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Fibronectin: A flexible image
CopyrIght
FIBRONECTIN: MATTI
VUENTO,
Departmenrs
KLAUS
c$ Biochemis/r.y,
A FLEXIBLE
HEDMAN,
TAP10
Virology and Pathology
Some investigators studying the structure of fibronectin by electron optical methods have pictured fibronectin molecules as globular structures. Others, however, have provided evidence for an elongated structure. It appears that both schools of thought have been right. Probably the first electron micrographs of fibronectin were those by Sadaaki Iwanaga, Koji Suzuki and Senichiro Hashimoto, published ten years ago in the Proceedings of the First International Conference on Fihrohlust Surface Protein (Iwanaga et al., 1978). The pictures, obtained after negative staining with phosphotungstic acid, showed very long filamentous structures whose thickness varied greatly, over a range of 20100 nm. In addition, some more finely-structured, negatively-stained material could be seen in the pictures that presumably could represent building blocks of the large protein filaments. In the years since then, more and more refined (although perhaps still controversial) images have been obtained of the originally only poorly visible building blocks, that is, fibronectin molecules. The assembly of fibronectin filaments out of the building blocks is still a mystery, although there is no shortage of models proposed to explain it.
STRUCTURE
In reviews written for non-afecionados, customary to start by introducing fibronectin
VAHERI
Helsinki,
Finland
341 341 349
large molecular weight protein present in a soluble form in blood and other body fluids of vertebrates and in an insoluble form in tissues, especially in the connective tissue where it is involved in the intricate organization of the extracellular matrix. In this role, fibronectin interacts through its multiple binding functions (Fig. 1): there are binding sites for fibrinogen, collagen, glycosaminoglycans and cell surfaces, among other things. The circulatory protein is not bound, as there is normally nothing in the blood to bind it. However, when there is a wound the situation changes: subendothelial collagen is exposed and new potential ligands, such as fibrin, become available for fibronectin, so that it participates in the process of wound healing and the final stages of blood clotting. A first good estimate for the size of fibronectin (called at the time “Cold Insoluble Globulin”, for its tendency to form precipitating complexes in the cold) was obtained by sedimentation equilibrium analysis, a method now familiar to but a few of the classical biochemistry breed. Fibronectin was found to have a molecular weight (M,) of 450,000 (Mosesson et al., 1975). Half-molecules, obtained by reduction, migrate in SDS-PAGE at a rate suggesting a M, of about 220,000. Limited proteolysis of the intact dimer produces fragments of about this size, showing that the two polypeptide chains are linked to each other at one end of the chains (Jilek and Hormann, 1977). Later studies employing small-angle X-ray scattering and neutron scattering methods have revealed slightly higher molecular weights (M, 510,000~530,000)
I. INTRODUCTION
II. FIBRONECTIN
and ANTTT
of the Unit~ersiry of Helsinki,
II. Fibronectinstructure .._..._.._.__._..._..___.._...___.._...__..__.....___.._...._._.._.....,...,,.,.,,,..,, References ._._.__._.._..._.._._.._..._..__..._...___.__...__.._......____._........,.,,,..,.,.,,,..,......
0892.0354,X8 $0.00 + 0.50 19X8 Pqamon Press plc
IMAGE
VARTIO
CONTENTS 1. Introduction.............................................................................................,,
c
it is as a 341
342
type
Jl
0
type
m
0
Fig. I. Domam structure of libroncctm. The two subunits of the protein are disulfde-linked at the C‘OOH-terminus. The locations of some of the binding functions are indicated. Black dots indicate regions that are variably present in fibronectin polypeptide iwforms depending on the alternative splicing of the precursor mRNA before translation.
for fibronectin (Sjoberg rr ul.. 1987). A protein molecule with a M, of 510,000 and partial specific volume of 0.72 cm3/g would have a volume of 610 nm3. Assuming that the molecule takes the form of a completely spherical non-hydrated particle, such a particle would have a diameter of about IO nm. Particles with approximately these dimensions have indeed been observed by electron microscopy. In a study by Koteliansky ef ul. (1981) compact, negatively stained (the sample was freeze-dried and shadowed with tungsten-tantalum) structures were observed, having ellipsoid axial ratios of 2: I : I, a length of 15.5 nm and a width of 8.8 nm. Such a particle would have an approximate dry volume of 630nm’. The dimensions obtained by Koteliansky et ul. (I 98 I) can be compared with the dimensions obtained for the hydrated fibronectin particles by Sjiiberg et ul. (1987), using small angle X-ray and neutron scattering methods. The results from the latter study indicated that fibronectin in solution under physiological pH and ionic strength conditions assumes the shape of an oblate ellipsoid (disc-shape) with an axial ratio of IO: I. Thus the molecule in solution would be compact, but rather asymmetric. On the other hand, not everyone looking at fibronectin by electron microscopy methods has come up with particles like those of Koteliansky et ul. (1981). Erickson and Carrel1 (1983) observed considerably more extended structures. Both intact fibronectin and half molecules were observed as extended curvilinear strands. At low ionic strength, the structures were more curved or bent. In an-
other study (Engel rf al.. 198 I) fibronectin was seen as two distinctly non-globular strands about 2 nm wide and 61 nm long, linked together at their ends enclosing a fixed angle of 70 Price et ul. (I 982) obtained randomly coiled images for intact fibronectin. Fibronectin fragments were visualized as linear rod-like structures, with the length varying according to the molecular size: the M, 200,000 fragments had a length of 57 nm. The images also suggested the presence of nodular structures along the rods. Finally, our own studies (Figs. 2 4) with negative staining revealed very long (up to several /lrn), thin (minimum width 2 3 nm) filamentous structures and also larger structures apparently assembled from the thin filaments by lateral association (Vuento rt ul.. 1980). These structures closely resembled those shown by lwanaga et d. (1978). In an interesting paper Tooney rt ul. (1983) showed that the method of sample preparation greatly affected the outcome of electron microscopic analysis of fibronectin. Fibronectin deposited at pH 7.0 7.4 on carbon films and subsequently freeze-dried showed relatively compact, irregular-shaped images about 16 nm in their shortest dimension and 24 nm in their longest dimension. Fibronectin deposited on carbon at pH 9.3 showed less compact and more extended images; even more extension was observed when the protein was deposited at pH 2.8. Interestingly. protein sprayed on a mica surface appeared much more extended than protein deposited on carbon. These results suggest that both the conformational state of fibronectin. and the polarity/other surface
Fibronectin:
Fig.
Electron
micrograph From
A Flexible
of negatively stained unpolymerized Vuento et al., 1980, with permission.
properties of the material it is deposited on, affect the degree of extension of the fibronectin molecules seen in the electron microscopic images. That fibronectin does undergo conformational changes with changing ionic milieu has been documented beyond question. Measurements of sedimentation coefficients (Alexander et al., 1979; Erickson and Carrell, 1983; Markovic et al., 1983; Tooney et al., 1983) have shown that the S,,,, values are increased when the pH is increased or lowered from the physiological values. The sedimentation coefficient was also decreased by increasing the ionic strength. These observations, supported by measurements of the diffusion coefficient by quasielastic light scattering and by measurements of intrinsic viscosity (Williams et al., 1982) indicate that the structure of fibronectin
Image
plasma fibronectin. Ammonium Scale marker indicates 100 nm.
343
molybdate
staining.
becomes more extended at extreme pH values or at high ionic strength. Binding of fibronectin to its ligands, collagen or heparan sulphate, has also been shown to cause conformational changes in the protein (Williams et al., 1982; ijsterlund et al., 1985). In the light of the above evidence, one may assume that extended structures observed in electron microscopy do not represent the native structure of fibronectin, but result from some kind of unfolding of the protein caused by interaction of the protein with surfaces or by the ionic environment. However, before rejecting the extended structures of fibronectin as an artifact, one should consider the phenomenon of fibronectin fibrillogenesis. While circulatory fibronectin, which is probably produced by hepatocytes (Tamkun and
344
M. Vuento
et ~1.
Fig. 3. Electron micrography of spontaneous filament formation by purified plasma fibronectin. Negative staining with ammonium molybdate. (A) Linear filamentous polymers. (B) Early bundle formation with a heavy background 01‘ unpolymerized fibronectin. (C) Extensive bundle formation. (D) Fibronectin incubated with affinity purified antibodies. From Vuento e/ al., 1980, with permission. Scale markers indicate 200 nm (A and D), 400 nm (B and C).
Hynes, 1983) is under normal conditions perfectly soluble, the so called cellular fibronectin, synthesized by a variety of cell types (Hynes, 1985) is not. Both types of fibronectin have a similar gross structure (Fig. I), but differ by an additional type III homology region found only in cellular fibronectin (Kornblihtt et al., 1984). The differences in protein structure derive from variable splicing of the fibronectin mRNA transcribed from a single copy gene (Kornblihtt et al., 1984; Schwartzbauer et al., 1983; Paul and Hynes, 1984). In
the pericellular matrix of cultured fibroblasts (Fig. 5 and 6; Hedman et al., 1978) fibronectin and collagen co-distribute, that is, the fibrillar immunofluorescence staining patterns for fibronectin and collagen are identical images that coincide (Vahcri et al., 1978). Subsequent studies showed that in the fibroblast matrix heparan sulphate proteoglycan also co-distributes with fibronectin (Hedman P/ (/I., 1982) and that in matrix assembly fibronectin fibrillogenesis is independent of the deposition of collagen (Hedman and Vaheri, 1988). Further-
Fibronectin:
Fig. 4. Phase-contrast micrographs of filamentous a network structure. (B) Linear polymers forming
A Flexible
Image
345
structures found in solutions of fibronectin. (A) Linear polymers forming a thick “cable”. From Vuento et al., 1980, with permission. Scale marker indicates 50 pm.
more, in the fibroblast matrix, enzymatic digestion of sulfated glycosaminoglycans leaves the fibronectin fibrils intact (Hedman et al., 1984). Thus, it is possible that in the matrix assembly fibronectin has the organizing role in fibrillogenesis. It is interesting to note that exogeneously added plasma fibronectin can also be incorporated into the protein fibrils (Hayman and Ruoslahti, 1979; Oh et al., 198 1). This co-distribution of fibronectin with collagen could be explained by the fact that fibronectin has a potent binding affinity for both collagen and for glycosaminoglycans (reviewed by Hynes, 1985). Such multiple binding interactions
could immobilize fibronectin in the pericellular matrix. There are, however, other possibilities; fibronectin has been shown to possess free, though cryptic, sulfhydryl groups, and to be able to form disulphide-bonded multimers (Mosher and Johnson, 1983). In addition to the free sulfhydryls, the intramolecular disulfide bridges of fibronectin molecules may exchange to form intermolecular ones, resulting in polymerization (McKeownLong0 and Mosher, 1984; Vartio, 1986). Formation of such multimers, possibly involving subsequent stabilization by transglutaminase (Mosher and Johnson, 1983) could contribute to
M. Vuento
(‘I I//
Fig. 5. Pericellular matrix isolated from human embryonic skin libroblast tibronectin as seen in a phase contrast micrograph shows a cell-free fihrillar
the immobilization of fibronectin to fibrillar structures. These models of fibronectin fibrillogenesis all rely (except, perhaps, for the involvement of sulfhydryls) on the special environmental conditions in the pericellular matrix. Further along this line of thought. a role for the cell surface itself in
culture. network.
Immunoperoxidase staining for Scale marker indicates 20 /cm.
fibronectin fibrillogenesis has been proposed in a series of interesting papers (McKeown-Longo and Mosher, 1983. 1984, 1985; Peters and Mosher, 1987). These authors showed that fibronectin was incorporated into two distinguishable pools in the pericellular matrix of cultured fibroblasts. Binding
Fibronectin:
A Flexible
Image
Fig. 6. Scanning electron micrograph of the isolated pericellular matrix. The diameter of the matrix fibers varies greatly. The thicker fibers can be resolved to have filamentous ultrastructure (arrow). From Hedman et al., 1979, with permission. Scale marker indicates 2pm.
to pool I had the characteristics of a receptormediated process. In a recent study, McDonald et al. (1987) used proteolytically derived fibronectin fragments and monoclonal antibodies against specific determinants in the fibronectin structure to map the sites involved in the assembly of fibronectin into matrix. They found that antibodies to an M, 25,000 amino terminal domain in fibronectin inhibited the assembly. They also identified another site, located towards the C-terminus and containing the cell adhesion site (Ruoslahti and Pierschbacher, 1986) that was also involved in the matrix assembly. These authors also presented evidence against the involvement of the collagen-binding domain of fibronectin in the matrix assembly. Antibodies to the collagenbinding domain did not inhibit the assembly, nor did purified collagen-binding domains themselves.
Two intriguing findings link the above findings on matrix assembly to the discussion on fibronectin structure. It has been shown that plasma fibronectin can be induced under certain conditions to form fibrillar polymers (Fig. 7) that resemble, superficially at least, matrix-borne fibronectin fibrils. The polyamines spermine and sperminide precipitate fibronectin and the precipitates have a filamentous structure as revealed by electron microscopy (Vuento et al., 1980). The thin filaments had a width of about 2 nm. Similar structures were observed by electron microscopy in fibronectin precipitated with heparin (Jilek and Hiirmann, 1979). Microscopic (Vuento et al., 1980; Richter et al., 1985) or even macroscopic (unpublished observations) fibrils of fibronectin are sometimes formed in fibronectin solutions without added polycations or polyanions. An in-
Fig. 7. Electron micrographs of filamentous polymers of libronectin induced by spermine. Negative staining with ammonium molybdate. Fibronectin (0 5 mg/ml in 5 mM Tris-HCI. pH 7.5. was incubated with spermine (I mM) for 30 min at room temperature before staining. (A) A field showing long linear filnments. (B) Extensive network formation. From Vucnto (21~1.. 1980. with permission. Scale markcrh indicate 200 nm 34x
Fibronectin:
terpretation of the images of the thin filaments is that apparently these represent fibronectin molecules in extended conformation and are linked to each other in an end-to-end manner. Apparently, the compact structure that fibronectin has in solution, as discussed above, can be induced to unfold under special conditions. Perhaps this unfolding to an extended structure favours end-to-end association of the molecules. An interesting mechanistic model for folding/unfolding of fibronectin has been proposed by Hiirmann and Richter (1986). According to their model, a key factor in the folding of fibronectin is an uneven charge distribution along the polypeptide chain. Folding to a compact structure is facilitated by attraction of regions with opposite charge. Neutralization of critical charges by anionic or cationic ligands could thus cause an opening of the structure similar to that found to occur with heparin or with polyamines. An important factor, therefore, in the fibrillogenesis of fibronectin may lie in the intrinsic structure of fibronectin itself. Further studies are needed to clarify whether fibronectin fibrillogenesis is basically a multiple liganding process or a self assembly process or both. In the light of the present data, we see emerging a real flexible image for this multifunctional protein.
REFERENCES Alexander, S. S.. Colonna, G. and Edelhoch, H., 1979. The structure and stability of human plasma cold-insoluble globulin. J. hiol. Chem. 254, 1501 1505. Engel, J.. Odermatt, E., Engel, A.. Madri, J. A., Furthmayr, H., Rohde, H. and Timpl, R.. 1981. Shapes. domain organization and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J. m&c. Biol. 150, 97 120. Erickson, H. P. and Carrell, N. A.. 1983. Fibronectin in extended and compact conformations. J. hiol. Chem. 258, 14539-14544. Hayman, E. G. and Ruoslahti, E., 1979. Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix. J. Cell Biol. 83, 2555259. Hedman, K. and Vaheri. A., 1988. Fibronectin and the pericellular matrix. In: Fihronrcrin, Mosher, D. (ed.), Academic Press (in press). Hedman, K., Vaheri. A. and Wartiovaara. J.. 1978. External fibronectin of cultured human tibroblasts is predominantly a matrix protein. J. Cell Biol. 76, 748 760.
A Flexible
Image
349
Hedman, K., Johansson, S., Vartio, T., Kjelltn, L., Vaheri. A. and Hook. M., 1982. Structure of the pericellular matrix: Association of heparan and chondroitin sulfates with fibronectin-procollagen fibers. Ceil 28, 663367 I. Hedman. K., Vartio, T., Johansson, S., Kjelltn, L., Hook, M., Linker, A., Salonen, E-M. and Vaheri, A., 1984. Integrity of the pericellular fibronectin matrix of tibroblasts is independent of sulfated glycosaminoglycans. EMBO J. 3, 58 I-584. Hynes, R. O., 1985. Molecular biology of tibronectin. A. Rev. Cell Biol. 1, 67790. Hormann, H. and Richter. H., 1986. Models for the subunit arrangement in soluble and aggregated plasma fibronectin. Biopolymers 25, 947-958. Iwanaga, S., Suzuki, K. and Hashimoto, S., 1978. Bovine plasma cold-insoluble globulin: Gross structure and function. Ann. N. Y. Acad. Sci. 312, 5673. Jilek, F. and Hormann, H., 1977. Cold-insoluble globulin: Plasminolysis of cold-insoluble globulin. Hoppe-Seyler’s Z. physiol. Chem. 358, 133-136. Jilek. F. and Hormann, H., 1979. Fibronectin (cold-insoluble globulin). VI. Influence of heparin and hyaluronic acid on the binding of native collagen. Hoppe-Seyler’s Z. physiol. Chem. 360, 597-603. Kornblihtt, A. R., Vibe-Pederson, K. and Baralle. F. E., 1984. Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain. EMBO J. 3, 221-226. Koteliansky, V. E., Glukhova, M. A., Bejanian, M. V.. Smirnov, V. N., Flimonov. V. V.. Zalite, 0. M. and Venvaminov. S. Y., 1981. A study of the structure of fibronectin. Eur. J: Biochem. 119, 619-624. Markovic, Z., Lustig, A., Engel, J., Richter, H. and Hiirmann, H., 1983. Shape and stability of fibronectin in solutions of different pH and ionic strength. Hoppe-Seyler’s Z. physiol. Chem. 364, 1795P1804. McDonald, J. A., Quade, B. J.. Broekelmann, T. J.. LaChance, R., Forsman, K., Hasegawa. E. and Akiyama, S., 1987. Fibronectin’s cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix. J. hiol. Chem. 262, 29572967. McKeown-Longo. P. J. and Mosher, D. F., 1983. Binding of plasma fibronectin to cell layers of human skin fibroblasts. J. Cell Biol. 97, 46&472. McKeown-Longo, P. J. and Mosher. D. F., 1984. Mechanisms of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fib&blasts. J. hiol. Chem. 259, 1221&12215. McKeown-Longo, P. J. and Mosher, D. F., 1985. Interaction of the 70,000-mol.wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts. J. Cell Biol. 100, 3644374. Mosesson. M. W.. Chen, A. B. and Huseby, R. M., 1975. The cold-insoluble globulin of human plasma: Studies of its essential structural features. Biochim. hiophys. Acra 386, 509-524. Mosher, D. F. and Johnson, R. B., 1983. In c>irro formation of disulfide-bonded fibronectin multimers. J. hiol. Chem. 258, 6595. 660 I. Oh, E., Pierschbacher, M. and Ruoslahti, E., 1981. Deposition of plasma fibronectin in tissues. Proc. nrrtn. Acad. Sci. U.S.A. 78, 3218 3221.
M. Vuento
350
ijsterlund, E., Eronen, I.. tjsterlund. K. and Vuento. M.. 1985. Secondary structure of human plasma fibronectin: co,,formational change induced by calf alveolar heparan sulfates. Biochem. 24, 2661-2667. Paul. J. I. and Hynes. R. O., 1984. Multiple fibronectin subunits and their post-translational modifications. J. hid. Chem. 259, 13477-I 3487. Peters, D. M. P. and Mosher, D. F.. 1987. Localization of cell surface sites involved in fibronectin fibrillogenesis. J. Cc,// Bid. 104, 121~~130. Price. T. M.. Rudee, M. L.. Pierschbacher, M. and Ruoslahti. E.. 1982. Structure of fibronectin and its fragments in electron microscopy. Eur. J. Bioc~hm~. 129, 359-363. Richter, H., Wendt. C. and H(irmann, H., 1985. Aggregation and fibril formation of plasma fibronectin by heparin. Bid. Chem.
Hoppc-Se_&r
366,
509%5 14.
Ruoslahti. E. and Pierschbacher, M. D.. 1986. Arg-Gly-Asp: A versatile cell recognition signal. Cell 44, 517 518. Schwartzbauer. J. E., Tamkun, J. W., Lemischka, I. R. and Hynes, R. 0.. 1983. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Crll 35, 421 431. Sjoberg. B., Pap, S., ijsterlund. E., &terlund, K., Vuento. M. and Kjems. J., 1987. Solution structure of human plasma fibronectin using small-angle X-ray and neutron scattering
1’1(11
at physiological Biophy.y.
255,
pH and
ionic
strength.
,4r&s
Biwhm~.
347 353.
Tamkun. J. W. and Hynes, R. 0.. 19X3. Plasma libronectm is synthesized and secreted by hepatocytes. J. hid. Chcv~r. 245,
5728 5736.
Tooney. N. M.. Mosesson. M. W., Amram. D. L.. tlainlkld, J. F. and Wall, J. S.. 1983. Solution and surface elf‘ects on plasma fibronectin structure. J. C‘cs//. Bid. 97, 16X6 1692. Vaheri. A.. Kurkinen. M.. Lehto. V. P.. Linder. E. and Timpl. R.. 1978. Codlstribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: libronectin and procollagen. Proc,. r~~/n. .~uu/. %i ( ‘..~.A. 75. 4944 4948. Vartio. T., 1986. Disulfide-bonded polymerization of plasma fibronectin in the presence of metal ions. J. hiol. (‘hc,nr. 261, 9433 9437. Vuento. M.. Vartio. T.. Saraste. M.. van Bonsdorff. C‘-H. and Vaheri. A.. 1980. Spontaneous and polyamine-induced formation of filamentous polymers from soluble libronectin. Eur. J. Biochem. 105, 33 42. Williams. E. C.. Janmcy. P. A.. Ferry. J. D. and Mosher, D. F.. 1982. Conformational states of fibronectin: Effects of pH, ionic strength and collagen binding. ./. hid ~‘/rcv~~ 257, 14973 14978.
FIBRONECTIN: MATTI
VUENTO,
Departmenrs
KLAUS
c$ Biochemis/r.y,
A FLEXIBLE
HEDMAN,
TAP10
Virology and Pathology
Some investigators studying the structure of fibronectin by electron optical methods have pictured fibronectin molecules as globular structures. Others, however, have provided evidence for an elongated structure. It appears that both schools of thought have been right. Probably the first electron micrographs of fibronectin were those by Sadaaki Iwanaga, Koji Suzuki and Senichiro Hashimoto, published ten years ago in the Proceedings of the First International Conference on Fihrohlust Surface Protein (Iwanaga et al., 1978). The pictures, obtained after negative staining with phosphotungstic acid, showed very long filamentous structures whose thickness varied greatly, over a range of 20100 nm. In addition, some more finely-structured, negatively-stained material could be seen in the pictures that presumably could represent building blocks of the large protein filaments. In the years since then, more and more refined (although perhaps still controversial) images have been obtained of the originally only poorly visible building blocks, that is, fibronectin molecules. The assembly of fibronectin filaments out of the building blocks is still a mystery, although there is no shortage of models proposed to explain it.
STRUCTURE
In reviews written for non-afecionados, customary to start by introducing fibronectin
VAHERI
Helsinki,
Finland
341 341 349
large molecular weight protein present in a soluble form in blood and other body fluids of vertebrates and in an insoluble form in tissues, especially in the connective tissue where it is involved in the intricate organization of the extracellular matrix. In this role, fibronectin interacts through its multiple binding functions (Fig. 1): there are binding sites for fibrinogen, collagen, glycosaminoglycans and cell surfaces, among other things. The circulatory protein is not bound, as there is normally nothing in the blood to bind it. However, when there is a wound the situation changes: subendothelial collagen is exposed and new potential ligands, such as fibrin, become available for fibronectin, so that it participates in the process of wound healing and the final stages of blood clotting. A first good estimate for the size of fibronectin (called at the time “Cold Insoluble Globulin”, for its tendency to form precipitating complexes in the cold) was obtained by sedimentation equilibrium analysis, a method now familiar to but a few of the classical biochemistry breed. Fibronectin was found to have a molecular weight (M,) of 450,000 (Mosesson et al., 1975). Half-molecules, obtained by reduction, migrate in SDS-PAGE at a rate suggesting a M, of about 220,000. Limited proteolysis of the intact dimer produces fragments of about this size, showing that the two polypeptide chains are linked to each other at one end of the chains (Jilek and Hormann, 1977). Later studies employing small-angle X-ray scattering and neutron scattering methods have revealed slightly higher molecular weights (M, 510,000~530,000)
I. INTRODUCTION
II. FIBRONECTIN
and ANTTT
of the Unit~ersiry of Helsinki,
II. Fibronectinstructure .._..._.._.__._..._..___.._...___.._...__..__.....___.._...._._.._.....,...,,.,.,,,..,, References ._._.__._.._..._.._._.._..._..__..._...___.__...__.._......____._........,.,,,..,.,.,,,..,......
0892.0354,X8 $0.00 + 0.50 19X8 Pqamon Press plc
IMAGE
VARTIO
CONTENTS 1. Introduction.............................................................................................,,
c
it is as a 341
342
type
Jl
0
type
m
0
Fig. I. Domam structure of libroncctm. The two subunits of the protein are disulfde-linked at the C‘OOH-terminus. The locations of some of the binding functions are indicated. Black dots indicate regions that are variably present in fibronectin polypeptide iwforms depending on the alternative splicing of the precursor mRNA before translation.
for fibronectin (Sjoberg rr ul.. 1987). A protein molecule with a M, of 510,000 and partial specific volume of 0.72 cm3/g would have a volume of 610 nm3. Assuming that the molecule takes the form of a completely spherical non-hydrated particle, such a particle would have a diameter of about IO nm. Particles with approximately these dimensions have indeed been observed by electron microscopy. In a study by Koteliansky ef ul. (1981) compact, negatively stained (the sample was freeze-dried and shadowed with tungsten-tantalum) structures were observed, having ellipsoid axial ratios of 2: I : I, a length of 15.5 nm and a width of 8.8 nm. Such a particle would have an approximate dry volume of 630nm’. The dimensions obtained by Koteliansky et ul. (I 98 I) can be compared with the dimensions obtained for the hydrated fibronectin particles by Sjiiberg et ul. (1987), using small angle X-ray and neutron scattering methods. The results from the latter study indicated that fibronectin in solution under physiological pH and ionic strength conditions assumes the shape of an oblate ellipsoid (disc-shape) with an axial ratio of IO: I. Thus the molecule in solution would be compact, but rather asymmetric. On the other hand, not everyone looking at fibronectin by electron microscopy methods has come up with particles like those of Koteliansky et ul. (1981). Erickson and Carrel1 (1983) observed considerably more extended structures. Both intact fibronectin and half molecules were observed as extended curvilinear strands. At low ionic strength, the structures were more curved or bent. In an-
other study (Engel rf al.. 198 I) fibronectin was seen as two distinctly non-globular strands about 2 nm wide and 61 nm long, linked together at their ends enclosing a fixed angle of 70 Price et ul. (I 982) obtained randomly coiled images for intact fibronectin. Fibronectin fragments were visualized as linear rod-like structures, with the length varying according to the molecular size: the M, 200,000 fragments had a length of 57 nm. The images also suggested the presence of nodular structures along the rods. Finally, our own studies (Figs. 2 4) with negative staining revealed very long (up to several /lrn), thin (minimum width 2 3 nm) filamentous structures and also larger structures apparently assembled from the thin filaments by lateral association (Vuento rt ul.. 1980). These structures closely resembled those shown by lwanaga et d. (1978). In an interesting paper Tooney rt ul. (1983) showed that the method of sample preparation greatly affected the outcome of electron microscopic analysis of fibronectin. Fibronectin deposited at pH 7.0 7.4 on carbon films and subsequently freeze-dried showed relatively compact, irregular-shaped images about 16 nm in their shortest dimension and 24 nm in their longest dimension. Fibronectin deposited on carbon at pH 9.3 showed less compact and more extended images; even more extension was observed when the protein was deposited at pH 2.8. Interestingly. protein sprayed on a mica surface appeared much more extended than protein deposited on carbon. These results suggest that both the conformational state of fibronectin. and the polarity/other surface
Fibronectin:
Fig.
Electron
micrograph From
A Flexible
of negatively stained unpolymerized Vuento et al., 1980, with permission.
properties of the material it is deposited on, affect the degree of extension of the fibronectin molecules seen in the electron microscopic images. That fibronectin does undergo conformational changes with changing ionic milieu has been documented beyond question. Measurements of sedimentation coefficients (Alexander et al., 1979; Erickson and Carrell, 1983; Markovic et al., 1983; Tooney et al., 1983) have shown that the S,,,, values are increased when the pH is increased or lowered from the physiological values. The sedimentation coefficient was also decreased by increasing the ionic strength. These observations, supported by measurements of the diffusion coefficient by quasielastic light scattering and by measurements of intrinsic viscosity (Williams et al., 1982) indicate that the structure of fibronectin
Image
plasma fibronectin. Ammonium Scale marker indicates 100 nm.
343
molybdate
staining.
becomes more extended at extreme pH values or at high ionic strength. Binding of fibronectin to its ligands, collagen or heparan sulphate, has also been shown to cause conformational changes in the protein (Williams et al., 1982; ijsterlund et al., 1985). In the light of the above evidence, one may assume that extended structures observed in electron microscopy do not represent the native structure of fibronectin, but result from some kind of unfolding of the protein caused by interaction of the protein with surfaces or by the ionic environment. However, before rejecting the extended structures of fibronectin as an artifact, one should consider the phenomenon of fibronectin fibrillogenesis. While circulatory fibronectin, which is probably produced by hepatocytes (Tamkun and
344
M. Vuento
et ~1.
Fig. 3. Electron micrography of spontaneous filament formation by purified plasma fibronectin. Negative staining with ammonium molybdate. (A) Linear filamentous polymers. (B) Early bundle formation with a heavy background 01‘ unpolymerized fibronectin. (C) Extensive bundle formation. (D) Fibronectin incubated with affinity purified antibodies. From Vuento e/ al., 1980, with permission. Scale markers indicate 200 nm (A and D), 400 nm (B and C).
Hynes, 1983) is under normal conditions perfectly soluble, the so called cellular fibronectin, synthesized by a variety of cell types (Hynes, 1985) is not. Both types of fibronectin have a similar gross structure (Fig. I), but differ by an additional type III homology region found only in cellular fibronectin (Kornblihtt et al., 1984). The differences in protein structure derive from variable splicing of the fibronectin mRNA transcribed from a single copy gene (Kornblihtt et al., 1984; Schwartzbauer et al., 1983; Paul and Hynes, 1984). In
the pericellular matrix of cultured fibroblasts (Fig. 5 and 6; Hedman et al., 1978) fibronectin and collagen co-distribute, that is, the fibrillar immunofluorescence staining patterns for fibronectin and collagen are identical images that coincide (Vahcri et al., 1978). Subsequent studies showed that in the fibroblast matrix heparan sulphate proteoglycan also co-distributes with fibronectin (Hedman P/ (/I., 1982) and that in matrix assembly fibronectin fibrillogenesis is independent of the deposition of collagen (Hedman and Vaheri, 1988). Further-
Fibronectin:
Fig. 4. Phase-contrast micrographs of filamentous a network structure. (B) Linear polymers forming
A Flexible
Image
345
structures found in solutions of fibronectin. (A) Linear polymers forming a thick “cable”. From Vuento et al., 1980, with permission. Scale marker indicates 50 pm.
more, in the fibroblast matrix, enzymatic digestion of sulfated glycosaminoglycans leaves the fibronectin fibrils intact (Hedman et al., 1984). Thus, it is possible that in the matrix assembly fibronectin has the organizing role in fibrillogenesis. It is interesting to note that exogeneously added plasma fibronectin can also be incorporated into the protein fibrils (Hayman and Ruoslahti, 1979; Oh et al., 198 1). This co-distribution of fibronectin with collagen could be explained by the fact that fibronectin has a potent binding affinity for both collagen and for glycosaminoglycans (reviewed by Hynes, 1985). Such multiple binding interactions
could immobilize fibronectin in the pericellular matrix. There are, however, other possibilities; fibronectin has been shown to possess free, though cryptic, sulfhydryl groups, and to be able to form disulphide-bonded multimers (Mosher and Johnson, 1983). In addition to the free sulfhydryls, the intramolecular disulfide bridges of fibronectin molecules may exchange to form intermolecular ones, resulting in polymerization (McKeownLong0 and Mosher, 1984; Vartio, 1986). Formation of such multimers, possibly involving subsequent stabilization by transglutaminase (Mosher and Johnson, 1983) could contribute to
M. Vuento
(‘I I//
Fig. 5. Pericellular matrix isolated from human embryonic skin libroblast tibronectin as seen in a phase contrast micrograph shows a cell-free fihrillar
the immobilization of fibronectin to fibrillar structures. These models of fibronectin fibrillogenesis all rely (except, perhaps, for the involvement of sulfhydryls) on the special environmental conditions in the pericellular matrix. Further along this line of thought. a role for the cell surface itself in
culture. network.
Immunoperoxidase staining for Scale marker indicates 20 /cm.
fibronectin fibrillogenesis has been proposed in a series of interesting papers (McKeown-Longo and Mosher, 1983. 1984, 1985; Peters and Mosher, 1987). These authors showed that fibronectin was incorporated into two distinguishable pools in the pericellular matrix of cultured fibroblasts. Binding
Fibronectin:
A Flexible
Image
Fig. 6. Scanning electron micrograph of the isolated pericellular matrix. The diameter of the matrix fibers varies greatly. The thicker fibers can be resolved to have filamentous ultrastructure (arrow). From Hedman et al., 1979, with permission. Scale marker indicates 2pm.
to pool I had the characteristics of a receptormediated process. In a recent study, McDonald et al. (1987) used proteolytically derived fibronectin fragments and monoclonal antibodies against specific determinants in the fibronectin structure to map the sites involved in the assembly of fibronectin into matrix. They found that antibodies to an M, 25,000 amino terminal domain in fibronectin inhibited the assembly. They also identified another site, located towards the C-terminus and containing the cell adhesion site (Ruoslahti and Pierschbacher, 1986) that was also involved in the matrix assembly. These authors also presented evidence against the involvement of the collagen-binding domain of fibronectin in the matrix assembly. Antibodies to the collagenbinding domain did not inhibit the assembly, nor did purified collagen-binding domains themselves.
Two intriguing findings link the above findings on matrix assembly to the discussion on fibronectin structure. It has been shown that plasma fibronectin can be induced under certain conditions to form fibrillar polymers (Fig. 7) that resemble, superficially at least, matrix-borne fibronectin fibrils. The polyamines spermine and sperminide precipitate fibronectin and the precipitates have a filamentous structure as revealed by electron microscopy (Vuento et al., 1980). The thin filaments had a width of about 2 nm. Similar structures were observed by electron microscopy in fibronectin precipitated with heparin (Jilek and Hiirmann, 1979). Microscopic (Vuento et al., 1980; Richter et al., 1985) or even macroscopic (unpublished observations) fibrils of fibronectin are sometimes formed in fibronectin solutions without added polycations or polyanions. An in-
Fig. 7. Electron micrographs of filamentous polymers of libronectin induced by spermine. Negative staining with ammonium molybdate. Fibronectin (0 5 mg/ml in 5 mM Tris-HCI. pH 7.5. was incubated with spermine (I mM) for 30 min at room temperature before staining. (A) A field showing long linear filnments. (B) Extensive network formation. From Vucnto (21~1.. 1980. with permission. Scale markcrh indicate 200 nm 34x
Fibronectin:
terpretation of the images of the thin filaments is that apparently these represent fibronectin molecules in extended conformation and are linked to each other in an end-to-end manner. Apparently, the compact structure that fibronectin has in solution, as discussed above, can be induced to unfold under special conditions. Perhaps this unfolding to an extended structure favours end-to-end association of the molecules. An interesting mechanistic model for folding/unfolding of fibronectin has been proposed by Hiirmann and Richter (1986). According to their model, a key factor in the folding of fibronectin is an uneven charge distribution along the polypeptide chain. Folding to a compact structure is facilitated by attraction of regions with opposite charge. Neutralization of critical charges by anionic or cationic ligands could thus cause an opening of the structure similar to that found to occur with heparin or with polyamines. An important factor, therefore, in the fibrillogenesis of fibronectin may lie in the intrinsic structure of fibronectin itself. Further studies are needed to clarify whether fibronectin fibrillogenesis is basically a multiple liganding process or a self assembly process or both. In the light of the present data, we see emerging a real flexible image for this multifunctional protein.
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