Journal of Molecular Biology
Volume 385, Issue 4, 30 January 2009, Pages 1277-1286
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Nanomechanical Properties of Tenascin-X Revealed by Single-Molecule Force Spectroscopy

https://doi.org/10.1016/j.jmb.2008.11.038Get rights and content

Abstract

Tenascin-X is an extracellular matrix protein and binds a variety of molecules in extracellular matrix and on cell membrane. Tenascin-X plays important roles in regulating the structure and mechanical properties of connective tissues. Using single-molecule atomic force microscopy, we have investigated the mechanical properties of bovine tenascin-X in detail. Our results indicated that tenascin-X is an elastic protein and the fibronectin type III (FnIII) domains can unfold under a stretching force and refold to regain their mechanical stability upon the removal of the stretching force. All the 30 FnIII domains of tenascin-X show similar mechanical stability, mechanical unfolding kinetics, and contour length increment upon domain unfolding, despite their large sequence diversity. In contrast to the homogeneity in their mechanical unfolding behaviors, FnIII domains fold at different rates. Using the 10th FnIII domain of tenascin-X (TNXfn10) as a model system, we constructed a polyprotein chimera composed of alternating TNXfn10 and GB1 domains and used atomic force microscopy to confirm that the mechanical properties of TNXfn10 are consistent with those of the FnIII domains of tenascin-X. These results lay the foundation to further study the mechanical properties of individual FnIII domains and establish the relationship between point mutations and mechanical phenotypic effect on tenascin-X. Moreover, our results provided the opportunity to compare the mechanical properties and design of different forms of tenascins. The comparison between tenascin-X and tenascin-C revealed interesting common as well as distinguishing features for mechanical unfolding and folding of tenascin-C and tenascin-X and will open up new avenues to investigate the mechanical functions and architectural design of different forms of tenascins.

Introduction

Tenascins are a family of highly conserved oligomeric glycoproteins in the extracellular matrix (ECM) of vertebrate organisms.1, 2, 3, 4 Tenascins are involved in the process of mechanotransduction and play important roles in modulating cell adhesion as well as cell–matrix interactions.2, 5, 6 A wide range of ECM molecules including proteins, glycosaminoglycans, and proteoglycans are found to bind to tenascins with high affinity.3 These adhesive properties of tenascins help knit structural ECM proteins with surrounding cells as well as other ECM components to form an interacting network to provide desired mechanical strength and elasticity to tissues. Mechanical forces are constantly involved during such processes. Thus, understanding how tenascins respond to mechanical stretching force is important to the understanding of biological and mechanical roles of tenascins in such mechanotransduction processes.

There are five known tenascins: tenascin-C, tenascin-R, tenascin-W, tenascin-X, and tenascin-Y.7 All tenascins share the same tandem modular structure, and the constituent domains can be divided into four structurally distinct classes: a tenascin-assembly domain, a stretch of epidermal-growth-factor (EGF)-like repeats, a fibronectin type III (FnIII) domain region that is composed of a series of FnIII domains, and a terminal knob domain that is homologous to the globular domain of fibrinogen. Tenascins typically exist as oligomers assembled from tenascin monomers via heptad repeats, further stabilized by disulfide bridges in the tenascin-assembly domain.8, 9 Despite the importance of the mechanical properties of tenascins in their biological functions, tenascin-C is the only form of tenascin whose mechanical properties have been studied in detail using single-molecule atomic force microscopy (AFM).10, 11, 12, 13, 14 Single-molecule AFM studies revealed that tenascin-C is an elastic protein, and its contour length can be extended to several times its resting one by mechanically unfolding its constituting FnIII domains under a stretching force.10 However, the mechanical properties of other forms of tenascins remain unexplored, and thus, it has not been possible to investigate the mechanical roles of other forms of tenascins or to directly compare the mechanical properties and mechanical design of different forms of tenascins. As a step towards understanding the mechanical properties and design of other forms of tenascins, here we use single-molecule AFM to characterize the mechanical properties of recombinant full-length bovine tenascin-X as well as its fragment.

Tenascin-X is expressed in a wide range of tissues including skin, joints, heart, and blood vessels.15 Tenascin-X binds a range of molecules in the ECM as well as on the cell membrane16, 17, 18 including collagen fibrils, decorin,19 and glycosaminoglycans17 and plays important roles in regulating the structure and mechanical properties of connective tissues. Recently, a recessive form of Ehlers–Danlos syndrome (EDS), an inherited connective disorder, has been found to result from deficiency as well as point mutations in the tenascin-X gene.20, 21 The typical symptoms of EDS are hypermobile joints, hyperelastic skin, and easy bruising. Tenascin-X is the first gene outside the collagen family that causes EDS, highlighting the importance of tenascin-X in the structure and mechanical properties of connective tissues. Similar to other tenascins, bovine tenascin-X has a modular structure (Fig. 1) and is composed of an N-terminal domain, an EGF domain region containing 18.5 EGF domains, an FnIII domain region containing 30 FnIII domains, and a C-terminal fibrinogen-like terminal domain.22 Using single-molecule AFM, we have stretched bovine tenascin-X molecules to measure their mechanical properties. Our results demonstrate that tenascin-X is an elastic protein, and its FnIII domains can unfold under a stretching force and refold to regain their mechanical stability upon removal of the stretching force. Our results show that tenascin-X and tenascin-C not only share some similar mechanical features but also exhibit distinguishing mechanical properties that are unique to either tenascin-X or tenascin-C. These results pave the way to investigating how the mechanical properties of tenascin-X influence the biological functions of tenascin-X and making it possible to carry out thorough comparative studies among different forms of tenascins to examine how the mechanical properties of tenascins are finely regulated at a molecular level by their similar yet distinctive modular design.

Section snippets

Mechanical unfolding of the full-length tenascin-X and all-FnIII fragment (rTNXΔEΔF)

Using single-molecule AFM,10, 23 we have investigated the mechanical properties of the full-length bovine tenascin-X. The full-length tenascin-X was deposited onto a clean glass coverslip and picked up by the AFM tip randomly along its contour. Stretching tenascin-X between the AFM tip and glass substrate resulted in force–extension curves exhibiting characteristic sawtooth pattern appearance (Fig. 2a), in which the individual force peaks correspond to the mechanical unfolding events of

Tenascins are a family of elastic proteins

Using single-molecule AFM, we have characterized the mechanical unfolding and folding kinetics of tenascin-X in detail. Our results showed that tenascin-X is an elastic protein and its constitutive FnIII domains can undergo reversible unfolding–folding transitions under a stretching force to effectively modulate the contour length of tenascin-X molecules. These results provide a general and average description of the mechanical properties of tenascin-X and provide the possibility to compare the

Conclusion

We have used single-molecule AFM to characterize the elastic behaviors of full-length tenascin-X as well as its fragment comprising all the FnIII domains. Our results revealed that tenascin-X is an elastic protein and its FnIII domains can undergo reversible unfolding–folding reactions. The folding and unfolding behaviors of FnIII domains exhibit intriguing similarities and distinct contrasts between FnIII domains from tenascin-C and tenascin-X. We hypothesize that different tenascins encode

Protein engineering

Recombinant full-length bovine tenascin-X and a truncated form comprising all its FNIII domains (rTNXΔEΔF) were prepared as described previously.18 Both proteins were produced by HEK293 cells and secreted in the culture medium. Purification was achieved by two chromatographic steps, the first one by affinity with heparin and the second one on Q-Sepharose.

TNXfn10 gene was amplified from a plasmid encoding TNXfn9–11 via standard polymerase chain reaction using sense primer (CGT GGA TCC CTG CTC

Acknowledgements

This work is supported by Canadian Institutes for Health Research Operating Grant MOP-81225, Michael Smith Foundation for Health Research, University of British Columbia Health Research Resources Office, Canada Research Chairs Program, and Canadian Foundation for Innovation. H.L. is a Michael Smith Foundation for Health Research Scholar.

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