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Ed-a sequence containing fibronectin in human amniotic fluid and amnion epithelial cells
Inr. J. Biochem. Vol. 21, No. 3, pp. 307-311, 1989 Printed in Great Britain. All rights reserved
Copyright 0
0020-7 I 1X/89 63.00 + 0.00 1989 Pergamon Press plc
ED-A SEQUENCE CONTAINING FIBRONECTIN IN HUMAN AMNIOTIC FLUID AND AMNION EPITHELIAL CELLS TAPIO VARTIO,’ HARRIETVON KCSKULL*and ISMOVIRTANEN”~ ‘Department of Pathology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland [ Tei. 90-43-46 I] *Departments I and II of Obstetrics and Gynaecology, Helsinki University Central Hospital, 00290 Helsinki, Finland 3Department of Anatomy, University of Helsinki, Siltavuorenpenger 20, 00170 Helsinki, Finland (Received 1I July 1988)
Abstract-i.
Human amniotic fluid fibronectin {aFn) was studied by using a monoclonal antibody 52DHl (DH) that recognizes the extra domain (ED-A) sequence of cellular Fn (cFn). 2. In immunoblotting the DH antibody reacted with a sharp polypeptide band at the top of the bulk of the diffuse aFn. Another monoclonal antibodv 52BF12 (BFj against the cell binding site of Fn. recognized the whole aFn. 3. The ED-A sequence containing cFn (EcFn) formed a constant proportion in aFns from all amniotic fluid preparations studied. 4. In amniotic membranes the DH antibody revealed bright subepithelial immunofluorescence. 5. Also isolated and cultured human amnion epithelial cells were strongly positive in immunofluorescence and secreted EcFn into the culture medium as revealed by immunoblotting. 6. The results indicate that aFn is a composition of at least two different Fn subtypes of which the EcFn most probably originates from amnion epithelial cells. I
_
~A~RIA~ Fibronectins (Fns) are adhesive glycoproteins found in connective tissue matrix and in soluble form in body fluids (Vartio and Vaheri, 1983; Furcht, 1983; Yamada et al., 1985). The ubiquitous distribution of Fns suggests an existence of several molecular forms of the protein. These variants are at least in part due to muttiple mRNAs produced by alternative splicing of the primary transcript encoded by a single gene [for references, see Hynes (1985)]. Depending on the splicing, different Fn polypeptides containing variable domain compositions may be generated. Among the alternative domains there is an extra domain (ED-A) sequence that is specific for at least certain types of cFns (Kornblihtt et al., 1984, 1985: Vibe-Pedersen et al., 1984) designated as EcFn in the present study. Human aFn has been thought to represent a distinct type of Fns. It has a little different electro-
phoretic mobility than plasma Fn (pFn) and under reducing conditions forms a diffuse band while pFn gives a sharp doublet of polypeptide bands (Ruoslahti et al., 1982). The major differences between aFn and pFn have been assigned to carbohydrate structures which differ quite considerably (Balian et al., 1979; Pande et al., 1981; Ruoslahti et al., 1981; Krusius et al., 1985). We now show that EcFn is a regular constituent of aFn that hence is a complex of at least two types of Fn which differ at ~lypeptide level. Immunofluorescence studies suggested that the EcFn component of aFn is most probably produced by amnion epithelial cells.
AND METHOD
Human amniotic fluids were obtained at 15th or 16th week of normal gestations. Fns were purified from amniotic fluid and human plasma by gelatin-Sepharose (Engvall and Ruoslahti, 1977). Fibroblast or human amnion epithelial cell cFns were isolated by the same method from serum free spent growth medium of cells to avoid copurification of serum Fn protein concentrations were measured according to Lowry et ul. (1951). Eiectrophoresis and ~~~nobiotttng Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) was performed by using 6% vertical slab gels. The samples were run nonreduced or reduced with 10% 2-mercaptoethanol. After electroDhoresis. the gels were either protein stained or immuioblotted (Towbin el ai., 1979) bv transferring SDS-PAGE senarated polypeptides electr~phbretically oito nitrocellulos; sheets (type I HAWP filter, Milli~re, Bedford, Mass., U.S.A.). Monoclonai antibodies (culture su~ma~nts), 52BF12 (BF), against the cell-binding site of Fn, and 52DHl (DH), against the ED-A sequence of EcFn, were as described in Vartio et al. (1987). Immunoreactions were detected by peroxidase-coupled sheep anti-mouse IgG antiserum (Dakopatts, Glostrup, Denmark). Cell cultures and tissue samples Human embryonic skin tibroblasts (F84-75) were established from skin biopsies. Human amnion epithelial cells were prepared as described (Regauer ef al., 1985). The cells were grown in RPM1 I640 medium, supplemented with 10% fetal calf serum to confluency. For purification of the cFns, the cultures were changed in serum-free RPM1 1640 medium.
307
TAPIO VARTIO et al.
308
For frozen sectioning, pieces of amniotic membrane were frozen ‘in melting Freon, cooled in liquid nitrogen. Indirect immuno~uorescence microscopy Human amnion epithelial cells grown on glass coverslips and cytocentrifuge preparations of freshly isolated amnion epithelial cells were fixed in methanol and frozen sections of amniotic membrane in acetone, both cooled to -20°C for 10 min. The cells and frozen sections were reacted with the monoclonal antibodies for 30 min. After washes, they were reacted with FITC-coupled goat anti-mouse 1%; antiserum (Cannel, Cooner Biochemical. Malvem. Pa. U.S.A.). A iei&*Axiophdt microscope equipped with filters for FITCfluorescence, was used.
RESULTS
Analysis of purtjied Fns
PFn, aFn and fibroblast cFn gave sharp nonreduced polypeptide bands in SDS-PAGE (Fig. 1A). Reduced polypeptides migrated to the position of about M, 220,000-260,000. PFn formed a doublet of polypeptides while aFn appeared as a broad diffuse band having its upper part at about the level of fibroblast cFn (Fig. 1B). Both nonreduced and reduced pFns migrated slightly faster than aFn and fibroblast cFn. In immunoblotting, the BF antibody recognized all these Fns (Fig. 2A), while the DH antibody failed to react with pFn (Fig. 2B). Furthermore, the DH antibody recognized only the top region of the reduced diffuse aFn band at about the same level as fibroblast cFn while the BF antibody reacted with the whole polypeptide band (Fig. 2C). The DH antibody detected a similar sharp polypeptide band in all aFn (n = 22) samples studied. Also in the nonreduced samples the DH antibody seemed to react with only the upper part of the aFn polypeptide band (cf. lanes 2 in Fig. 2A,B). Immunofuorescence
studies
with the DH antibody a strong reactivity was seen, especially beneath the epithelial cell layer of amniotic membranes (Fig. 3a,b). In frozen section it was difficult to judge whether the epithelial cell layer would also be distinctly positive. However, immunostaining of both cytocentrifuged freshly isolated (Fig. 3c,d) as well as cultured amnion epithelial cells (Fig. 3e,f) revealed bright intra- and extracellular fluorescence with the DH antibody. The staining results were identical with the BF antibody (not shown). Isolated cFn from culture medium of amnion epithelial cells formed a distinct polypeptide band comigrating with the top region of aFn (Fig. 4A). Figures 4B and 4C show the immunoblottings of similar samples with the BF and DH antibodies, respectively. The amnion epithelial cell cFn comigrated with the DH-positive part of aFn. In immunostaining
DISCU!SSlON
As stated in the Introduction, the major differences between pFn and aFn have been assigned to variable carbohydrate compositions of these two forms of the protein. However, due to multiple mRNAs Fns from various sources differ also at the polypeptide level and
in certain situations nonidentical Fn polypeptides may coexist. In the present study we have used a monoclonal antibody against the ED-A sequence of EcFn. This antibody, that is very similar to the previously characterized IST-9 antibody (Borsi et al., 1987; Carnemolla et al., 1987), revealed that human aFn is composed of at least two different polypeptide forms of Fns: one contains the ED-A sequence and the other one lacks it. The diffuse band of aFn may, however, not be just a sum of pFn and EcFn which both form sharp polypeptide bands. Thus, it is possible that different carbohydrate compositions or other Fn variants, not detected so far, account for further heterogeneity in aFn. On the other hand, the fact that the latter is composed of at least two forms of Fn may in part explain the carbohydrate differences detected between aFn and pFn. In SDS-PAGE the EcFn of amniotic fluid had very similar mobility as that from fibroblasts suggesting a close relationship between these two forms of Fn. The EcFn seems to be a regular constituent of aFn since the immunoperoxidase reactions were constantly seen at about the same strength in the samples analysed. All the amnion fluids used in this study were from normal pregnancies. It remains, however, to be seen whether the ratios of the components of aFn may change in some pathologic conditions. Immunofluorescence staining of the frozen sections of amniotic membrane showed bright subepithelial staining with the DH antibody, while the epithelial layer itself was virtually negative. However, in spite of the difficulty of verifying EcFn in the epithelial cells of the frozen sections they may contribute to its presence in the subepithelial tissue. This assumption is supported by the fact that immunofluorescence staining of both freshly isolated and cultured amnion epithelial cells showed abundant staining with the DH antibody. Furthermore, cultured amnion epithelial cells also secreted the EcFn. These results suggest that the EcFn in aFn preparations originates, if not exclusively, at least partially from the epithelial cells lining the amniotic membrane. The negative staining results on amnion epithelial cells in frozen sections may be due to the general difficulty of detecting intracellular Fn in tissue sections, thus, e.g. hepatocytes, known to produce pFn (Tamkun and Hynes, 1983), do not contain intracellular positivity in immunostaining (Stenman and Vaheri, 1978; Hahn et al., 1980). The present results indicate that at least in certain situations soluble Fn is a mixture of variable amounts of different Fn subtypes. It is evident that local environmental conditions have a great influence in the composition of Fn subtypes in a given situation. Thus, it is expected that also in other body fluids or tissues different Fn compositions will be found depending on the Fn-producing cell types involved. Acknowledgements-The skilful technical assistance of MS Tuija JBrvinen, MS Pipsa Kaipainen, MS Saija Roine, MS Anna Maria Siivonen and MS Raili Taavela is kindly acknowledged. This study was supported by a research contract with the Academy of Finland and by grants from the Sigrid Juselius Foundation, the Paul0 Foundation, the Rinnekoti Foundation and the Finnish Cancer Foundation.
Fig. 1. SDS-PAGE of Fns. Fns (46 pg) isolated by gelatinSepharose from human plasma, amniotic fluid and fibroblast culture medium were run in 6% SDS-PAGE under nonreducing (A) and reducing (B) conditions and the gels were protein stained. Lanes: 1, pFn; 2, aFn; 3, fibroblast cFn. dFn = dimeric Fns; mFn = monomeric Fns.
mFn
aFn cFn Fig. 2. Immunoblotting of Fns. Fns (4-6 pg) isolated by gelatin-Sepharose from plasma, amniotic fluid and fibroblast culture medium were run in 6% SDS-PAGE under nonreducing conditions and immunoblotted with the BF (A) or DH (B) antibodies. Lanes: 1, pFn; 2, aFn; 3, fibroblast cFn. (C) aFn (IOpg, lanes I-3) and fibroblast cFn (8 lug, lane 4) were run under reducing conditions and the gels were either protein stained (lane 1) or immunoblotted with the BF (lane 2) or DH (lanes 3 and 4) antibodies. Note tire immunoperoxidase reaction at the top region of the aFn band in lane 3. st = protein stained lane; dFn = dimeric Fns; mFn = monomeric Fns. 309
Fig. 3. I~unol~~tion of EcFn in amniotic membr~es and epithelial cells with the DH antibody. In frozen sections of amniotic membrane (1 lth week of pregnancy) (a) a brigbt positivity was seen especially beneath the epithelial cell layer (E). Cytoeentrifuge preparations of freshly isolated amnion epithelial ceils show bright cytoplasmic DH-positivity (c), seen also in cultured amnion epithelial cells (e) that contain also spotty pericellular positivity. b. d and f show the corresponding phase contrast views. a, b x 200; c-f, x 500.
mF
Fig. 4. Comparison of aFn and amnion epithelial cell cFn. APn (5 pg, lanes 1) and amnion epithelial cell cFn (3 ~8, lanes 2) were isolated by the ~latin-Sharon method, run under reducing conditions in 6% SD!&PACE and either protein stained (A) or ~munobiott~ with the BF (B) or DH (C) antibodies. mFn = monomeric Fns. 310
Amniotic fluid fibronectin REFERENCES Balian G., Crouch E., Click E. M., Carter W. G. and Bomstein P. (1979) Comparison of the structures of human fibronectin and plasma cold insoluble globulin. J. supramolec. Struct. 12, 505-516. Borsi L., Camemolla B., Castellani P., Rosellini C., Vecchio D., Allemanni G., Chang S. E., Taylor-Pa~dimitriou J., Pande H. and Zardi L. (1987) Monoclonal antibodies in the analysis of fibronectin isofonns generated by alternative splicing of mRNA precursors in normal and transformed human cells. J. Cell Biol. 104, 595-600. Carnemolla B., Borsi L., Zardi L., Owens R. J. and Baralle F. E. (19871Localization of the cellular gbronectin snecific epitope r&ognized by the monoclonal antibody *IST-9 using fusion proteins expressed in E. co/i. FEBS Lett. 255, 269-273. Engvall E. and Ruoslahti E. (1977) Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Inr. J. Cancer 20, l-5. Furcht L. T. (1983) Structure and function of the adhesive glycoprotein fibronectin. Mod. Cell Biol. 1, 53-l 17. Hahn E., Wick G., Pencev D. and Timpl R. (1980) Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin and fibronectin Gut 21, 63-71. Hynes R. (1985) Molecular biology of libronectin. A. Rev. Ceii Biol. 1, 67-90. Komblihtt A. R., Vibe-Pedersen 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 JI 3, 221-226. Kornblihtt A. R., Umezawa K., Vibe-Pedersen K. and Baralle F. E. (1985) Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO JI 4, 1755-1759. Krusius T., Fukuda M., Dell A. and Ruoslahti E. (1985) Structure of the carbohydrate units of human amniotic fluid fibronectin. J. biol. Chem. 260, 41 IO-41 16. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&683. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall
311
R. J. (1951) Protein measurement with the Folin phenol reagent. J. bioi. Chem. 193,265-275. Pande H., Corkill J., Sailor R. and Shively E. (1981) COmparative structural studies of human plasma and amniotic fluid fibronectins. Biochem. biophys. Res. Commun. 101, 265-272. Regauer S., Franke W. W. and Virtanen I. (1985) Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium. J. Ceil Biol. loo, 997-1009. Ruoslahti E., Engvall E., Hayman E. G. and Spiro R. (1981) Comparative studies on amniotic fluid and plasma fibronectins. Biochem. J. 193, 295-299. Ruoslahti E., Hayman E. G., Pierschbacher M. and Engvall E. (1982) Fibronectin: purification, immunochemical prop erties, and biological activation. Meth. Enzym. 82,803-83 1. Stenman S. and Vaheri A. (1978) Distribution of major connective tissue protein, fibronectin, in normal human tissues. J. exp. Med. 147, 1054-1066. Tamkun J. W. and Hynes R. 0. (1983) Plasma fibronectin is synthetized and secreted by hepatocytes. J. biof. Chem. 258,4641-4647. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Froc. natn. Acod. Sci. U.S.A. 76, 435&4354. Vartio T. and Vaheri A. (1983) Fibronectin: chains of domains with diversified functions. Trends biochem. Sci. 8, 442-444. Vartio T., Laitinen L., NLrvlnen O., Cutolo M., Thornell L.-E., Zardi L. and Virtanen I. (1987) Differential expression of the ED sequence confining form of cellular fibronectin in embryonic and adult human tissues. J. CeU Sci. 88,419-430. Vibe-Pedersen K., Kornblihtt A. R. and Baralle F. E. (1984) Expression of a human a-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing. EMBQ Jl 2511-2516. Yamada K., Akiyama S. K., Hasegawa T., Hasegawa E., Humphries M. J., Kennedy D. W., Nagata K., Urushihara H., Olden K. and Chen W.-T. (1985) Recent advances in research on fibronectin and other cell attachment proteins. J. cell. Biochem. 23, 77-98.
Copyright 0
0020-7 I 1X/89 63.00 + 0.00 1989 Pergamon Press plc
ED-A SEQUENCE CONTAINING FIBRONECTIN IN HUMAN AMNIOTIC FLUID AND AMNION EPITHELIAL CELLS TAPIO VARTIO,’ HARRIETVON KCSKULL*and ISMOVIRTANEN”~ ‘Department of Pathology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland [ Tei. 90-43-46 I] *Departments I and II of Obstetrics and Gynaecology, Helsinki University Central Hospital, 00290 Helsinki, Finland 3Department of Anatomy, University of Helsinki, Siltavuorenpenger 20, 00170 Helsinki, Finland (Received 1I July 1988)
Abstract-i.
Human amniotic fluid fibronectin {aFn) was studied by using a monoclonal antibody 52DHl (DH) that recognizes the extra domain (ED-A) sequence of cellular Fn (cFn). 2. In immunoblotting the DH antibody reacted with a sharp polypeptide band at the top of the bulk of the diffuse aFn. Another monoclonal antibodv 52BF12 (BFj against the cell binding site of Fn. recognized the whole aFn. 3. The ED-A sequence containing cFn (EcFn) formed a constant proportion in aFns from all amniotic fluid preparations studied. 4. In amniotic membranes the DH antibody revealed bright subepithelial immunofluorescence. 5. Also isolated and cultured human amnion epithelial cells were strongly positive in immunofluorescence and secreted EcFn into the culture medium as revealed by immunoblotting. 6. The results indicate that aFn is a composition of at least two different Fn subtypes of which the EcFn most probably originates from amnion epithelial cells. I
_
~A~RIA~ Fibronectins (Fns) are adhesive glycoproteins found in connective tissue matrix and in soluble form in body fluids (Vartio and Vaheri, 1983; Furcht, 1983; Yamada et al., 1985). The ubiquitous distribution of Fns suggests an existence of several molecular forms of the protein. These variants are at least in part due to muttiple mRNAs produced by alternative splicing of the primary transcript encoded by a single gene [for references, see Hynes (1985)]. Depending on the splicing, different Fn polypeptides containing variable domain compositions may be generated. Among the alternative domains there is an extra domain (ED-A) sequence that is specific for at least certain types of cFns (Kornblihtt et al., 1984, 1985: Vibe-Pedersen et al., 1984) designated as EcFn in the present study. Human aFn has been thought to represent a distinct type of Fns. It has a little different electro-
phoretic mobility than plasma Fn (pFn) and under reducing conditions forms a diffuse band while pFn gives a sharp doublet of polypeptide bands (Ruoslahti et al., 1982). The major differences between aFn and pFn have been assigned to carbohydrate structures which differ quite considerably (Balian et al., 1979; Pande et al., 1981; Ruoslahti et al., 1981; Krusius et al., 1985). We now show that EcFn is a regular constituent of aFn that hence is a complex of at least two types of Fn which differ at ~lypeptide level. Immunofluorescence studies suggested that the EcFn component of aFn is most probably produced by amnion epithelial cells.
AND METHOD
Human amniotic fluids were obtained at 15th or 16th week of normal gestations. Fns were purified from amniotic fluid and human plasma by gelatin-Sepharose (Engvall and Ruoslahti, 1977). Fibroblast or human amnion epithelial cell cFns were isolated by the same method from serum free spent growth medium of cells to avoid copurification of serum Fn protein concentrations were measured according to Lowry et ul. (1951). Eiectrophoresis and ~~~nobiotttng Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) was performed by using 6% vertical slab gels. The samples were run nonreduced or reduced with 10% 2-mercaptoethanol. After electroDhoresis. the gels were either protein stained or immuioblotted (Towbin el ai., 1979) bv transferring SDS-PAGE senarated polypeptides electr~phbretically oito nitrocellulos; sheets (type I HAWP filter, Milli~re, Bedford, Mass., U.S.A.). Monoclonai antibodies (culture su~ma~nts), 52BF12 (BF), against the cell-binding site of Fn, and 52DHl (DH), against the ED-A sequence of EcFn, were as described in Vartio et al. (1987). Immunoreactions were detected by peroxidase-coupled sheep anti-mouse IgG antiserum (Dakopatts, Glostrup, Denmark). Cell cultures and tissue samples Human embryonic skin tibroblasts (F84-75) were established from skin biopsies. Human amnion epithelial cells were prepared as described (Regauer ef al., 1985). The cells were grown in RPM1 I640 medium, supplemented with 10% fetal calf serum to confluency. For purification of the cFns, the cultures were changed in serum-free RPM1 1640 medium.
307
TAPIO VARTIO et al.
308
For frozen sectioning, pieces of amniotic membrane were frozen ‘in melting Freon, cooled in liquid nitrogen. Indirect immuno~uorescence microscopy Human amnion epithelial cells grown on glass coverslips and cytocentrifuge preparations of freshly isolated amnion epithelial cells were fixed in methanol and frozen sections of amniotic membrane in acetone, both cooled to -20°C for 10 min. The cells and frozen sections were reacted with the monoclonal antibodies for 30 min. After washes, they were reacted with FITC-coupled goat anti-mouse 1%; antiserum (Cannel, Cooner Biochemical. Malvem. Pa. U.S.A.). A iei&*Axiophdt microscope equipped with filters for FITCfluorescence, was used.
RESULTS
Analysis of purtjied Fns
PFn, aFn and fibroblast cFn gave sharp nonreduced polypeptide bands in SDS-PAGE (Fig. 1A). Reduced polypeptides migrated to the position of about M, 220,000-260,000. PFn formed a doublet of polypeptides while aFn appeared as a broad diffuse band having its upper part at about the level of fibroblast cFn (Fig. 1B). Both nonreduced and reduced pFns migrated slightly faster than aFn and fibroblast cFn. In immunoblotting, the BF antibody recognized all these Fns (Fig. 2A), while the DH antibody failed to react with pFn (Fig. 2B). Furthermore, the DH antibody recognized only the top region of the reduced diffuse aFn band at about the same level as fibroblast cFn while the BF antibody reacted with the whole polypeptide band (Fig. 2C). The DH antibody detected a similar sharp polypeptide band in all aFn (n = 22) samples studied. Also in the nonreduced samples the DH antibody seemed to react with only the upper part of the aFn polypeptide band (cf. lanes 2 in Fig. 2A,B). Immunofuorescence
studies
with the DH antibody a strong reactivity was seen, especially beneath the epithelial cell layer of amniotic membranes (Fig. 3a,b). In frozen section it was difficult to judge whether the epithelial cell layer would also be distinctly positive. However, immunostaining of both cytocentrifuged freshly isolated (Fig. 3c,d) as well as cultured amnion epithelial cells (Fig. 3e,f) revealed bright intra- and extracellular fluorescence with the DH antibody. The staining results were identical with the BF antibody (not shown). Isolated cFn from culture medium of amnion epithelial cells formed a distinct polypeptide band comigrating with the top region of aFn (Fig. 4A). Figures 4B and 4C show the immunoblottings of similar samples with the BF and DH antibodies, respectively. The amnion epithelial cell cFn comigrated with the DH-positive part of aFn. In immunostaining
DISCU!SSlON
As stated in the Introduction, the major differences between pFn and aFn have been assigned to variable carbohydrate compositions of these two forms of the protein. However, due to multiple mRNAs Fns from various sources differ also at the polypeptide level and
in certain situations nonidentical Fn polypeptides may coexist. In the present study we have used a monoclonal antibody against the ED-A sequence of EcFn. This antibody, that is very similar to the previously characterized IST-9 antibody (Borsi et al., 1987; Carnemolla et al., 1987), revealed that human aFn is composed of at least two different polypeptide forms of Fns: one contains the ED-A sequence and the other one lacks it. The diffuse band of aFn may, however, not be just a sum of pFn and EcFn which both form sharp polypeptide bands. Thus, it is possible that different carbohydrate compositions or other Fn variants, not detected so far, account for further heterogeneity in aFn. On the other hand, the fact that the latter is composed of at least two forms of Fn may in part explain the carbohydrate differences detected between aFn and pFn. In SDS-PAGE the EcFn of amniotic fluid had very similar mobility as that from fibroblasts suggesting a close relationship between these two forms of Fn. The EcFn seems to be a regular constituent of aFn since the immunoperoxidase reactions were constantly seen at about the same strength in the samples analysed. All the amnion fluids used in this study were from normal pregnancies. It remains, however, to be seen whether the ratios of the components of aFn may change in some pathologic conditions. Immunofluorescence staining of the frozen sections of amniotic membrane showed bright subepithelial staining with the DH antibody, while the epithelial layer itself was virtually negative. However, in spite of the difficulty of verifying EcFn in the epithelial cells of the frozen sections they may contribute to its presence in the subepithelial tissue. This assumption is supported by the fact that immunofluorescence staining of both freshly isolated and cultured amnion epithelial cells showed abundant staining with the DH antibody. Furthermore, cultured amnion epithelial cells also secreted the EcFn. These results suggest that the EcFn in aFn preparations originates, if not exclusively, at least partially from the epithelial cells lining the amniotic membrane. The negative staining results on amnion epithelial cells in frozen sections may be due to the general difficulty of detecting intracellular Fn in tissue sections, thus, e.g. hepatocytes, known to produce pFn (Tamkun and Hynes, 1983), do not contain intracellular positivity in immunostaining (Stenman and Vaheri, 1978; Hahn et al., 1980). The present results indicate that at least in certain situations soluble Fn is a mixture of variable amounts of different Fn subtypes. It is evident that local environmental conditions have a great influence in the composition of Fn subtypes in a given situation. Thus, it is expected that also in other body fluids or tissues different Fn compositions will be found depending on the Fn-producing cell types involved. Acknowledgements-The skilful technical assistance of MS Tuija JBrvinen, MS Pipsa Kaipainen, MS Saija Roine, MS Anna Maria Siivonen and MS Raili Taavela is kindly acknowledged. This study was supported by a research contract with the Academy of Finland and by grants from the Sigrid Juselius Foundation, the Paul0 Foundation, the Rinnekoti Foundation and the Finnish Cancer Foundation.
Fig. 1. SDS-PAGE of Fns. Fns (46 pg) isolated by gelatinSepharose from human plasma, amniotic fluid and fibroblast culture medium were run in 6% SDS-PAGE under nonreducing (A) and reducing (B) conditions and the gels were protein stained. Lanes: 1, pFn; 2, aFn; 3, fibroblast cFn. dFn = dimeric Fns; mFn = monomeric Fns.
mFn
aFn cFn Fig. 2. Immunoblotting of Fns. Fns (4-6 pg) isolated by gelatin-Sepharose from plasma, amniotic fluid and fibroblast culture medium were run in 6% SDS-PAGE under nonreducing conditions and immunoblotted with the BF (A) or DH (B) antibodies. Lanes: 1, pFn; 2, aFn; 3, fibroblast cFn. (C) aFn (IOpg, lanes I-3) and fibroblast cFn (8 lug, lane 4) were run under reducing conditions and the gels were either protein stained (lane 1) or immunoblotted with the BF (lane 2) or DH (lanes 3 and 4) antibodies. Note tire immunoperoxidase reaction at the top region of the aFn band in lane 3. st = protein stained lane; dFn = dimeric Fns; mFn = monomeric Fns. 309
Fig. 3. I~unol~~tion of EcFn in amniotic membr~es and epithelial cells with the DH antibody. In frozen sections of amniotic membrane (1 lth week of pregnancy) (a) a brigbt positivity was seen especially beneath the epithelial cell layer (E). Cytoeentrifuge preparations of freshly isolated amnion epithelial ceils show bright cytoplasmic DH-positivity (c), seen also in cultured amnion epithelial cells (e) that contain also spotty pericellular positivity. b. d and f show the corresponding phase contrast views. a, b x 200; c-f, x 500.
mF
Fig. 4. Comparison of aFn and amnion epithelial cell cFn. APn (5 pg, lanes 1) and amnion epithelial cell cFn (3 ~8, lanes 2) were isolated by the ~latin-Sharon method, run under reducing conditions in 6% SD!&PACE and either protein stained (A) or ~munobiott~ with the BF (B) or DH (C) antibodies. mFn = monomeric Fns. 310
Amniotic fluid fibronectin REFERENCES Balian G., Crouch E., Click E. M., Carter W. G. and Bomstein P. (1979) Comparison of the structures of human fibronectin and plasma cold insoluble globulin. J. supramolec. Struct. 12, 505-516. Borsi L., Camemolla B., Castellani P., Rosellini C., Vecchio D., Allemanni G., Chang S. E., Taylor-Pa~dimitriou J., Pande H. and Zardi L. (1987) Monoclonal antibodies in the analysis of fibronectin isofonns generated by alternative splicing of mRNA precursors in normal and transformed human cells. J. Cell Biol. 104, 595-600. Carnemolla B., Borsi L., Zardi L., Owens R. J. and Baralle F. E. (19871Localization of the cellular gbronectin snecific epitope r&ognized by the monoclonal antibody *IST-9 using fusion proteins expressed in E. co/i. FEBS Lett. 255, 269-273. Engvall E. and Ruoslahti E. (1977) Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Inr. J. Cancer 20, l-5. Furcht L. T. (1983) Structure and function of the adhesive glycoprotein fibronectin. Mod. Cell Biol. 1, 53-l 17. Hahn E., Wick G., Pencev D. and Timpl R. (1980) Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin and fibronectin Gut 21, 63-71. Hynes R. (1985) Molecular biology of libronectin. A. Rev. Ceii Biol. 1, 67-90. Komblihtt A. R., Vibe-Pedersen 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 JI 3, 221-226. Kornblihtt A. R., Umezawa K., Vibe-Pedersen K. and Baralle F. E. (1985) Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO JI 4, 1755-1759. Krusius T., Fukuda M., Dell A. and Ruoslahti E. (1985) Structure of the carbohydrate units of human amniotic fluid fibronectin. J. biol. Chem. 260, 41 IO-41 16. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&683. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall
311
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