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

Castellani, Patrizia; Siri, Annalisa; Rosellini, C; Infusini, Edmondo; Borsi, Laura; Zardi, Luciano

doi:10.1083/jcb.103.5.1671

Cited by 120 publications

(54 citation statements)

References 27 publications

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“…Molecular mass pattern of isolated proteins corresponded to known FN fragments, often reported as a result of in vivo or in vitro proteolytic digestion [30][31][32][33][34], and this was confirmed by the immunoreactivity with distinct mono clonal antihuman FN Abs. Thus, protein bands in the region of 50-200 kDa were identified as the main human plasma FN immunoreactive fragments and, specifically, in the high molecular mass region, anti-EDA reactivity typical of cellular FN was also detected.…”

Section: Discussionsupporting

confidence: 52%

Molecular heterogeneity of gelatin-binding proteins from human seminal plasma

Kosanović¹,

Janković²

2010

Asian J Androl

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Defining the molecular characteristics of seminal plasma proteins is essential for understanding their function in physiological and pathological conditions. Starting from the predicted importance of human seminal plasma gelatin-binding proteins, comprising fibronectin (FN) and FN-related molecules, for male fertility, this study aims at gaining insight into their immuno-glycobiochemical properties. Human seminal plasma from subjects with normal semen parameters were separated on a gelatin-Sepharose column and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and immunoblotting using antibodies against distinct FN forms. Heterogeneity of the isolated molecular species was examined by protein chip arrays combined with surface-enhanced laser desorption/ionization time of flight mass spectrometry, on normal, metal and hydrophobic surfaces. Carbohydrate composition was investigated using mannose-, fucose-and sialic acid-specific plant lectins and galectin-1. The results obtained indicated a pattern of isolated proteins corresponding to that of known FN fragments, as confirmed by immunoreactivity. Among them heparin-binding ability was preferentially associated with low molecular mass species. As for posttranslational modifications, phosphorylation and glycosylation of distinct fragments were revealed. Lectin binding to fragments containing the gelatin-binding domain, particularly with Ricinus communis agglutinin I, was stronger than to fragments containing the cell-binding site of FN. A low level of sialylation and distinctive concanavalin A-and Lens culinaris agglutinin-reactive species were also observed. Galectin-1 did not interact with the isolated preparation. Resolving the molecular heterogeneity of normal human seminal plasma FN and gaining initial insight into possible similarities/differences with known FN molecular species may be considered a prerequisite step preceding challenging the clinical usefulness of these molecular properties.

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Section: Discussionsupporting

confidence: 52%

Molecular heterogeneity of gelatin-binding proteins from human seminal plasma

Kosanović¹,

Janković²

2010

Asian J Androl

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“…In fact, we have previously demonstrated that some FN molecules are sialylated in this region (3). The 52-kD fragment also contains the last type III homology repeat (positive reaction with IST-7); this is due to the fact that this fragment originates from FN subunits in which the IIICS sequence is completely deleted, conferring to this polypeptide resistance to thermolysin (3). Because the determinant recognized by IST-9 is very sensitive to thermolysin while on the contrary, the heparin-binding domain is extremely resistant (3), we mildly digested the three purified fragments with thermolysin.…”

Section: Ricinus Communis Agglutinin Comparison Of the Nhz-mentioning

confidence: 99%

“…The characterization of the mAbs IST-2, IST-7, and IST-4 has been previously reported (3,28,33). They are specific both for plFN and cell media-cultured FN (cFN).…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

“…In particular, the epitope recognized by IST-2 is localized within the first three type III homology repeats of the heparinbinding domain (see Fig. 3) (3,28); the epitope recognized by IST-7 within the last type III homology repeat of the FN molecule (3) (see Fig. 3), and the epitope of IST-4 within the first four type III homology repeats of the FN molecule (28).…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

“…Only clones showing a negative reaction for plFN and a positive reaction for cFN were selected for further characterization. The solid-phase double-antibody radioimmunoassay method was also used for the quantitative competition binding assay (3,36). It was carried out using the mAbs IST-9 and FN-3 (both specific for the ED sequence), and IST-4 (specific for a sequence common to all FN variants).…”

Section: Radioimmunoassaymentioning

confidence: 99%

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Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells.

Borsi¹,

Bárbara²,

Castellani³

et al. 1987

The Journal of Cell Biology

Self Cite

194

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Abstract. Recent results showing that a singlefibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered. F IBRONECT1NS (FNs) ~ are high-molecular-mass, adhesive glycoproteins present in the soluble form in plasma and other body fluids and in insoluble form in the extracellular matrices and basement membranes. FN molecules act as bridges between the cell surface and extracellular material. In fact, the FN molecules contain a cellbinding site and binding sites for collagen, heparin, gangliosides, and fibrin. Because of their multiple interactions, FNs play an important role in diverse biological phenomena, including cell adhesion, cell migration, hemostasis and thrombosis, wound healing and the ability to induce a more normal phenotype in transformed cells (for reviews on distribution, structure, and biological functions, see references 1,7,8,18,24).It has been demonstrated that FN polymorphism may be at least partially due to alternative splicing schemes in two regions (ED and IIICS) because as many as 10 different mRNAs may originate from the primary transcript of a single gene (9, 12-15, 25, 26, 29) localized on chromosome 2 (11, 34). In fact, Schwarzbauer et al. (25) have shown that an antiserum specific for the rat fibronectin IIICS sequence recognizes the larger subunit of rat plasma FN (plFN), but not the smaller one.Here we report the characterization of mAbs for the ED 1. Abbreviations used in this paper: cFN and plFN, fibronectin from cultured cell media and plasma, respectively. fragment of fibronectin. Using these mAbs in a quantitative assay, we have demonstrated that this sequence is not present in plFN and that tumor-derived and SV-40-transformed human cells release a population of FN molecules in which the percentage of subunits containing the ED sequence is about 10 times higher than in the FN released by normal human fibroblasts.

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