Proteoglycan Mechanics Studied by Single-molecule Force Spectroscopy of Allotypic Cell Adhesion Glycans
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m507878200
ISSN1083-351X
AutoresSergi Garcia-Manyes, Iwona Bucior, Robert Ros, Dario Anselmetti, Fausto Sanz, Max M. Burger, Xavier Fernàndez‐Busquets,
Tópico(s)Silk-based biomaterials and applications
ResumoEarly Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history. Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history. Specific carbohydrate-carbohydrate interactions are rarely reported in biologically relevant situations such as cell recognition (1Bucior I. Burger M.M. Curr. Opin. Struct. Biol. 2004; 14: 631-637Crossref PubMed Scopus (114) Google Scholar). However, carbohydrate structures have immense structural diversity (2Dietrich C.P. Nader H.B. Straus A.H. Biochem. Biophys. Res. Commun. 1983; 111: 865-871Crossref PubMed Scopus (94) Google Scholar), a ubiquitous distribution in vertebrate and invertebrate tissues (3Cássaro C.M. Dietrich C.P. J. Biol. Chem. 1977; 252: 2254-2261Abstract Full Text PDF PubMed Google Scholar), and are associated with the cell surface (4Hook M. Kjellén L. Johansson S. Annu. Rev. Biochem. 1984; 53: 847-869Crossref PubMed Scopus (460) Google Scholar, 5Roseman S. J. Biol. Chem. 2001; 276: 41527-41542Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), as required of cell recognition molecules. Carbohydrate-carbohydrate interactions are characterized by relatively weak forces that, when multimerized, can be easily potentiated by orders of magnitude, representing a highly versatile form of cell adhesion given the extraordinary plasticity of their structures (6Spillmann D. Burger M.M. Carbohydrates in Chemistry and Biology: A Comprehensive Handbook. Wiley-VCH Verlag GmbH, Weinheim, Germany2000: 1061-1091Crossref Scopus (2) Google Scholar). Among the few known examples of carbohydrate self-recognition proposed to be specific in biological processes are the multivalent binding of Lewisx epitopes involved in the first steps of embryogenesis (7Eggens I. Fenderson B. Toyokuni T. Dean B. Stroud M. Hakomori S. J. Biol. Chem. 1989; 264: 9476-9484Abstract Full Text PDF PubMed Google Scholar, 8Pincet F. Le Bouar T. Zhang Y. Esnault J. Mallet J.M. Perez E. Sinaÿ P. Biophys. J. 2001; 80: 1354-1358Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), glycolipid-glycolipid interactions controlling cell adhesion, spreading, and motility (9Kojima N. Hakomori S. J. Biol. Chem. 1991; 266: 17552-17558Abstract Full Text PDF PubMed Google Scholar, 10Dicko A. Heng Y.M. Boggs J.M. Biochim. Biophys. Acta. 2003; 1613: 87-100Crossref PubMed Scopus (12) Google Scholar, 11Wang X. Sun P. Al Qamari A. Tai T. Kawashima I. Paller A.S. J. Biol. Chem. 2001; 276: 8436-8444Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and self-interactions of the glycan portion of sponge proteoglycans leading to species-specific cell adhesion (12Bucior I. Scheuring S. Engel A. Burger M.M. J. Cell Biol. 2004; 165: 529-537Crossref PubMed Scopus (112) Google Scholar, 13Bucior I. Burger M.M. Glycoconj. J. 2004; 21: 111-123Crossref PubMed Scopus (66) Google Scholar). Sponge cells associate in a species-specific process through multivalent interactions of carbohydrate structures on a type of extracellular proteoglycan termed aggregation factor (14Humphreys S. Humphreys T. Sano J. J. Supramol. Struct. 1977; 7: 339-351Crossref PubMed Scopus (33) Google Scholar, 15Fernàndez-Busquets X. Burger M.M. Cell Mol. Life Sci. 2003; 60: 88-112Crossref PubMed Scopus (57) Google Scholar, 16Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar). Based on their molecular structure, aggregation factors have been related to hyalectans (15Fernàndez-Busquets X. Burger M.M. Cell Mol. Life Sci. 2003; 60: 88-112Crossref PubMed Scopus (57) Google Scholar): large, extracellular aggregating modular proteoglycans. However, unlike hyalectans, aggregation factors do not possess common glycosaminoglycans; instead, they have complex and repetitive acidic carbohydrate motives different from those found in classical proteoglycans and mucins (17Misevic G.N. Guerardel Y. Sumanovski L.T. Slomianny M.C. Demarty M. Ripoll C. Karamanos Y. Maes E. Popescu O. Strecker G. J. Biol. Chem. 2004; 279: 15579-15590Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), which include novel acid-resistant and acid-labile carbohydrate domains, large and branched pyruvylated oligosaccharides (18Guerardel Y. Czeszak X. Sumanovski L.T. Karamanos Y. Popescu O. Strecker G. Misevic G.N. J. Biol. Chem. 2004; 279: 15591-15603Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and other previously unknown structures (16Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar, 19Misevic G.N. Burger M.M. J. Biol. Chem. 1990; 265: 20577-20584Abstract Full Text PDF PubMed Google Scholar, 20Spillmann D. Thomas-Oates J.E. van Kuik J.A. Vliegenthart J.F. Misevic G. Burger M.M. Finne J. J. Biol. Chem. 1995; 270: 5089-5097Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 21Spillmann D. Hård K. Thomas-Oates J. Vliegenthart J.F. Misevic G. Burger M.M. Finne J. J. Biol. Chem. 1993; 268: 13378-13387Abstract Full Text PDF PubMed Google Scholar). In the marine sponge Microciona prolifera the proteoglycan molecule, Microciona aggregation factor (MAF 3The abbreviations used are: MAF, Microciona aggregation factor; AFM, atomic force microscope; CMF, calcium- and magnesium-free artificial seawater; CSB, chondroitin sulfate B; g6, 6-kDa glycan; g200, 200-kDa glycan; nN, nanonewton; pN, piconewton; SPR, surface plasmon resonance. ; Mr = 2 × 107; Fig. 1A), binds cell membrane receptors via Ca2+-independent interactions of small 6-kDa glycans (g6) on a 400-kDa protein (MAFp4) that corresponds to one MAF "arm" (Fig. 1B). Larger 200-kDa glycans (g200), bound to each of the ∼20 units of the MAFp3 protein that forms the ring of MAF, self-interact through calcium-dependent associations (Fig. 1C). This model has been demonstrated using cell-free techniques that include bead aggregation experiments (12Bucior I. Scheuring S. Engel A. Burger M.M. J. Cell Biol. 2004; 165: 529-537Crossref PubMed Scopus (112) Google Scholar, 16Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar, 22Jarchow J. Burger M.M. Cell Adhes. Commun. 1998; 6: 405-414Crossref PubMed Scopus (10) Google Scholar), membrane blot assays (23Popescu O. Misevic G.N. Nature. 1997; 386: 231-232Crossref PubMed Scopus (29) Google Scholar, 24Jarchow J. Fritz J. Anselmetti D. Calabro A. Hascall V.C. Gerosa D. Burger M.M. Fernàndez-Busquets X. J. Struct. Biol. 2000; 132: 95-105Crossref PubMed Scopus (43) Google Scholar), and force spectroscopy studies (12Bucior I. Scheuring S. Engel A. Burger M.M. J. Cell Biol. 2004; 165: 529-537Crossref PubMed Scopus (112) Google Scholar, 25Dammer U. Popescu O. Wagner P. Anselmetti D. Güntherodt H.J. Misevic G.N. Science. 1995; 267: 1173-1175Crossref PubMed Scopus (377) Google Scholar). Recent data have shown that the binding forces recorded between g200 from the same species were significantly stronger than those observed between g200 from different species (12Bucior I. Scheuring S. Engel A. Burger M.M. J. Cell Biol. 2004; 165: 529-537Crossref PubMed Scopus (112) Google Scholar, 13Bucior I. Burger M.M. Glycoconj. J. 2004; 21: 111-123Crossref PubMed Scopus (66) Google Scholar), indicating that the measurement of adhesive forces mediated by surface glycans may contribute to the study of molecular systems having determinant implications in cellular recognition. Single-molecule force spectroscopy approaches have successfully dealt with other investigations related to cell adhesion molecules in general (26Benoit M. Gabriel D. Gerisch G. Gaub H.E. Nat. Cell Biol. 2000; 2: 313-317Crossref PubMed Scopus (484) Google Scholar, 27Oberhauser A.F. Badilla-Fernandez C. Carrion-Vazquez M. Fernandez J.M. J. Mol. Biol. 2002; 319: 433-447Crossref PubMed Scopus (328) Google Scholar, 28Oberhauser A.F. Marszalek P.E. Erickson H.P. Fernandez J.M. Nature. 1998; 393: 181-185Crossref PubMed Scopus (748) Google Scholar, 29Zhang X. Wojcikiewicz E. Moy V.T. Biophys. J. 2002; 83: 2270-2279Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and to polysaccharide interactions in particular (30Abu-Lail N.I. Camesano T.A. J. Microsc. 2003; 212: 217-238Crossref PubMed Scopus (127) Google Scholar, 31Tromas C. Rojo J. de la Fuente J.M. Barrientos A.G. García R. Penadés S. Angew. Chem. Int. Ed. Engl. 2001; 40: 3052-3055Crossref PubMed Scopus (115) Google Scholar, 32Seog J. Dean D. Plaas A.H.K. Wong-Palms S. Grodzinsky A.J. Ortiz C. Macromolecules. 2002; 35: 5601-5615Crossref Scopus (96) Google Scholar, 33De La Fuente J.M. Penadés S. Glycoconj. J. 2004; 21: 149-163Crossref PubMed Scopus (113) Google Scholar). MAF has been found to be polymorphic in both its protein and carbohydrate moieties, and different individuals within the species possess allotypic forms of the molecule (34Fernàndez-Busquets X. Gerosa D. Hess D. Burger M.M. J. Biol. Chem. 1998; 273: 29545-29553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This raises the question of whether the species-specific carbohydrates could also have individual-specific structures and interactions, particularly in view of the fact that allotype rejection could be shown in this lowest extant Metazoan phylum (35Fernàndez-Busquets X. Burger M.M. J. Biol. Chem. 1997; 272: 27839-27847Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 36Müller W.E.G. Müller I.M. Integr. Comp. Biol. 2003; 43: 281-292Crossref PubMed Scopus (84) Google Scholar). To explore that issue, we have performed force spectroscopy and surface plasmon resonance (SPR) studies of the self-interactions of MAF and MAF subunits purified from different individual sponges. Dissecting MAF into its active adhesive components has allowed us to track the individual self-binding units down to the circular core of the sunburst-like molecule and further down to the g200 glycan itself. The identification of different allelic-like g200 forms suggests a role for carbohydrate-carbohydrate interactions in sponge allogeneic recognition. The results obtained reveal surprisingly high forces and selectivity for this most ancient cell adhesion system and open new perspectives on proteoglycan function-structure relationships. Preparation of Samples and Biochemical Analyses—Specimens of the marine sponge M. prolifera were collected by the Marine Resources Department at the Marine Biological Laboratory in Woods Hole. Isolation of cell surface proteoglycans and purification of the g200 glycan were performed as described previously (37Misevic G.N. Finne J. Burger M.M. J. Biol. Chem. 1987; 262: 5870-5877Abstract Full Text PDF PubMed Google Scholar). g200 stock solutions were prepared by dissolving ≥10 mg of the purified lyophilized molecule in water. Calcium- and magnesium-free artificial seawater (CMF: 20 mm Tris, pH 7.4, buffered Ca2+-, Mg2+-free artificial seawater) was made according to the standards of the Marine Biological Laboratory (see www.mbl.edu). MAF rings were prepared according to established protocols (14Humphreys S. Humphreys T. Sano J. J. Supramol. Struct. 1977; 7: 339-351Crossref PubMed Scopus (33) Google Scholar). SDS-PAGE, Alcian blue stainings, Western blots, and restriction fragment length polymorphism analysis were done as described previously (35Fernàndez-Busquets X. Burger M.M. J. Biol. Chem. 1997; 272: 27839-27847Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Atomic Force Microscope (AFM) Imaging and Force Spectroscopy— AFM visualization in air of proteoglycans was done as specified elsewhere (24Jarchow J. Fritz J. Anselmetti D. Calabro A. Hascall V.C. Gerosa D. Burger M.M. Fernàndez-Busquets X. J. Struct. Biol. 2000; 132: 95-105Crossref PubMed Scopus (43) Google Scholar). Force spectroscopy studies were performed with a Molecular Force Probe one-dimensional microscope (Asylum Research, Santa Barbara, CA). Gold-coated pyramidal Si3N4 tips mounted on triangular 100-μm long cantilevers (k = 0.09 newtons/m) were purchased from Olympus (Tokyo, Japan). The spring constant of every tip was measured individually through the equipartition theorem using the thermal noise of the cantilever (38Florin E.L. Rief M. Lehmann H. Ludwig M. Dornmair C. Moy V.T. Gaub H.E. Biosensors Bioelec. 1995; 10: 895-901Crossref Scopus (417) Google Scholar) with an absolute uncertainty of ∼10%. For the preparation of gold surfaces we followed the template-stripped gold method (39Hegner M. Wagner P. Semenza G. Surface Sci. 1993; 291: 39-46Crossref Scopus (497) Google Scholar). The biomolecules were attached through their endogenous sulfate groups to gold-coated surfaces and tips by overlaying the surface with or floating the tip on a drop of a solution of the molecule (1 mg/ml for chondroitin sulfate B (CSB) and 0.1 mg/ml for MAF, rings, and g200) in the corresponding solvent (CMF + 2 mm CaCl2 for MAF and H2O for all others) for 1 h, followed by a wash with the respective solvents. Tips so prepared were used immediately and performed well for up to 24 h when washed with deionized H2O and kept dry at 4 °C. Surfaces were stored by floating them on CMF + 2 mm CaCl2, at 4°C, and in these conditions they yielded reproducible results for up to 96 h. For pulling experiments, the gold surface was mounted in the AFM and overlaid with 100 μlofCMF +10 mm CaCl2. The cantilever was manually lowered to the surface, and the AFM was operated such that it moved away from and then toward the sample surface during the course of each pull. Between consecutive pulls the tip was allowed a surface dwell time of 1 s. All of the results presented here were obtained with a pulling velocity of 1 μm/s. For data analysis we used the software provided by the AFM manufacturer (IgorPro version 4.09A, WaveMetrics, Inc.). Peaks were selected manually setting a minimum length threshold of 40 nm and considering only the last adhesion event of force-extension curves except where otherwise indicated. Force peaks below 100 piconewtons (pN) were rare, and thus this value was generally chosen as lower force threshold. Calculations done automatically with different minimum force thresholds between 0 and 100 pN gave results essentially identical to those of the manual analysis. Curves that extended beyond the expected contour length of two fully stretched interacting molecules and force peaks above 1 nanonewton (nN) were not considered, and experiments containing more than 1% of either were discarded. Exceptionally, for data analysis of control experiments we considered all force peaks regardless of their intensity or distance from the contact point. This was justified because the adhesion of the immobilized molecules to gold results in a large peak above 1 nN and less than 40 nm from the contact point. For statistical treatments the Origin 6.0 software package was used. p values were calculated with the Mann-Whitney test. As a consequence of the uncertainty inherent to the cantilever calibration described above, changing the AFM tip between two consecutive experiments might result in significant quantitative variations (up to ±10%) in the mean binding force recorded between any given pair of molecules, whereas changing only the surface did not have such an effect. For this reason p values were calculated always for the comparison of data from consecutive experiments where the tip was maintained and only the surface was changed. SPR Studies—SPR studies were carried out with a BIAcore 1000 instrument (BIAcore AB, Uppsala, Sweden) using plain gold surfaces in Au sensor chips. When the surface was functionalized with ligand before docking the chip into the SPR system, this was done by depositing on the gold surface of the sensor 40 μl of a solution of the molecule (1 mg/ml for CSB and 0.1 mg/ml for MAF and g200) in the corresponding solvent (CMF + 2 mm CaCl2 for MAF and water for g200 and CSB), followed by an overnight incubation at 4 °C in a wet chamber. Before docking the chip was washed extensively with the same solvent in which it was incubated. For each assay 60 μl of analyte solution was injected with a flow of 5 μl/min. Single-molecule Force Spectroscopy of Native MAF Reveals the Existence of Multiple Intermolecular Binding Sites—MAF was bound to gold-coated surfaces and AFM tips by deposition of a 0.1 mg/ml solution of the molecule in CMF supplemented with 2 mm Ca2+ (CMF + 2 mm Ca2+). Binding is provided by the affinity for gold of the sulfate groups present on both g6 and g200 (12Bucior I. Scheuring S. Engel A. Burger M.M. J. Cell Biol. 2004; 165: 529-537Crossref PubMed Scopus (112) Google Scholar). Sponge cell aggregation experiments have shown that MAF purified from sulfate-depleted cells had an aggregative activity identical to that of sulfated MAF (40Kuhns W.J. Popescu O. Burger M.M. Misevic G. J. Cell. Biochem. 1995; 57: 71-89Crossref PubMed Scopus (9) Google Scholar), indicating that sulfate groups are not involved in MAF self-binding and thus can be confidently used to immobilize the molecule on surfaces without affecting the functional groups. Sulfate accounts for about 4% of MAF by weight (40Kuhns W.J. Popescu O. Burger M.M. Misevic G. J. Cell. Biochem. 1995; 57: 71-89Crossref PubMed Scopus (9) Google Scholar), an amount similar to that found in glycosaminoglycans such as CSB (41Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell.2nd Ed. Garland Publishing, Inc., New York1989: 805Google Scholar). At the concentration and incubation time used MAF covered the gold surface with a film ∼5 nm thick (Fig. 2A), consistent with the deposition of a single layer of proteoglycans spread out and flat, in agreement with the molecular dimensions shown in Fig. 1A. Incubation with a 0.2 mg/ml MAF solution for an equal time resulted in the deposition of a layer about twice as deep (Fig. 2B), but the surface covering was not improved. Schematically, the setup for our force spectroscopy experiments is depicted in Fig. 2C. For clarity, in the illustration we have represented the molecules standing up. Although most likely several molecules are bound to the tip we assume that on average only one will be participating in the interaction because of the respective mean radii (∼200 nm for MAF and 0.2 mg/ml) could reach a length of several microns (Fig. 3I). These long curves maintained the typical jagged profile (Fig. 3J) and are the consequence of pulling concatamers made up of several MAF molecules. After hundreds of cycles the sawtooth pattern of the curves remained unaltered, indicating repeated zipping and unzipping of the intermolecular bonds. The arms of MAF have a modular structure resulting from the presence of a multiplicity of globular domains (15Fernàndez-Busquets X. Burger M.M. Cell Mol. Life Sci. 2003; 60: 88-112Crossref PubMed Scopus (57) Google Scholar, 24Jarchow J. Fritz J. Anselmetti D. Calabro A. Hascall V.C. Gerosa D. Burger M.M. Fernàndez-Busquets X. J. Struct. Biol. 2000; 132: 95-105Crossref PubMed Scopus (43) Google Scholar), and the shape of MAF force-extension curves could also be consistent with the unfolding of such intramolecular protein domains (45Rief M. Gautel M. Oesterhelt F. Fernandez J.M. Gaub H.E. Science. 1997; 276: 1109-1112Crossref PubMed Scopus (2620) Google Scholar). To explore this possibility, force spectroscopy experiments had to be performed with MAF molecules devoid of arms. Single-molecule Force Spectroscopy of MAF Ring Interactions—The arms of MAF, representing about three-fourths of the total mass of the molecule (24Jarchow J. Fritz J. Anselmetti D. Calabro A. Hascall V.C. Gerosa D. Burger M.M. Fernàndez-Busquets X. J. Struct. Biol. 2000; 132: 95-105Crossref PubMed Scopus (43) Google Scholar), can be easily eliminated by removing calcium from the medium with a treatment in the presence of 1 mm EDTA (14Humphreys S. Humphreys T. Sano J. J. Supramol. Struct. 1977; 7: 339-351Crossref PubMed Scopus (33) Google Scholar) (Fig. 4A). AFM imaging on mica of ring preparations reveals a characteristic beaded appearance (Fig. 4C), where each of the ∼20 beads in a ring is a MAFp3 unit (24Jarchow J. Fritz J. Anselmetti D. Calabro A. Hascall V.C. Gerosa D. Burger M.M. Fernàndez-Busquets X. J. Struct. Biol. 2000; 132: 95-105Crossref PubMed Scopus (43) Google Scholar). In hyalectans, the glycosylated link protein interacts with the proteoglycan monomer core protein, which is heavily substituted with glycosaminoglycan chains. In MAF, each glycosylated MAFp3 interacts with one arm that corresponds to the glycosylated MAFp4 protein (15Fernàndez-Busquets X. Burger M.M. Cell Mol. Life Sci. 2003; 60: 88-112Crossref PubMed Scopus (57) Google Scholar). Despite the topological analogy of MAFp3 and the hyalectan link protein, their amino acid sequences do not have significant homology. In high resolution AFM images, structures supposed to represent g200 can be seen as one or two short chains up to 30 nm long protruding from the MAFp3 beads (Fig. 4C, arrowheads). The resulting ring preparations yield reproducible force-distance curves in single-molecule force spectroscopy experiments performed in CMF + 10 mm Ca2+ (Fig. 4, B and D). The maximum length of ring
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