Transformed human cells release different fibronectin variants than do normal cells.

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.

of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA.
Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts.
Furthermore, we observed that 45-65 % of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparinbinding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.

Interactions:
Domain:  III III III lU III III III III III II1" II1 III III III   28kD  56kD  155kD   with thermolysin of FN subunit a (containing the IIICS sequence) and 13 (without the IIICS sequence) are schematically presented based on previous reports (19)(20)(21)28), and the results reported are here. White arrows indicate the sites where the two monoclonal antibodies IST-2 and IST-7 react. Arrowheads indicate the thermolysin cleavage sites. The figure also indicates the internal homologies (I-III) (15).
The Journal of Cell Biology, Volume 103, 1986 bound polypeptides were eluted using 25 mM Tris-HCl, pH 7.6 buffer containing 0.5 mM EDTA, 0.7 M NaCI. All operations were carried out at a flow rate of 20 ml/h. 1.5-ml fractions were collected.

Radioimmunoassay (RIA )
The solid-phase double-antibody RIA method was used for the quantitative competition binding assay (24). Competition reactions were carded out in PBS containing 1 mg/ml of bovine serum albumin (BSA), 5 U/ml aprotinin, and 0.2 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO).
The heparin-bound and unbound FN fractions (obtained as described above) were, after dialysis, diluted with PBS m identical volumes (10 ml), and BSA and apmtinin were added to give a final concentration of 1% and 10 U/ml, respectively. The competition binding assay was carried out on these fractions using the monoclonal antibody IST-7.
Various dilutions of the heparin-bound and heparin-unbound fractions were incubated with a monoclonal antibody (at a dilution that had given 50% maximum binding in titration experiments) for 1 h at 37°C.
The mixtures were then transferred into the wells of polyvinyl plates (Cooke Labs, Alexandria, VA) previously coated with purified FN, and incubated for 2 h. Then the mixture was removed and the wells were washed with PBS. The amount of antibody bound was detected by adding specific 125I-labeled rabbit anti-mouse Ig and incubating for 2 h at room temperature. The dilutions of heparin-bound and heparin-unbound fractions that could give a 50% inhibition were used to calculate the ratio between the Ix-and [i-subunits.

Characterization of Monoclonal Antibodies IST-2 and IST-7
The monoclonal antibody IST-2 has previously been partially characterized (19,21). It reacts with the heparinbinding domain 5 which is comprised of the COOH-terminal four type III homology repeats (21) (see Fig. 1). This domain is present in the different FN molecules with differences in the primary structure since different portions of a 120 amino acid segment (IIICS) may be inserted between the last two type HI homology repeats; this is due to alternative patterns of RNA splicing (7-10, 17).
After thermolysin digestion of plasma FN, IST-2 reacts with two fragments of 38 and 29 kD which represent the heparin-binding domain 5 (19, 21, 28). Both the 38 and 29-kI) fragments are extremely resistant to thermolysin. Digestion of the purified 38-kD fragment by 50 gg of thermolysin/mg of 38-kD fragment did not generate any 29-kD fragment or other degradation peptides. Identical resistance to thermolysin was shown by the 29-kD fragment. Purification (28) and primary structure studies (14; and Pande, H.,   Borsi, and L. Zardi, manuscript submitted for publication) of these two fragments demonstrated that the 38-kD fragment (domain 513) contains the four repeats of type HI homology with the deletion of the entire 120 amino acid IIICS sequence, whereas the 29-kD fragment (domain 5(x0 contains the first three repeats of type III homology plus a small portion of the connecting segment (IIICS) (see Fig. 1). These data indicate that the monoclonal antibody IST-2 reacts with an antigenic determinant present on the first three type III homology repeats (Fig. 1) and that the presence of the IIICS sequence introduces into the FN molecule a site that is very sensitive to the proteolytic enzyme thermolysin. The monoclonal antibody IST-7 reacts with the 38-kD (domain 513) but not the 29-kD fragment (domain 5a~) (Fig.  2). It also reacts with a smaller fragment of ~12 kD (domain 5a2) (Fig. 2), which represents the last type III homology repeat, as also confirmed by the NH2-terminal amino acid sequence of the purified fragment (data not shown). This 12-kD fragment (domain 5a2) does not have heparin-binding activity (data not shown), which is the characteristic of domains 5a~ and 513.

Domain 5 May Be Sialylated
The monoclonal antibody IST-2, which reacts with the 38and 29-kD fragments of plasma FN, also reacts with FN from the spent media of different human cell lines with an extra band having a molecular mass of '~33 kD (see Fig. 2). This band does not react with the monoclonal IST-7, indicating that this fragment lacks the last type III homology repeat. Furthermore, this fragment shows as great an affinity for heparin as do the 29-and 38-kD fragments (data not shown). All three of these fragments were purified and treated with different amounts of neuraminidase (Fig. 3). The 38-and 29-kD fragments did not show any change in molecular mass, whereas that of the 33-kD fragment decreased to '~30 kD. The fact that neuraminidase treatment does not reduce the 33-kD fragment exactly to 29 kD is very likely due to either incomplete desialylation or the presence of other carbohy-

Determination of the Expression of the IIICS Sequence in FN from Different Sources
To estimate the presence of the IIICS sequence in FN from different sources we used two different approaches, using the monoclonal antibodies IST-2 and IST-7, and the different sensibilities of the two FN subunits to thermolysin: (a) Since the 38-kD fragment originates from the FN subunit in which the IIICS sequence is completely deleted and the 29-and 33-kD fragments originate from the FN subunit in which the mCS sequence is at least partially expressed (14; and Pande, H., J. Calaycay, T. Lee, K. Legesse, J. E. Shively, A. Siri, L. Borsi, and L. Zardi, manuscript submitted for publication), the ratio between the 38-kD fragment and the 33 + 29 kD fragments represents the ratio between the 13-and ct-subunits in the different FNs. To estimate the relative amount of each of these fragments we performed a densitometric scanning of immunoblots stained using the monoclonal antibody IST-2 (Fig. 4). The percentage of the area of each peak is reported in Table I. (b) The second procedure involved the use of the monoclonal antibody IST-7 (specific for domains 513 and 5(t2) and is based on the observation that when the IIICS sequence is deleted, the proteolytic enzyme thermolysin cannot separate the determinant recognized by IST-7 from the se-quence with heparin affinity. On the contrary, the expression of the IIICS sequence introduces into the FN molecule a site extremely sensitive to thermolysin and after digestion the fragment containing the determinant recognized by IST-7 cannot bind heparin (see Fig. 5). Fig. 6 shows competition RIA experiments for the binding of the monoclonal antibody IST-7 using the heparin-bound and heparin-unbound fractions of the thermolysin digests of different FNs. The data are summarized in Table I (for the  procedure see Materials and Methods).
The results obtained with the two procedures were nearly identical (Table I) and demonstrated that the percentage of the a-subunit (containing the IIICS sequence) is higher in virus-transformed or tumor-derived cells with respect to normal fibroblasts or plasma.
Furthermore, the percentage of the glycosylated domain 5~t seems to be higher in FNs from embryonic or transformed cells with respect to normal cells (Table I).

Discussion
In this work we provide evidence that tumor-derived or transformed human cells release into the culture media different FN variants than do normal cells.
It has previously been demonstrated that the FN polymorphism is due to alternative splicing schemes in at least two regions (IIICS and ED) of the pre-mRNA (7-10, 17, 18, 22). Previous studies (8,9) have suggested that the ED segment seems to be absent in the hepatocyte mRNAs, which are the source of plasma FN, and it is probable that the inclusion of Here we show that the expression of the IIICS sequence introduces into the FN molecule a region that is susceptible to thermolysin cleavage. This agrees with previous indications of a different susceptibility to proteolytic enzymes in the COOH-terminal region of the two FN subunits (2,11,15,20,23,24,26). Thus, thermolysin digestion of subunits containing the IIICS sequence produces fragments different with respect to those originating from FN molecules in which the IIICS sequence is deleted. Using two monoclonal antibodies (IST-2 and IST-7), the different susceptibilities to thermolysin of the two FN subunits, and the affinity for heparin of the different FN thermolysin fragments, we have set up two procedures for the quantitative determination of the expression of the IIICS sequence in FN from different sources. However, five splicing variants within the I/ICS have been demonstrated in human FN (10). In fact, the 120 amino acids of the IIICS segment may be present or deleted, or portions of 95, 89, or 64 amino acids may be expressed in the FN molecules (10). The two procedures described here can only discriminate whether the HICS sequence is completely deleted or at least partially expressed.
Using these procedures we have observed that: (a) the percentage of molecules containing at least part of the IIICS sequence varies depending on the FN source; and (b) expression of the IIICS sequence is higher in FN from tumor- Percentage of a-and 13-FN subunits as estimated using the RIA method with the IST-7 monoclonal antibody and by densitometric scanning of immunoblots with the IST-2 monoclonal antibody. FN was from plasma or from the spent media of the different cell lines. * The percentages of subunits in which domain 5 is sialylated are indicated in parentheses, nd, not determined. derived and SV-40-transformed cells with respect to normal fibroblasts and plasma. These results suggest a specifically programmed splicing mechanism in different cell types and that in transformed or tumor-derived cells these mechanisms are altered. Recently Matsuura and Hakomori, using monoclonal antibodies, found a domain that is present in FNs from tumors and fetal tissues but absent in those from adult tissues and plasma (12). We can exclude that the monoclonal antibodies prepared by Matsuura and Hakomori (12) recognize some antigenic determinant on the IIICS domain since this is also present in plasma FN.
Furthermore, we demonstrated that the FN domain 5a~ (which contains part of the HICS sequence) is sialylated in some FN molecules from spent media of culture cells but not in plasma FN. The percentage of the sialylated domain 5cq in FN from the spent media of embryonic fibroblasts is about twice that in FN from the spent media of adult fibroblasts. The percentage of sialylated domain 5ct~ is even higher in FN from the spent media of transformed cells. We did not observe any sialylation of domain 513 (from which the IIICS sequence is deleted).
Future research should be directed towards establishing the biological consequences of these structural modifications.