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Stretching Single Molecules of Connective Tissue Glycans to Characterize Their Shape-Maintaining Elasticity Richard G. Haverkamp,*,† Martin A. K. Williams,‡ and John E. Scott§ Institute of Technology and Engineering, Massey University, Palmerston North 5331, New Zealand, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand, and Injury Repair and Rehabilitation Group, School of Medicine, The University of Manchester, Manchester M13 9PT, United Kingdom Received January 18, 2005 Revised Manuscript Received February 27, 2005
Introduction Structural polymers help maintain body shape against mechanical stress. Collagen, a fibrous protein, is the defining structural polymer in animals, supporting life in the fast lane. Cellulose is characteristic of the static lifestyles of plants and chitin overlaps both, being present in, e.g. fungi and crustaceans. Recent extensive electron histochemical evidence1 showed that the animal anionic glycosaminoglycans (AGAGs) dermochondan sulfate (DS) and keratan sulfate play a key role in organising connective tissue extracellular matrixes (ECMs), which maintain bodily shape.2 [Dermochondan is the recommended term indicating the copolymeric nature of DS, replacing dermatan Arch. Biochem. Biophys. 1997, 344, 245; Eur. J. Biochem. 1997, 247, 734.] ECM collagen fibrils are bridged and tied in ordered arrays by the AGAG chains of proteoglycans such as decoran. These bridges occur at regular (∼65 nm) intervals along collagen fibrils, anchored noncovalently via proteoglycan proteins to specific binding sites on fibril surfaces. A pair of segments on neighboring collagen fibrils linked by AGAG chains was termed a shape module since it determines interfibrillar separation as well as mutual orientation, thereby defining tissue shape.1 ECMs are thus held together by carbohydrate strings.2,3 Shape modules must deform reversibly to preserve the shape of the organism against stress. It was proposed recently that interfibrillar AGAG bridges extend elastically2,3 by utilizing [a] the interconversion of DS L-iduronate (IdoUA) conformers under tensile stress, and [b] reversible slippage within AGAG aggregates of shape module bridges. Electron microscopy, NMR, and molecular modeling indicate that the tapelike 2-fold helical AGAG chains are probably layered on top of each other, head-to-tail, which allows hydrophobic and H-bonds to form between neighboring AGAGs. Thus, aggregated AGAGs can be pulled apart in a slippage process, during which the low energy hydrophobic and H-bonds are broken. Slippage occurs along the AGAG bridge axis, in the * Corresponding Author. Tel: 6463504167. E-mail: r.haverkamp@ massey.ac.nz. † Institute of Technology and Engineering. ‡ Institute of Fundamental Sciences. § The University of Manchester.
direction of applied tensile stress. On releasing the stress, slippage reverses as the bonds originally present are reformed into the optimal (lowest energy) structure. 13C rheo-NMR evidence6 demonstrated reversible, specific disaggregation of tertiary structures of a prototypical AGAG, hyaluronan (HA), under shear stress, compatible with this mechanism. Mechanism [a] was based on NMR of AGAGs in solution, molecular models2 and calculations showing that the relevant IdoUA conformations had similar energies.5 We now consider mechanisms of this type for which we provide the first direct evidence. Elasticity in C-C bonds and glycosidic linkages of AGAGs has been examined computationally.7 In another context, glycan elasticities were measured by stretching single molecules of pectin, dextran, etc. using AFM.4,8 Two sorts of glycans were identified: [i] in which all glycosidic bonds are equatorial and [ii] where some are axial. Tensile forces are conveyed along the chain in [i] (e.g., cellulose) without reorienting glycosidic bonds, which are aligned with the force along the molecular chains. In [ii] axial glycosidic bonds introduce kinks into the chain that straighten into the chain axis under tension, causing conformational changes in the associated sugar units. Total glycan length thereby increases reversibly, providing additional elastic extension compared to polymers of type [i]. Using these guidelines,4,8 it was predicted that ECM AGAGs HA, chondroitan sulfate (CS), and keratan sulfate would be less elastically extensible since their glycosidic bonds are equatorial, whereas DS would exhibit greater elastic extensibility, since the preferred IdoUA 1C4 and 2S0 forms contain axial glycosidic bonds3. These forms are shorter along the chain than C1 chairs, so that the reversible change from compact to longer conformers provides elastic extension.2 This hypothesis was tested directly by singlemolecule stretching. Experimental Methods DS was prepared from defatted and dried pigskin by digesting with papain, precipitating with cetylpyridium chloride at 0.3 M NaCl, reprecipitating with ethanol from aqueous solution, and drying with ethanol and ether. Chondroitan-4-sulfate and hyaluronan were similarly prepared from bovine nasal septa and human mesothelioma fluid, respectively. Their 1H and 13C NMR12,13 spectra were reported. Molecular masses were DS ∼20 kDa, CS, ∼20 kDa and HA, ∼1 MDa, corresponding to lengths of 40, 40, and 2500 nm. Methylesterified HA samples were prepared as described previously.13 These were used to assess the significance of 3° structures, present in native HA but not in solutions of the methyl-esters.13 Methylcellulose of molecular mass ∼86 kDa (Aldrich) was used without further purification. It is a uniformly equatorially linked neutral polymer. Chitosan was from Seikagaku, Tokyo, Japan a cationic analogue of cellulose. Pectinate from citrus peel, anydrogalacturonate content ∼90%, and methylesterification
10.1021/bm0500392 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/08/2005
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Figure 2. Force/extension curves of (a) bovine nasal septal CS and (b) pigskin DS. The pigskin DS had >90% uronate as IdoUA. The CS slope (a) increases smoothly while in DS (b) an inflection is seen at ∼200 pN. We attribute the DS inflection to IdoUA 1C f C1, since no inflection occurs in CS (which is identical with DS except for the D-glucuronate f L-iduronate epimerisation) the change is not in galactosamine; and no inflection occurs in HA (Figure 1), implying that D-glucuronate does not stretch conformationally. Figure 1. Force/extension curves of (a) polyanionic (unmodified) HA; (b) 25% methylated HA (c) neutral methyl cellulose, (d) polycationic chitosan. Force/extension curves are without clicks shown by glycans with axial glycosidic bonds (Figures 2b and 3). All have solely equatorial glycosidic links. Lengths of HA stretched segments center around 60-100 nm, equivalent to 25-40 kD. This is much lower than estimates based on bulk solution properties. HA of widely differing mol. mass (120-2000 kD) showed similar short force/extension lengths, emphasising that only a portion of the polymer is being sampled so that the data obtained from a stretch may not represent the whole molecule.
of ∼35% was a gift from Unilever Research, Bedford, U.K. Sodium alginate from Hopkins and Williams, London, was purified by precipitation from 2.0% w/v aqueous solution by adding NaCl to 0.3 M. The precipitate was redissolved in water and precipitated by dropwise addition of ethanol to 66% v/v, washed with pure ethanol then ether, and dried in vacuo. Stock solutions (1%) were made in deionized water with gentle shaking for 24 h, with the exception of chitosan which was prepared in 100 mM acetate buffer at pH 3. For the AFM experiments, solutions were diluted to give final concentrations of 0.001% w/v in 0.1 M Na acetate solution with pH ∼ 7. The AFM measurements were performed in solution from the outset. We stretched glycans between gold plated mica and a 10 µm radius gold tipped cantilever (Ultrasharp CSG11/Au, NT MDT Co, Moscow, Russia), using an Asylum Research MFP 3D AFM Instrument. The AFM tip was lowered into a 100 µL solution on the support, contacting the gold surface and retracting at a rate of 500 nm/s every few seconds, recording the force vs extension relationship of the stretched single molecule. This was repeatable up to several hundred times. This simple technique allows conditions to be varied widely, approaching those in vivo. Typically 30% of tip-surface contacts resulted in no pulloff features, 30% yielded a relatively low and constant force corresponding to peeling molecules from the surface, and 30% gave complex multiple features, leaving some 10% deemed to be single-molecule stretching events. If the data from several experiments could be normalized onto the same curve by dividing the distances by the contour lengths, this was taken as evidence for single-molecule stretching events. Each curve on all of the plots (Figures 1-3) could be reproduced multiple times; however, for
Figure 3. Force/extension curves of (a) citrus pectinate and (b) alginate. The alginate was 0.3 M NaCl-insoluble, of unknown Lguluronate/D-mannuronate ratio. Pectinate (containing axial glycosidic bonds) exhibits two plateau regions in the force/extension curve when stretched.16 Inflections are seen at ∼200 pN, similar to that in DS (Figure 2b). The measured pectinate elongation was ∼8% and ∼9% for the two conformational changes, and ∼11% for alginate.
clarity, we have plotted single data sets in Figure 1 and two or three data sets in Figures 2 and 3. Tensile force was measured by cantilever deflection as a function of separation distance. Force constants were measured for each cantilever.9 Data were normalized by dividing measured lengths (l) by the straightened unstretched length, (lc), known as the contour length. Experiments were carried out at 293 K. Force/extension curves were fitted to the extensible wormlike chain model10 F)
[(
)
kT 1 l F 1- + lp 4 lc Φ
-2
+
]
l F 1 - lc Φ 4
where F is the force, l is the distance, T is the temperature, k is the Boltzmann constant, lp is the persistence length, lc is the contour length, and Φ is the specific stiffness, using a nonlinear regression method based on the LevenburgMarquardt algorithm. Results and Discussion Single polymer molecules adsorbed to a support were induced to stick simultaneously to an AFM cantilever probe which was moved away from the support, while measuring the tension and the extension of the molecule.4,8 Two features of force/extension curves are relevant: (1) the specific stiffness, indicating the amount of “give” in the stretched polymer and (2) clicks, revealing the presence of units which undergo discrete elastic extensions above a critical loading.
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The term “click” is synonymous with force-induced conformational transition, describing the inflection in the stretching curves during which little or no increase in force is required to produce a significant elongation of the polymer, producing plateaus parallel to the x axis. The glycans HA, methyl cellulose, and chitosan (Figure 1a,c,d) have solely equatorial glycosidic bonds. HA, which is ubiquitous in animal ECMs, and present in some bacterial coats, is β-linked (1e,4e; 1e,3e) with many features of cellulose, being based on cellobiose units (as is also chitosan). The backbones of these glycans should stretch mainly within chemical bonds and no β T R clicks are expected.8 Our data confirm this (Figure 1a,c,d) and demonstrate that our methodology is applicable to anionic, neutral, and cationic polymers. HA forms 3° structures stabilized by intermolecular H-bonds from acetamido NH to carboxylate. To assess the importance of these, we studied methyl-esterifed HA (Figure 1b) in which such interactions are blocked.13 The stretching behavior of methyl esterified HA and unmodified HA were identical implying that neither was influenced by complex formation but was based on single molecules. The two animal AGAGs CS and DS were compared. Clicks were absent from the force/extension curves of 1e,3e; 1e,4e CS (Figure 2a) which, as predicted, cannot undergo the β T R conformational change (and this should apply to the 1e,3e; 1e,4e keratan sulfate). In contrast, an inflection in the force/extension curve of DS (Figure 2b) was as predicted since the alternative IdoUA 1C, C1, and 2So conformers have similar energies but different lengths in the polymer backbone. The reversible elongation of the R1C to the βC1 form thus endows the chain with increased elastic extensibility3 compared with CS. The maximum observed contour length of DS was 46 nm (with associated mol. mass ∼23 kD) compared with previous estimates (15-20 kD14), suggesting that ∼100% of the polymer chain was stretched in this instance. Since the elongation per uronate unit on clicking is ∼15% (∼0.08 nm/ sugar residue)2,15 and the DS uronate comprises ∼50% of the molecular length, a plateau of ∼7.5% stretch was expected since 15% × 50% ) 7.5%. The plateau length (3.5 nm) represents ∼7% stretch, in accord with this calculation. The energy of the conformational change for IdoUA 1C f C1 in DS from the area under the inflection was estimated at 4 kJ mol-1 in agreement with Casu.5 These calculations further validate the conclusions. The suggestion that AGAG aggregates hold ECM collagen fibrils together elastically during strains of up to 10%2 is therefore directly supported by our data. The Hookean polymers CS, keratan sulfate (in cartilages and corneas), and HA (in liquid or semi-liquid tissues) are characteristic of connective tissues in which relative inextensibility of polymers is permissible and/or desirable. On the other hand DS is a defining glycan in tendon, skin, sclera, and blood vessel walls etc. where subtle and varied elastic responses to stress are required in rapidly varying circumstances.2 Analogous clicks in R-linked pectinate16 (Figure 3a, a major structural glycan of land plants) and in carageenan (a structural polymer in red seaweeds (Rhodophyceae)) have been observed.17 A similar conformational change, probably
Notes
in L-guluronate, was seen in alginate (a structural glycan in brown seaweeds (Phaeophyceae)) (Figure 3b). Glycans containing predominantly D-mannuronate or L-guluronate, which are the main components of alginate, provided confirmatory data, with a click in polyguluronate but not in polymannuronate. Although a signaling role16 has been speculatively attributed to conformational changes in tensioned pectates, our work on animal AGAGs suggests that clicks in the structural polymers of land and marine plants might have similar mechanical functions. Conclusions DS-containing aggregates can stretch elastically by e10% using force-induced conformational transitions not available to the almost-identical CS. For the first time, this gives a rationale for their tissue-specific distribution. In general, our work suggests that the nanomechanics of force-induced conformational transitions of pyranose rings play a role in controlling macroscopic tissue properties. Such clicks allow greater strain with less stress at low-to-moderate tensile stress, manifest at similar tensile stresses (∼200 pN) in the three clicking glycans (DS, alginate and pectinate) we examined. We see this as a hitherto unrecognized general strategy in biology. Acknowledgment. Supported by The MacDiarmid Institute for Advanced Materials and Nanotechnology, NZ and the Injury Repair and Regenerative Medicine Group, School of Medicine, University of Manchester, U.K. We thank Doug Hopcroft, Hort Research, NZ, for preparing the gold coated mica. References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
Scott, J. E.; Thomlinson A. M. J. Anat. 1998,192, 391-405. Scott, J. E. J. Physiol. 2003, 553, 335-343. Scott, J. E. Physiol. News 2004, 56, 22-24. Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 1295-1297. Casu, B.; Petitou, M.; Provasoli, M.; Sinay, P. Trends Biochem. Sci. 1998, 13, 221-225. Fischer, E.; Callaghan, P.; Heatley, F.; Scott J. E. J. Mol. Struct. 2002, 602-603, 303-311. Redaelli, A. Vesentini, S.; Soncini, M.; Vena, P.; Mantero, S.; Montevecchi F. M. J. Biomech. 2003, 36, 1555-1569. Marszalek, P. E.; Li, H., Oberhauser; A. F., Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4278-4283. Hutter, J. L.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64, 1868-1873. Wang, M. D.; Yin, H.; Landick, R.; Gelles, J.; Block, S. M. Biophys. J. 1997, 72, 1335-1346. Fujii, T.; Sun, Y. L.; An, K. N.; Luo Z. P. J. Biomech. 2002, 35, 527-531. Scott, J. E., Heatley, F.; Wood, B. Biochemistry 1995 34, 1546715474. Scott, J. E.; Heatley, F. Pro. Natl. Acad. Sci. U.S.A. 1999, 96, 48504855. Scott, J. E. J. Anat. 1992, 180, 155-164. Marszalek, P. E.; Oberhauser, A. F.; Li, H., Fernandez, J. M. Biophys. J. 2003, 85, 2696-2704. Marszalek, P. E. Li, H.; Oberhauser, A. F.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7894-7898. Xu, Q. B.; Zhang, W.; Zhang, X. Macromolecules 2002, 35, 871-876.
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