C6: A Monoclonal Antibody Specific for a Fibronectin Epitope Situated at the Interface between the Oncofoetal Extra-Domain B and the Repeat III8

Background

Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN isoform that contains the “extra-domain-B” (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues in vivo. We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B.

Methodology

We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6.

Conclusion

The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon.

Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high-level expression of an extra type III homology repeat (ED-B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.     

Detection of multiple fibronectin isoforms in fetal fibroblast monolayer culture: a novel method for the qualitative and quantitative detection of multiple antigens

 Analyzing the expression of multiple distinct antigens within a single monolayer culture involves cumbersome immunostaining techniques. We describe a simple and economical procedure for the detection and quantification of multiple antigens within a single monolayer. By generating an immunohistochemical grid which divides a monolayer in a standard tissue culture dish into 20 distinct areas, we were able to detect and quantify four individual fibronectin (FN) isoforms within a single fibroblast monolayer culture. Quantification of each isoform was performed using a modified enzyme-linked immunoassay. In addition, within the same monolayer, each FN isoform was detected using standard immunohistochemical detection with DAB visualization. Using this novel approach to immunohistochemical analysis we determined that within the first 4 days of culture, the quantity of all FN isoforms increases faster than the number of cells. However, upon reaching confluency, the quantity of FN/cell drops dramatically. After reaching confluency, the amount of FN/cell levels off and remains constant within the postconfluent monolayer. Statistical analysis of the quantity of FN/cell indicates that a significant reduction in the amount of FN/cell occurs in the 2 days prior to reaching confluency. The distribution of all the FN isoforms, with the exception of B-FN, was found along the length of the cell body. In contrast, the distribution of B-FN was altered in postconfluent monolayers where it was detected only in distinct locations within the monolayer. Accepted: 27 October 1998     

Endothelins: regulators of extracellular matrix protein production in diabetes

Fibronectin (FN), a key extracellular matrix protein, is upregulated in target organs of diabetic angiopathy and in cultured cells exposed to high levels of glucose. FN has also been reported to undergo alternative splicing to produce the extra domain-B (ED-B) containing isoform, which is exclusively expressed during embryogenesis, tissue repair, and tumoral angiogenesis. The present study was aimed at elucidating the role and mechanism of endothelins (ETs) in FN and ED-B FN expression in diabetes. We investigated vitreous samples for ED-B FN expression from patients undergoing vitrectomy for proliferative diabetic retinopathy. Our results show increased FN and ED-B FN expression in the vitreous of diabetic patients in association with augmented ET-1. Using an antibody specific to the ED-B segment of FN, we show an increase in serum ED-B FN levels in patients with diabetic retinopathy and nephropathy. We further examined retinal tissues, as well as renal and cardiac tissues, from streptozotocin-induced diabetic rats. Diabetes increased FN and ED-B FN in all three organs, which was prevented by ET antagonist bosentan. To provide insight into the mechanism of glucose-induced and ET-mediated ED-B FN upregulation, we assayed endothelial cells (ECs). Inhibition of mitogen-activated protein kinase with pharmacological inhibitors and protein kinase B with dominant negative transfections prevented glucose- and ET-1-mediated FN and ED-B FN expression. Furthermore, treatment of cells exposed to high levels of glucose with ET antagonist prevented the activation of all signaling pathways studied and normalized glucose-induced ED-B FN expression. We then determined the functional significance of ED-B in ECs and show that ED-B FN is involved in vascular endothelial growth factor expression and cellular proliferation. These studies show that glucose-induced and ET-mediated FN and ED-B FN expressions involve complex interplays between signaling pathways and that ET may represent an ideal target for therapy in chronic diabetic complications.     

Alternative Splicing of the Angiogenesis Associated Extra-Domain B of Fibronectin Regulates the Accessibility of the B-C Loop of the Type III Repeat 8

Background

Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulated around neovasculature during angiogenic processes, thus making ED-B one of the best markers and targets of angiogenesis. The functions of ED-B are still unclear; however, it has been postulated that the insertion of an extra-domain such as ED-B modifies the domain-domain interface and may unmask loops that are otherwise cryptic, thus giving FN new potential activities.

Methodology

We used the mAb C6, which reacts with ED-B containing FN, but not with ED-B-free FN and various recombinant FN fragments containing mutations, to precisely localize the epitopes recognized by the mAb C6.

Conclusion

We formally demonstrated that the inclusion of the alternatively spliced angiogenesis-associated ED-B leads to the unmasking of the FNIII 8 B-C loop that is cryptic in FN molecules lacking ED-B. Thus, the mAb C6, in addition to providing a new reagent for angiogenesis targeting, represents a new tool for the study of the potential biological functions of the B-C loop of the repeat FNIII 8 that is unmasked during angiogenic processes.     

Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment

Background

Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. We hypothesized that the specific targeting of TNF-alpha antagonists to inflamed tissues could increase drug efficacy and reduce side effects.

Results

Using uteroglobin (UG), a potent anti-inflammatory protein, as a scaffold, we prepared a bispecific tetravalent molecule consisting of the extracellular ligand-binding portion of the human TNF-alpha receptor P75 (TNFRII) and the scFv L19. L19 binds to the ED-B containing fibronectin isoform (B-FN), which is expressed only during angiogenesis processes and during tissue remodeling. B-FN has also been demonstrated in the pannus in rheumatoid arthritis. L19-UG-TNFRII is a stable, soluble homodimeric protein that maintains the activities of both moieties: the immuno-reactivity of L19 and the capability of TNFRII to inhibit TNF-alpha. In vivo bio-distribution studies demonstrated that the molecule selectively accumulated on B-FN containing tissues, showing a very fast clearance from the blood but a very long residence time on B-FN containing tissues. Despite the very fast clearance from the blood, this fusion protein was able to significantly improve the severe symptomatology of arthritis in collagen antibody-induced arthritis (CAIA) mouse model.

Conclusions

The recombinant protein described here, able to selectively deliver the TNF-alpha antagonist TNFRII to inflamed tissues, could yield important contributions for the therapy of degenerative inflammatory diseases.     

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.     

Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule that is highly expressed on the surface of endothelial cells and some hematopoietic cells. Its cytoplasmic domain is encoded by multiple exons, which undergo alternative splicing. Here, we demonstrate that the human PECAM-1 cytoplasmic domain undergoes alternative splicing, generating six different isoforms. RT-PCR cloning and DNA sequence analysis indicated that human tissue and endothelial cells express multiple isoforms of PECAM-1, including the full-length PECAM-1 and five other isoforms, which lack exon 12, 13, 14, or 15 or exons 14 and 15. The full-length PECAM-1 is the predominant isoform detected in human tissue and endothelial cells. This is in contrast to murine endothelium, in which the PECAM-1 isoform lacking exons 14 and 15 is the predominant isoform. The PECAM-1 isoform lacking exon 13 detected in human tissue and endothelial cells is absent in murine endothelium. The expression pattern of PECAM-1 isoforms changes during tube formation of endothelial cells on Matrigel, which may indicate specialized roles for specific isoforms of PECAM-1 during angiogenesis. The data presented here demonstrate that human PECAM-1 undergoes alternative splicing, generating multiple isoforms in vascular beds of various tissues. Therefore, the regulated expression of these isoforms may influence endothelial cell adhesive properties during angiogenesis and/or vasculogenesis.     

NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis

BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.     

A single human myosin light chain kinase gene (MLCK; MYLK)

The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garcia et al., 1997, Am. J. Respir. Cell Mol. Biol. 16, 489-494) except for a deletion of nucleotides 1428-1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081-5233 (MLCK 3a, D3), double deletions of nucleotides 1428-1634 and 5081-5233 (MLCK 3b), and nucleotide deletions 4534-4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5' and 3' neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.     

Human mucin MUC1 RNA undergoes different types of alternative splicing resulting in multiple isoforms

MUC1 is a transmembrane mucin with important functions in normal and transformed cells, carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat, respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5′ splice site but variable 3′ splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3′ splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1^+/?Kras^G12D/+Pten^loxP/loxP mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice.     

The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.