Jul 25, 1991 - D. D. (1990) J. Cell Biol. 111, 1685-1699. 7. Engvall, E. ... Conway, J. F. & Parry, D. A. D. (1988) Int. J. Biol. Macromol. 10,79-98. 20. Wu, C.
Extracellular Matrix in Health and Disease
In conclusion, we have provided experimental and theoretical evidence that the a-helical, coiledcoil domain of laminin is responsible for chain assembly and chain selectivity. None of the laminin chains appears to be designed for formation of homodimers, although these are common in many other fibrous proteins [ 111. Only selected heteroassociations are stable in laminin isoforms. The data on fragment E8 support the notion that dimeric laminins, composed of B1 and B2 chains only, may be stable intermediates of biosynthesis [ 101 or they may be isoforms [4], and that double-stranded, coiled-coil structures can form a triple-stranded structure by addition of an A chain. The work was supported by a grant of the Swiss National Science Foundation to J.E. 1. Timpl, K. (1989) Eur. J. Hiochern. 180,487-502 2. Beck, K., Hunter, I. & Engel, J. (1990) FASEB J. 4, 148-160 3. Martin, G. K., Timpl, It. & Kuhn, K. (1988) Adv. Protein Chem. 39, 1-50 4. Ekblom, P. (1989) FASEH J. 3 , 2 141-2 145 5. Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E. & Engvall, E. (1990) I’roc. Natl. Acad. Sci. USA. 87, 3264-3268 6. Sanes, J. R., Engvall, E., Butkowski, R. & Hunter, D. D. (1990) J. Cell Biol. 111, 1685-1699 7. Engvall, E., Earwicker, D., Haarparanta, T., Ruoslahti, E. & Sanes, J. R. (1990) Cell Regul. 1,731-740
8. Paulsson, M., Saladin, K. & Engvall, E. (1991) J. Biol. Chem. in the press 9. Hunter, D. D., Shah, V., Merlie, J. P. & Sanes, J. R. (1989) Nature (London) 338,229-234 10. Tokida, Y., Aratani. Y., Morita, A. & Kitagawa, Y. (1990) J. Biol. Chem. 265,18123-18129 11. Cohen, C. & Parry, D. A. D. (1990) Proteins: Struct., Funt. Genet. 7, 1- 15 12. Hunter, I.. Schulthess, T., Bruch, M., Beck, K. & Engel, J. (1990) Eur. J. Hiochem. 188,205-21 1 13. Turner, R. & Tjian, K. (1989) Science 243, 1689-1694 14. Doolittle, R. F. (1984) Annu. Rev. Biochem. 53, 195-229 15. Hartwig, R. & Danishefsky, K. J. (1991) J. Riol. Chem. 266,6578-6585 16. Patel, I,., Abate, C. & Curran. T. (1990) Nature (London) 347, 572-575 17. Weiss, M. A,, Ellenberger, T., Wobbe, C. R., Lee, J. P., Harrison, S. C. & Stuhl, K. (1990) Nature (London) 347, 575-578 18. Sauer, T. (1990) Nature (London) 347,s 14-5 15 19. Conway, J. F. & Parry, D. A. D. (1988) Int. J. Biol. Macromol. 10,79-98 20. Wu, C., Friedman, K. & Chung, A. E. (1988) Biochemistry 27,8780-8787 21. Paulsson, M., L)eutzmann, R.,Timpl, K., Dalzoppo, D., Odermatt, E. & Engel. J. (1985) EMBO J. 4, 309-3 16
Received 25 July 1991
~~
Cell adhesion to type-VI collagen Monique Aurnailley, Ulrich Specks and Rupert Tirnpl Max-Planck lnstitut fur Biochemie, 8033 Martinsried bei Munchen, Germany ~
~
Introduction A large variety of cells are embedded in interstitial connective tissues which provide them with a structural scaffold and can modulate their behaviour. This biological function is achieved through cellsurface receptors which transmit signals from the connective tissue components into the intracellular compartment, and specify to the cells whether to move or rest, or whether to divide or synthesize. Connective tissues have a complex composition, the main structural components being the collagens. Several of them, in particular collagen VI, have been shown to develop specific interactions with cells.
Collagen-VI composition and structure Collagen VI is composed of three different polypeptide chains [l-41; al(VI), a 2 (VI) (both of 140
kDa) and a 3 (VI) (250 kDa) which all have been completely sequenced [ S ] . They are the products of single-copy genes located on human chromosome 21 for a 1 (VI) and a2 (VI) chains and on human chromosome 2 for a 3 (VI) chain [6]. In human fibroblasts they are encoded by mRNA species of 4.2 kb for a 1 (VI), 3.5 kb for a 2 (VI) and 8.5 kb for a 3 (VI) [ 3 ] . The three polypeptides are folded together to form a collagen with unique structural features. The collagenous part represents only a fifth of its total mass and consists of a 105 nm-long triple helix [7] formed by 335-336 amino acids contributed by all three a chains [8]. This triplehelical domain contains several copies of the ArgGly-Asp (RGD) tripeptide sequence, known to be involved in cell recognition of fibronectin and other proteins [9]. Three copies are located on a 1 (VI) and
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on a 2 (VI) and five on a 3 (VI) chain. The rest of the polypeptide chains are folded into large globular domains at both ends of the triple helix. The N- and C-terminal junctions of the triple helix are rich in cysteine residues, which very likely are involved in intramolecular disulphide bridges, thus providing high thermal stability and protease resistance to collagen VI [ l , lo]. They are also involved in intermolecular bridges that stabilize collagen-VI oligomers and microfibrils and cause the notorious insolubility of these structures [ 1, 101. The globular domains of type-VI collagen are a mosaic of several types of subdomains [5, 8, 111. Similar repeats of 200 residues are located on the N- and C-terminal globular parts of a 1 (VI), a 2 (VI) and a 3 (VI). These are homologous to the A domain of von Willebrand factor, and similar domains found on some integrin a chains complement components B and C2 and a cartilage matrix protein. Other sequences have similarities with type-I11 repeats present in fibronectin and tenascin. A 50-residue sequence at the C-terminus of a 3 (VI) chain shows 40-50% identity to several serine protease inhibitors of the Kunitz type [ 51.
Collagen-VI interactions with cells: observations in situ Collagen VI is an ubiquitous component of interstitial connective tissues [ 121. By immuno-electron microscopy it has been shown to be the major component of a 100 nm-banded microfibrillar network, which shows close associations with cell surfaces both in vivo and in vitro [ 12-14]. At some anatomical sites, the location of collagen VI is particularly remarkable. This is the case in cartilage where collagen VI is concentrated in areas surrounding chondrocytes [15, 161, or in the synovium where it is observed in the extracellular matrix immediately adjacent to the lining-cell layer [ 171. In addition, it has also been shown that collagen VI is closely associated with the layer of cultured cells in a rather insoluble form [ 18, 191.
preferentially to collagen VI (Table 1) when compared with collagen IV. A further major difference between both collagens is that adhesion to collagen IV requires an intact triple-helical structure [22], while triple-helical as well as unfolded collagen VI promote cell adhesion in a similar fashion [21]. This property has allowed us to determine the cellbinding activity of isolated collagen-VI chains: a 1 (VI) is devoid of activity, while a 2 (VI) and a3 (VI) are active. The cell-adhesion activity has been localized on the part of the chains forming the collagen triple helix, which contains several copies of the RGD sequences [21]. Cell adhesion to isolated a 2 (VI) chains and a 3 (VI) chains and to unfolded collagen VI is RGDdependent; as was shown by inhibition assays using synthetic peptides such as GRGDS, which comprise the fibronectin cell-adhesion sequence [21]. However, cell adhesion to intact collagen VI is only slightly affected by the presence of such peptides [ZO, 211. Therefore it is still an open question whether or not the RGD sequences present in collagen VI are involved in cell adhesion to the native structure. These sequences are part of the collagen helix and are therefore likely to be folded into a constrained conformation which might not exist in linear synthetic RGD peptides. Alternatively, other sequences or neighbouring sequences might be necessary for cell adhesion to native collagen VI. T o address the latter possibility we have synthesized six peptides (12- or 18-mers) covering the region of a total of seven RGD sites present in the a 1 (VI), a 2 (VI) and a3 (VI) chains. None of the peptides had an appreciable inhibiting activity for cell adhesion to native collagen VI (ICso>100 pg/ml). Surprisingly, only one of these peptides was an inhibitor of cell adhesion to denatured collagen VI comparable in activity with GRGDS. This active peptide (GPRGNRGDSIDQ) was designed according to the most C-terminal sequence of the triplehelical segment of a 3 (VI) chain. The results therefore indicate a selective recognition of some, but not all, RGD sites present on denatured collagen VI.
Collagen VI as a cell-adhesion substrate Studies in vitro have corroborated the morpho-
Cellular receptors for collagen VI
logical observations by showing that collagen VI is indeed a very potent substrate for promoting cell adhesion. This was first demonstrated for baby hamster kidney cells adhering to a partially purified preparation of collagen VI [20]. More extensive studies have demonstrated that collagen VI induces adhesion of several types of normal and transformed cells [21]. Interestingly, some cells adhere
Like many other extracellular matrix molecules, integrins play a role in cell adhesion to collagen VI. This was first suggested by experiments using monoclonal antibodies developed against the surface of H T 1080 cells [23]. Two sets of antibodies inhibited cell adhesion to several collagens, including collagen VI. One set ('Class )'I immunoprecipitated bands at 147 and 125 kDa (130 and 135 kDa
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Table I
Cell adhesion to collagen VI: comparison with other substrates Multiwell plates (tissue-culture brand, Costar, Cambridge, MA) were coated with solutions of ftbronecttn, collagen IV or collagen VI at concentrations ranging from 0.5-40 pgl ml and then blocked with I % (w/v) BSA Cell suspension were seeded onto the coated surfaces and incubated for 30 rnin Extent of cell adhesion was determined using a colortmetric assay as previously described [21] Maximal adhesion on fibronectin was taken as 100% and maximal adhesion to collagens IV and VI were calculated as % adhesion to fi bronectin
84 5
Substrates Cell lines Pam 21 2, mouse epithelial Larnm kidney
F9. mouse teratocarcinoma Pys-2, rat parietal yolk sac
A375, human melanoma T47D, human mammary carcinoma Mouse skin fibroblasts NR6, transformed mouse fibroblasts
R N 22, rat schwannoma Rugli, rat glioma We, transformed human epithelial B 16F 10, mouse melanoma
SV 40 transformed human fibroblasts HT 1080, human fibrosarcoma A43 I , human epidermoid Human fibroblasts
after reduction) and the other set (‘Class 11’) immunoprecipitated bands at 145 and 125 kDa (140 and 135 kDa after reduction), which comprise sizes usually observed for a and p integrin subunits [23]. It was subsequently shown that these receptors correspond to the a 3 p l and a2p1 integrins respectively [241. The integrin recognition of collagen VI was confirmed by using polyclonal antibodies raised against the fibronectin receptor (a5p1). This antiserum has a strong titer against the /?1 subunit [25], and prevented cell attachment to intact collagen VI of human melanoma cells A375 (Fig. l), human fibrosarcoma cells HT 1080 and normal human fibroblasts (not shown). Affinity chromatography of human placenta Extracts on intact and denatured collagen VI leads n both cases to the retention of material which can ‘3e eluted with 10 msi-EDTA (Fig. 2). However, SDS/PAGE analysis demonstrated that the material ,:luted from the native-collagen-VI column was different from that eluted from the denatured-collagenVI column (Fig. 2, insert). The sizes of the major
Fibronectin Collagen IV Collagen VI
100 100 100 I00 I00 I00
100 100 I00 I00 100 I00 I00 I00 I00 100
0 18 23 8 53 260 90 78 100 100 100 82 I07 97 I06 98
0 6 7 13 29 12 49 35 82 89 100 77 97 93 98 95
bands eluted from the native collagen VI (170 and 125 kDa) indicate that they originate from a l p 1 integrin. This suggests that at least three different integrins bind to triple-helical collagen VI. The material eluted from the denatured-collagen-VI column was more complex (Fig. 2, insert lane 2), particularly as no band could be seen in the a1 subunit region (170 kDa). These results demonstrate that the cellular receptors involved in the recognition of native collagen VI are different from those recognizing denatured collagen VI, in agreement with their different sensitivities for inhibition by RGD peptides. In addition to integrins, lowmolecular-mass components (38, 42 and 45 kDa), membrane-associated but not intercalated into the plasma membrane, could also play a role in cellular interactions with collagen VI, as they were shown to bind to collagen-VI-affinity columns [23]. Although it is now well documented that collagen VI interacts with cells and can induce their adhesion and spreading in vitro, it is not known yet whether other cellular activities such as proliferation
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Fig. I Inhibition of cell adhesion to collagen VI by anti-integrin antibodies
846
Plastic-tissue-culturesurfaces were coated with native collagen VI and blocked with I % BSA as previously described [21] Human melanoma cells, A375, were seeded onto the coated surface in the absence (0)or in the presence ( b ) of polyclonal antibodies raised against the fibronectin receptor which showed a strong titer against the P I subunit [25] After 30 min incubation period, the unattached cells were removed by washing and the attached cells were fixed with 70% (v/v) ethanol Photographs of the cells were taken using a phasecontrast microscope The antibodies were kindly given by G Tarone, Torino, Italy, and used at a I 100 dilution
or differentiation can be modified by collagen VI. A role for collagen VI in gastrulation has been suggested [26]. Other data have shown that cellular contacts with the extracellular matrix can influence collagen VI synthesis [27]. The expression of the collagen-VI a chains depends on cell density and, in contrast to other interstitial collagens, is not down-regulated when cells are cultivated within collagen gels. Since these situations are correlated to a low proliferation rate of the cells, it indicates that resting cells continue to produce high levels of collagen VI as they need it for their anchoring to the surrounding matrix. U.S. was a recipient of a research fellowship from the Mayo Foundation, Rochester, MN, U.S.A. 1. Jander, K., Kauterberg, J & Glanville, K. W . (1983) Eur. J. Biochem. 133.39-46 2. Trueb, H. & Winterhalter, K. (1986) EMHO J. 3, 281 5-2819 3. Chu, M.-I+ Mann, K.. Deutzmann, K., I’ribulaConway. D., Hsu-Chen, C. C.. Bernard, M. I-’. & Timpl, K. (1987) Fur. J. Hiochem. 168,300-317 Fig. 2
Affinity chromatography of human placenta extracts on native and denatured human collagen VI Native and denatured collagen VI were coupled t o CNBr-activated Sepharose. Human placenta extracts were prepared according to [28] with minor modifications [29]. The extracts were chromatographed on the affinity columns equilibrated in 50 mM-Tris/HCI buffer, pH 7.4 containing 150 mM-NaCt and 0.1% reduced Triton X-100. After loading, the columns were washed with 300 mM-NaCt and then eluted with 10 mM-EDTA, all in the equilibration buffer. Absorbance was monitored at 280 nm and peak fractions were analysed by SDS/PAGE. The gels were silver stained. The insert shows electrophoretic analysis of the peak fractions eluted with 10 mM-EDTA from the native- (lane I ) and denatured-collagen-VI(lane 2) columns
0.10
kDa
+ Native ( I ) 0.08 -
+ Unfolded (2)
A&
200 150-
0
h
0.060
oa
cy, al
2
0.04-
f? 51
9
0.02 -
0.00
!
10
1
I
1
20
30
40
Fraction numbers
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4. Colornbatti, A,. Bonaldo, P.. Ainger, K., Hressan, G. M. & Volpin. D. (1987) J. Biol. Chem. 262, 14459-14460 5. Chu, M.-I+ Zhang, R.-Z., Pan, T.-C., Stokes, D., Conway, D., Kuo. H.-J., Glanville, R. W.. Mayer. U., Mann, K.. Deutzrnann, R. & Timpl. R. (1990) EMHO J. 9,385-393 6. Weil, D., Mattei, M.-G., Passage, E., Van Cong, N., Pribula-Conway, D. Mann, K., Deutzmann. R., Timpl, R. & Chu, M.4,. (1988) Am. J. Human Genet. 42.435-445 7. Furthmayr. H., Wiedernann. H., Timpl. K., Odermatt. E. & Engel, J. (1983) Hiochern.J. 211,303-31 1 8. Chu, M.-I,., Conway, D., Pan. T.-C., Haldwin, C., Mann. K.. Deutzmann, R. & Tirnpl, K. (1988) J. Biol. Chem. 263.18601-18606 9. Ruoslahti. E. & I’ierschbacher, M. D. (1987) Science 238.43 1-437 10. Odermatt, E., Risteli, J., van Delden. V. & Timpl, K. (1983) Hiochern. J. 21 1,295-302 11. Bonaldo, P. & Colombatti, A. (1989) J. Hiol. Chern. 264,20235-20239 12. von der Mark, H., Aumailley. M., Wick, G., Fleischmajer, R. & Timpl, R. (1984) Eur. J. Hiochem. 142. 493-502 13. Hruns, R. R., Press. W.. Engvall. E., Timpl, K. & Gross, J. (1986) J. Cell Biol. 103,393-404 14. Bruns, R. R. (1984)J. Ultrastr. Kes. 89, 136-145 15. Keene, D. R., Engvall, E. & Glanville. K. W. (1988) J. Cell Hiol. 107, 1995-2006 16. Poole, C. A.. Ayad. S. & Schofield, J. R. (1988) J. Cell Sci. 90,635-643 17. Okada. Y.. Naka. K., Minarnoto. T.. Ueda. Y., Oda, Y., Nakanishi, I. & Timpl. R. (1990) Lab. Invest. 63,
647-656 18. Carter, W. G. (1984)J. Cell Biol. 99, 105-1 14 19. Heckmann, M., Aurnailley, M., Hatamochi, A., Chu, M.-I,., Timpl, R. & Krieg, T. (1989) Eur. J. Hiochem. 182,719-726 20. Carter, W. G. (1982) J. Hiol. Chem. 257,3249-3257 21. Aurnailley, M., Mann. K., von der Mark, H. & Timpl, R. (1989) Exp. Cell Res. 181. 463-474 22. Aumailley, M. & Timpl, K. (1986) J. Cell Biol. 103, 1569-1 575 23. Wayner. E. A. & Carter, W. G (1987) J. Cell Hiol. 105. 1873-1884 24. Takada. Y.. Wayner, E. A., Carter, W. G. & Hernler, M. E. (1988) J. Cell Hiochern. 37, 385-393 25. Conforti, G., Zanetti, A., Colella, S., Abbadini, M., Marchisio, 1’. C., I’ytela. R., Giancotti. P., Tarone, G., Languino, I,. K. & Dejana, E. (1989) Blood 73. 1576- 1585 2h. Otte, A. P., Roy. L)., Siemerinck, M., Koster, C. H.. Hochstenbach, F., Timrnermans, A. & Lhrston, A. J. ( 1990)J. Cell Biol. 111, 27 1-278 27. Hatamochi, A,, Aumailley. M., Mauch, C., Chu, M.-L, Tirnpl, R. & Krieg, T. (1989) J. Hiol. Chern. 264, 3494-3499 28. I’ytela, K., Pierschbacher, M. D., Argraves, S., Suzuki. S. & Ruoslahti, E. (1987) Methods Enzymol. 144, 475-499 29. Vandenberg, P.. Kern, A., Kies, A., Luckenbill-Edds, I,., Mann, K. & Kuhn. K. (1991) J. Cell Hiol. 113, 1475-1483
Received 25 July 1991
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