time” in contact, the maximum load before retracting the surfaces, and the previous history of the contacts (i.e., the number of previous approachseparation cycles at the same contact position). These observations are consistent with the dynamic, that is, nonequilibrium, nature of bridging forces, as described above.39,40 A more systematic (39) Leckband, D.; Israelachvili, J. Q. ReV. Biophys. 2001, 34 (2), 105-267. (40) Israelachvili, J. Q. ReV. Biophys. 2005, 38 (4), 331-337.
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Figure 8. Effect of chymotrypsin digestion. Normalized forces, F/R, as a function of the mica-mica distance, D, in the presence of a solution of D-LUB in PBS solution. (a) Forces measured during the first approach/retraction cycle (9/0), during (+) and after ([/]) shearing the surfaces at the same contact position. The solid line is the force measured between bare mica surfaces in pure PBS (no LUB). (b) Forces measured at another contact position (first cycle, 9/0; during shear, ×; and after shearing, [/]). (c) Force obtained during the first (9/0), second (b/O), and third (2/4) approach/retraction cycle at the same contact position after rinsing the surfaces of panels (a) and (b) in pure PBS buffer and replacing one of them with a bare mica surface. (d) Detail of panel (c) showing the small adhesive forces (F < 0) for distance D < 70 nm. The arrows indicate distances where the surfaces “jump-out” from adhesive contact due a mechanical instability of the forcemeasuring cantilever spring for dF/dD > K, where K is the spring stiffness.
investigation would be needed to determine the exact dependence of the changing molecular configurations, the adhesion, and the other force characteristics of this system. In situ digestion of the protein (SD-LUB, see Table 1), after adsorption of the native form on the mica surface, led to results similar to the ones presented for D-LUB. First, we adsorbed LUB on mica (Figure 9a) and then rinsed the surfaces in pure PBS (Figure 9b) or PBS containing 5 mg/100 mL Ca2+ (Figure 9c). The forces measured after adsorption of LUB (Figure 9a) were similar to the ones previously obtained8 and presented in Figure 6. The force curves did not change significantly after rinsing in pure PBS (Figure 9b) or in a PBS solution containing a 0.45 mM physiological concentration of Ca2+ (Figure 9c). The presence of Ca2+ slightly reduced the range and amplitude of the repulsion compared to LUB, but it did not produce any measurable adhesion or hysteresis in the force curves. When chymotrypsin was added to the PBS/Ca2+ solution, the force remained repulsive and reversible during an approach/ retraction cycle, but the range and amplitude of the repulsion were greatly reduced. The force was close to the one measured
after adsorbing predigested LUB (Figure 8a and b, solid curve). The digestion was completed within 1 h as indicated by the fact that we did not observe any difference in the forces measured 1, 3, and 5 h after injection of the chymotrypsin. The normal force curve measured during shear always showed a large shearinduced thickening (Figure 9d). D-LUB and SD-LUB were ineffective in lubricating the surfaces: their coefficients of friction were found to be in the range µ ) 1.1-1.9, whereas those for LUB never exceed µ ) 0.6.8 The adsorbed layers of D-LUB and SD-LUB were irreversibly rearranged during shear. D-LUB developed a few “ripples”, roughly parallel to each other and perpendicular to the shearing direction, corresponding to a local thickening of the layer. The normal force measured during shearing also occasionally showed a thickening (Figure 8a) of the layer. However, the presence of ripples did not significantly affect the normal forces after shearing, which were comparable to the ones measured before shearing (Figure 8a and b). The shear-induced rearrangement of the D-LUB layers did not affect the underlying solid mica surface. On the contrary, the mica surfaces were severely
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Figure 9. Effect of calcium ions and chymotrypsin digestion on a preadsorbed layer of LUB. Normalized forces, F/R, are shown as a function of the mica-mica distance, D. Meaning of the symbols: (9/0) first, (b/O) second, and (2/4) third approach/retraction cycles at the same position before shearing; (×), (+), and (f) normal forces during shearing; and ([/]) forces after shear. (a) Forces measured after adsorption from a 0.29 mg/mL solution of LUB in PBS. (b) Forces after rinsing in pure PBS buffer (no LUB). (c) Forces after adding 0.45 mM CaCl2 to the PBS buffer. (d) Forces after adding 10 units of chymotrypsin/1 mL to the solution. The first, second, and third approach/retraction cycles were done 1, 3, and 15 h, respectively, after the beginning of the digestion. The mica surfaces are damaged during shearing, and a wear track appears across the contact position. In all panels, the dotted curve represents the second cycle of panel (a) (native LUB). The solid curve in panel (d) is the same as that in Figure 8b (D-LUB).
damaged during shearing of the SD-LUB layers. Wear tracks appeared across the contact position even for moderate loads, without the evidence of a critical load for the wear to occur. The mica-mica separation at the contact position strongly increased during shearing (Figure 9d). After shearing, the normal forces (not shown) were strongly repulsive at separations of a few micrometers. This indicated that wear of the mica surfaces produced microscopic flakes that became mixed with the remains of the SD-LUB layers. For both D-LUB and SD-LUB, the sliding motion was smooth (no stick slip) at a speed of V ) 1 µm/s.
Discussion Structure of the LUB Aggregates. Native LUB contains 1404 AA residues divided into 12 exons (Figure 1), three of which (exons 2, 4, and 5) are subjected to alternative splicing.1,24 Exon 6 (the “mucin” domain) is heavily glycosylated by negatively charged NeuAc(R2,3) or polar -Gal(β1-3)-GalNAc- sugar chains, in a proportion of about 2:1. The sugar chains are O-linked to threonine residues in 76 threonine-rich AA repeats.41 Glycosylation occurs for almost all eligible residues and accounts for almost 50% of the protein molecular weight, giving the mucin domain a strong hydrophilic character and a net negative charge.25 The LUB molecule has an isoelectric point (41) Jay, G. D.; Britt, D. E.; Cha, C. J. J. Rheumatol. 2000, 27 (3), 594-600.
(IEP) of 7.8-8.1.42 Therefore, in the range of pH ) 7.2-7.6 found in the synovial fluid,27 the LUB molecule carries a slight net positive charge, which is predominantly located in the nonglycosylated end domains. These contain subdomains analogous to two globular proteins: somatomedin-B (SMB) at the N-terminal and homeopexin (HPX) at the C-terminal (Figure 1). The end domains also contain most of the hydrophobic residues which, by analogy with globular SMB and HPX, are expected to be hidden in the core of a globule with an outer shell of hydrophilic, charged, and polar residues. Because of its particular molecular structure, LUB binds on negatively charged and hydrophobic substrates preferentially in the form of “tails” or “loops”, that is, by adsorbing one or both extremities to the substrate.8 Our AFM measurements on mica and HOPG show that the proteins are easily displaced by the AFM tip during a scan, because the adsorption of the extremities is too weak to resist the shearing action of the AFM tip. When divalent Ni2+ ions replace the monovalent K+ ions on mica, localized sites with an excess positive charge are created which can now attract the negative mucin residue. As a result, both the mucin domain and the end domains (also containing negatively charged residues) adsorb on the surface. The adsorption is now (42) Jay, G. D. Joint lubrication: a physico-chemical study of a purified lubricating factor from bovine synovial fluid. Ph.D. Thesis, State University of New York, Stony Brook, NY, 1990.
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Figure 11. Suggested conformations of dimeric LUB and monomeric R-LUB when adsorbed nonspecifically to negatively charged surfaces such as mica in the form a loop. The height, L, of the loop and the surface coverage, Γ, are similar in both cases, which implies that the average distance between neighboring molecules is larger for LUB (a) than for R-LUB (a′).
Figure 10. Friction force, f, as a function of the normal force (load), F, or the pressure, P, between two mica surfaces bearing layers of LUB digested with chymotrypsin. The sliding speed was V ≈ 1 µm/s. Straight lines are the best linear fits to the data, giving the friction coefficient µ ) df/dF. Symbols (+) and (×) correspond to D-LUB adsorbed on mica. The data come from the same experiment as that in Figure 8a and b, with the same meaning of the symbols. Symbol (f) corresponds to SD-LUB (data from Figure 9d). Wear tracks progressively appear across the contact region during shearing starting from small loads, F ) 0.3-0.4 mN. Error bars are only shown for the last point of each measurement. The error bars on F are much smaller than the sizes of the data points.
much stronger, and AFM imaging becomes stable. LUB molecules adsorbed on Ni2+-mica have a filamentous appearance, which is due to the adsorption of the longest, most extended, and most negatively charged portion of the molecule (the mucin domain) flat on the surface. The molecules associate in double-stranded filaments where the strands are well-separated but twisted around each other (Figure 2A and B) and the extremities of the strands are never observed as free ends. The filaments show a large polydispersity with a peak (most frequent) length at l ) 130140 nm and a smaller peak at roughly 2l ≈ 255 nm (Figure 3a). All objects that do not show a double-stranded structure have a ring-shaped or globular appearance, which suggests that the extremities of the strands have a strong tendency to associate to each other, probably even two extremities of the same strand. After removing the intra- and intermolecular disulfide bonds between Cys, the R-LUB filaments showed a peak length, l, similar to that of LUB, but the secondary peak around 2l was much smaller or nonexistent and filaments with l > 250 nm became very rare (Figure 3b). Inside the R-LUB filaments, the strands were less twisted around each other and long singlestranded portions appeared along the filaments, in particular, the strand extremities (Figure 2D). These observations indicate that LUB molecules tend to aggregate via disulfide bonds. The aggregation occurs already in solution, as shown by the DLS results: the z-averaged hydrodynamic radius, Rh, for LUB was almost half the value for R-LUB (Figure 4), clearly indicating a net decrease of the average size of the scattering objects. Aggregation of LUB molecules by disulfide bonds has been recently reported,2 and it is common for mucins, where endto-end aggregation creates long linear chains.17 Most of the 22 Cys residues of the LUB molecules are indeed contained in exons 2 and 3 (16 Cys) at the N-terminal and in exons 7-12 (5 Cys)
at the C-terminal, while only one Cys residue is present in the mucin domain (Figure 1). It has been shown that protein constructs comprising only exons 1-5 of the LUB sequence form dimers in nonreducing conditions while constructs comprising exons 7-12 form intrachain disulfide bonds without aggregating.2 Therefore, disulfide bonds are likely to form inside and between the end domains of the LUB molecules, while disulfide bonds involving the mucin domain are rare. A tendency toward end-to-end association could explain the open double-stranded and loosely twisted structure observed for LUB. The peak length l ) 130-140 nm, found for both LUB and R-LUB (Figure 3), could be the total length of an LUB dimer folded on itself or the length of a R-LUB monomer. This idea is illustrated in Figure 11. The presence of a secondary peak around 2l ≈ 255 nm can be attributed to the formation of higherorder aggregates (trimers, tetramers, etc.), which become rare after reduction and alkylation. Consider R-LUB filaments, which are expected to be prevalently monomeric. The length of a filament measured by AFM is shorter than the contour length, lc, of a single strand, because the strands form kinks, bends, and (circular) loops in the filaments, which were only partially resolved in the AFM images. The larger is the measured length of a filament, the less is the portion of the curved contour that was not resolved. Therefore, the upper end of the length distribution of the R-LUB filaments is our best estimate of the contour length of a fully extended monomer: lc ) 230 ( 20 nm (Figure 3b). The small ring-shaped objects with diameter ≈ 25 nm observed in the AFM images (Figure 2) are most likely proteins or protein fragments looped on themselves one or more times. The value of lc determined from the AFM images agrees with the value 220 ( 50 nm previously reported.43 The AFM images also show that LUB molecules are extended and flexible: they randomly bend and loop on a length scale of a few tens of nanometers. If we consider the tangent vectors of two small portions of a filament, the statistical correlation between the directions of the vectors falls to zero as the (partial) contour length between the portions exceeds a few tens of nanometers. By definition, this implies that the persistence length of the filaments is lp < 10 nm. This value is high compared to that for nonglycosylated proteins (lp < 1 nm), but it is typical for mucinous proteins,17 where the sugar side-chains of the mucin domains repel each other via steric and electrostatic interactions, thereby (43) Swann, D. A.; Slayter, H. S.; Silver, F. H. J. Biol. Chem. 1981, 256 (11), 5921-5925.
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resisting the bending of the polypepetide chain.44 The LUB molecule appears much more flexible than previously observed from electron micrographs,43 where LUB molecules appeared as fully extended, slightly kinked rods with a persistence length, lp, comparable to or longer than lc which was probably due to the preparation method (platinum rotary shadow-casting on mica). Because of the small value of lp , lc, LUB is expected to form random coils in solution rather than behave like an elastic “worm-like chain”. Our DLS measurements show a z-averaged hydrodynamic radius of Rh ≈ 40 nm for R-LUB and almost double that, Rh ≈ 80 nm, for LUB. These values are large compared to the typical value of ∼30 nm for mucins with 1400 AA,44,45 suggesting that the LUB molecules were still partly aggregated after the reduction and alkylation treatment, probably because of an incomplete or unstable removal of the disulfide bonds. Effect of the Reduction and Alkylation of LUB on its Lubricating Performance. Reduction and alkylation are known to reduce the ability of LUB to specifically bind to the cartilage surface2,6 and its ability to lubricate cartilage against glass in macroscopic in Vitro experiments.6 The N- and C-terminals of the LUB molecule are analogous to proteins SMB and HPX, respectively, which mediate cell-cell and cell-extracellular matrix interactions.1 An intermolecular disulfide bond between the last Cys residue of exon 12 and the first Cys residue of exon 7 (Figure 1) creates a loop in the C-terminal, which is naturally subjected to a cleavage event, leaving a bifurcated structure.2,16 The release of the branch after reduction and alkylation strongly reduces the ability of LUB to bind to the cartilage.2 Therefore, the composition and structure of the end domains of the LUB molecule seem to play a critical role in the binding of the molecule to the cartilage, although the exact binding mechanism is still unknown. Binding to the substrate is not the sole requirement for LUB to act as an efficient boundary lubricant. LUB adsorbs on mica in both its native8 and reduced forms, with a similar surface coverage of Γ ≈ 5 mg/m2. For both LUB and R-LUB, the adsorption is driven by nonspecific electrostatic and van der Waals attractions of the end domains to the mica substrate, while loops and tails comprising the longest portion of the protein (the mucin domain) are exposed to the aqueous solution. When two such brush-like layers are brought into contact, they repel each other due to steric-entropic interactions, similar to the repulsion between two layers of end-grafted neutral polymers of the Alexander-de Gennes theory (Figure 6). The electrostatic repulsion plays a minor role here due to the high salinity and, therefore, short Debye length of the solution (∼1 nm) compared to the thickness (>65 nm) of two layers. Similar repulsive forces are obtained when LUB is adsorbed on a monolayer of hydrophobic alkanethiol,8 with the difference that the end domains are now attached to the substrate by hydrophobic interactions, while the hydrophilic mucin domain is again exposed to the solution. It is because of this ability to form brush-like layers on a variety of surfaces that LUB is able to act as an effective antiadhesive in the synovial cavity and on cartilage surfaces.12,13 However, despite the similar mechanisms of adsorption and normal forces, R-LUB and LUB adsorbed on mica show some quite different lubricating properties, that is, lateral or frictional forces, as well as resistance to degradation and wear. (44) Shogren, R. L.; Jamieson, A. M.; Blackwell, J.; Jentoft, N. Biopolymers 1986. (45) Mueller, H.; Butt, H. J.; Bamberg, E. Biophys. J. 1999, 76 (2), 10721079.
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Two LUB layers adsorbed on mica and sheared at a sliding speed of ∼1 µm/s show low friction coefficients of µ ) 0.020.04 for loads up to Fc ) 0.4 mN (pressures up to P ) 6 atm). The friction coefficient suddenly increases to 0.35-0.63 at higher loads, and it is accompanied by a visible rearrangement of the protein layers under shear in the form of a bump localized at the center of the contact. In comparison, under similar testing conditions, R-LUB shows a relatively high friction coefficient of µ ) 0.13-1.17 already at low loads (Figure 7). Shearing of the R-LUB layer usually does not produce any visible rearrangement of the protein layers. In only one case, we observed a shear-induced rearrangement in the form of a bump, and this corresponded to a sudden decrease of the friction and of the friction coefficient, which was not accompanied by an increased wear of the mica surface. This could be interpreted as a beneficial property of LUB molecules subjected to high stress: aggregating into clusters so as to improve their tribological efficiency. In any case, both LUB and R-LUB layers protected the underlying mica surface from wear for loads up to about 8 mN (P ≈ 500 atm).8 The differences in the tribology (lubrication and wearprotection) between LUB and R-LUB must originate from differences in the state of aggregation and surface configuration of the protein forms. It is well-established that while the normal (e.g., repulsive or adhesion) forces between two surfaces are related to the lateral (frictional or lubrication) forces, small differences in the former can lead to large changes in the latter.46 Thus, hints and correlations may be expected to be found in the differences in the normal forces of the two systems. First, R-LUB shows a single thickness of the adsorbed layer of L ≈ 75 nm, while LUB forms layers of thickness L ) 65 or 100 nm (Figure 6). This was attributed to the coexistence of two adsorbed protein conformations: one predominantly of tails and the other of loops. Therefore, the relatively short value of L ≈ 75 nm measured for R-LUB indicates that loops are predominant over tails. Second, the brush-like behavior of R-LUB layers appears to have a firmly adsorbed and incompressible layer of thickness 1/2D0 ) 4 nm that does not get easily squeezed out even under a very high compressive force. In contrast, the LUB brushes appear to follow the Alexander-de Gennes equation without any finite offset distance, implying that the chains behave as if they had no excluded volume. Third, the average distance between grafting sites, as determined by the fit to the Alexander-de Gennes model, is larger for R-LUB layers (s ) 18 nm) than for LUB layers (s ) 14 nm). A possible explanation for these observations is illustrated in Figure 11. In its native, preferentially dimeric form, LUB adsorbs as tails and loops by binding one or both globular domains to the mica. Not all of the positive charges of the globular domain can face the mica surface, because of the conformational restrictions imposed by the disulfide bonds. When these bonds are broken in R-LUB, the end domain becomes more deformable and can bind to mica with an increased number of positively charged AA residues. The increase in the binding strength of the R-LUB extremities renders the loop conformation energetically more favorable than the tail conformation. This effect has been already reported for brushes of symmetric ABA triblock copolymer when the ratio of the lengths of the adsorbing portion A and nonadsorbing portion B is increased.47 In our experiments, loop-rich brushes of LUB and R-LUB have a similar height, L, surface coverage, Γ (in mg/m2), and average grafting distance, (46) Israelachvili, J.; Maeda, N.; Rosenberg, K. J. J. Mater. Res. 2005, 20, 1952-1972. (47) Dai, L.; Toprakcioglu, C. Macromolecules 1992, 25, 6000-6006.
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s. The latter can be considered as the average distance between (single-stranded) mucin domains inside the brush. Therefore, dimeric LUB and monomeric R-LUB loops tend to form brushes with a similar Volume density of mucin strands. This also implies that the extremities of the R-LUB molecules adsorb with a surface density that is about twice that of (dimeric) LUB extremities. If we suppose that the binding energy of a single R-LUB extremity is higher than the binding energy of a dimeric LUB extremity, we can also explain the relative stability during shearing of the R-LUB layers compared to LUB layers. In fact, it is more difficult to pull out both extremities of a R-LUB molecule by shearing, so that shear-induced rearrangements of the adsorbed layers are less likely. On the other hand, it is now more difficult to detach even one end of a R-LUB loop to create a tail. The protein layer is thus less mobile and more gel- or solid-like, which at the sliding speed considered could give rise to a higher friction force. A similar behavior has been observed for LUB adsorbed on hydrophobized mica.8 Also, in this case, the protein binds to the surface with the end domains which contain most of the hydrophobic residues. In this case, the predominance of loops, high resistance to shear-induced rearrangements, and high friction are due to the greater strength of the hydrophobic interactions compared to electrostatic interactions. Effect of Chymotrypsin Digestion. In joints affected by inflammatory arthritis, LUB is expressed at a lower concentration and exposed to increased proteolytic activity.48 The question arises whether this reduces the ability of LUB to act as an antiadhesive and lubricating agent for the cartilage surface. When inflammatory arthritis is induced in rat joints, LUB is degraded and the friction of the joint, as measured ex ViVo, increases.48 Enzymatic digestion is also known to reduce the efficiency of LUB as a boundary lubricant of latex-glass contacts in Vitro.28 Chymotrypsin cleaves peptide bonds at the carboxyl side of aromatic AA, which are rare in the mucin domain (1 tyrosine and 3 phenylalanine) and located close to the extremity of the domain.24 Therefore, chymotrypsin digestion is particularly interesting for our study, as it is expected to separate the lubricating element, namely, the mucin domain, from the rest of the protein. However, AFM imaging of D-LUB adsorbed on Ni2+-mica did not show any of the filamentous structures observed for LUB and R-LUB and attributed to the binding of the negatively charged mucin domain. This, together with the impossibility of isolating the mucin domain from the D-LUB solution by electrophoresis and ELISA tests, indicates that chymotrypsin had an unexpected ability to digest nonaromatic AA in the mucin domain, leaving only short fragments of the protein in the D-LUB solution. Most likely, the LUB fragments coadsorb on the mica surface together with other species present in the D-LUB solution (chymotrypsin, aprotinin, and leupeptin; see Table 1). The adsorption produces a layer that is much less dense than the ones considered so far for undigested LUB, with a surface coverage below the detection limit of Γ ∼ 1 mg/m2. Although the normal forces between the mica coated with D-LUB were still purely repulsive, the mechanism generating this repulsion is mainly due to the electrostatic double-layer force between the charged surfaces. The low adsorption to mica of species carrying positive charges, in particular, fragments from the end domain of the LUB molecule, produced attractive (adhesive) and hysteretic bridging forces with a range of ∼70 nm (Figure 8b and c), which is significantly shorter than the range of ∼200 nm measured under similar conditions with LUB.8 The attractive range of 70 (48) Elsaid, K. A.; Jay, G. D.; Chichester, C. O. Arthritis Rheum. 2007, 56 (1), 108-116.
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nm gives a rough estimate of the extended length of the molecules causing the bridging. The coefficient of friction with adsorbed D-LUB as the “boundary lubricant” is µ ) 1.1-1.4 at all loads, almost 2 orders of magnitude higher than the average coefficient of friction measured at low loads for LUB on mica. During shearing, the mica surface progressively wears with time, starting from small loads F ) 0.3-0.4 mN (pressures P ) 6-20 atm). This is unlike any other previous measurement with undigested LUB, where only the protein layer is seen to rearrange during shearing, while the underlying surface remains unaffected up to a load of F ) 8 mN (P ) 640 atm). Therefore, degradation of the mucin domain is sufficient to completely remove the lubricating ability of the protein layer. When LUB was preadsorbed on the mica surfaces and then digested with chymotrypsin, the normal forces (Figure 9d) were similar to the ones observed for D-LUB and also the friction behavior was similar, showing wear tracks and an even larger value of the friction coefficient, µ ) 1.9 (Figure 10). The similarities between D-LUB and SD-LUB show that the friction behavior does not depend on the presence of protease inhibitors or Ca2+ but it is due to the adsorption of digested LUB and probably chymotrypsin on the mica surface. Most important, SD-LUB shows a behavior similar to D-LUB already 1 h after starting the digestion, which is a delay too short for the enzyme to completely digest the (normally undigested) mucin domain. Most likely, the enzyme rapidly digests the end domains and separates them from the mucin domain, releasing the nonadsorbing mucin domain in the solution. The detachment of the mucin domain from the surface is sufficient to reduce the lubrication to a level comparable to that measured for D-LUB, regardless of the extent of digestion. The anchoring of the mucin domain on the surface is thus critical for the LUB molecule to express its full lubricating capability (results observed for HA in solution).23 Effect of Divalent Ions in the Solution. The concentration of ionized calcium, Ca2+, in healthy synovial fluid is about 0.05 mg/mL, which is close to the solubility limit.49 At higher concentrations, calcium precipitates on the cartilage surface as pyrophosphate crystals, triggering an inflammatory disease known as pseudogout. Ca2+ is known to influence the adsorption of negatively charged polyelectrolytes and biopolymers on negatively charged surfaces such as mica as well as to affect the forces between the adsorbed layers.50 It is thus important to know how physiological concentrations of Ca2+ affect the results presented so far. We have found that a concentration of 0.05 mg/mL Ca2+ in PBS does not produce any major alteration of the normal forces between layers of LUB adsorbed on mica (Figure 8c). The only difference is a slight decrease of the layer thickness from the value measured in pure PBS (Figure 8b), which can be ascribed to the increased screening (decreased Debye length) of the electrostatic double-layer repulsion between the charged residues and surfaces.
Conclusions • Lubricin has an extended contour length of about 220 nm and aggregates end-to-end preferentially in the form of dimers due to disulfide bonds. The N- and C-terminals of the protein bind to negatively charged surfaces, while the central mucin domain does not. (49) Bennett, R. M.; Lehr, J. R.; McCarty, D. J. J. Clin. InVest. 1975, 56, 1571-1579. (50) Claesson, P. M.; Poptoshev, E.; Blomberg, E.; Dedinaite, A. AdV. Colloid Interface Sci. 2005, 114, 173-187. (51) Gao, J.; Luedtke, W. D.; Gourdon, D.; Ruths, M.; Israelachvili, J.; Landman, U. J. Chem. Phys. 2004, 108, 3410-3425.
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• The necessary condition for lubricin to be a good wearprotector is that the mucin domain must be end-tethered to the surface. In this condition, lubricin protects the surface at pressures exceeding 500 atm at sliding speeds of 1 µm/s. Detachment of the mucin domain from the surface due to proteolytic digestion results in a dramatic increase of both friction and wear. • End-anchoring of the mucin domain is not sufficient to guarantee low friction. The adsorption must be optimal: strong enough to promote the formation of dense brush-like surface layers resistant to shear but without imposing a strong conformational restriction to the adsorbed proteins. The breaking of intraprotein disulfide bonds during shear opens the terminal
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globular domains and stregthens the binding of the terminals to the surfaces. The enhanced adhesion reduces the mobility (fluidity) of the molecules which in turn can enhance the friction coefficient by more than 1 order of magnitude. Acknowledgment. This work was funded by the McCutchen Foundation and by NIH Grant AR050180. We are grateful to Dr. Charles W. McCutchen for his comments and suggestions. We thank Prof. Helen Hansma, UCSB Physics Department, for letting us use her AFM setup and Prof. G. Stucky, UCSB Department of Chemistry and Biochemistry, for the use of the DLS setup. LA702383N
