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.     

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.     

The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts.

Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl-terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that recognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor-like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phenotypic changes.     

Fibronectin splice variants: understanding their multiple roles in health and disease using engineered mouse models

The extracellular matrix (ECM) is a highly dynamic network of proteins, glycoproteins, and proteoglycans. Numerous diseases result from mutation in genes coding for ECM proteins, but only recently it has been reported that mutations in the fibronectin (FN) gene were associated with a human disorder. FN is one of the main components of the ECM. It generates protein diversity through alternative splicing of a single pre-mRNA, having at least 20 different isoforms in humans. The precise function of these protein isoforms has remained obscure in most cases. Only in the recent few years, it was possible to shed light on the multiple roles of the alternatively spliced FN isoforms. This substantial progress was achieved basically with the knowledge derived from engineered mouse models bearing subtle mutations in specific FN domains. These data, together with a recent report associating mutations in the FN gene to a form of glomerulopathy, clearly show that mutations in constitutive exons or misregulation of alternatively spliced domains of the FN gene may have nonlethal pathological consequences. In this review, we focus on the pathological consequences of mutations in the FN gene, by connecting the function of alternatively spliced isoforms of fibronectin to human diseases.     

Tx-TnT在不同发育时期的艾维茵鸡和洪山鸡肌肉组织中的表达分析

以艾维茵鸡和湖北省地方鸡种洪山鸡为实验材料 ,借助特异性识别Tx残基肽的单克隆抗体 6B8,采用Western杂交方法 ,检测Tx TnT异构体在洪山鸡和艾维茵鸡 7个发育时期 (孵化第 14d、初生 1日龄、7、14、2 1、2 8和35日龄 )的胸肌和腿肌中的表达差异 ,并与胸肌重进行相关分析。结果表明 ,Tx TnT在腿肌和孵化第 14d的胸肌中均不表达 ,在初生 1日龄后胸肌中的表达随发育逐步增长 ,统计分析发现 ,Tx TnT在艾维茵鸡和洪山鸡胸肌中的表达量具有显著差异 (P <0 0 5 ) ,与胸肌重具有显著相关 (P <0 0 5 )。     

Expression of the ED B fibronectin isoform in adult human articular cartilage

The most recently described region of alternate splicing of fibronectin mRNA results in expression of an isoform which includes the type III repeat termed ED B (EIII B). To date this isoform has been detected in transformed cells in culture, in the synovial membrane and ovary but in no other adult tissue, and in embryonic chick cartilage to a much greater extent than in other cells of mesenchymal origin. Monoclonal antibody BC-1, which recognizes an epitope within the ED B segment, was used in two different assays and provided evidence that adult human articular cartilage contains ED B fibronectin. The extent of expression of this isoform, however, was variable and less than that found in fibronectin produced by the transformed fibroblast cell line WI-38VA13.     

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.     

Immunohistochemical studies on regulation of alternative splicing of fast skeletal muscle troponin T: non-uniform distribution of the exon x3 epitope in a single muscle fiber

Troponin T (TnT) isoforms of chicken fast skeletal muscle are classified into two types, breast-muscle-type (B-type) and leg-muscle-type (L-type) isoforms. These isoforms are produced from a single gene by differential alternative splicing of pre-mRNA. We investigated immunohistochemically the distribution of B-type TnT isoforms in chicken leg muscle (musculus biceps femoris), using anti-exon x3 that was raised against a synthetic peptide corresponding to exon x3 and recognized B-type, but not the L-type, TnT isoforms. Mosaic patterns of immunostaining showing locally different expression of B-type TnT isoforms in a single fiber were observed among fibers, and the non-uniform distribution of the isoforms was also detected in sectioned fibers and myofibrils from the muscle. The results indicated that regulation of pre-mRNA splicing of fast skeletal muscle TnT was different not only among the muscle fibers but also within a single fiber, suggesting that heterogeneous myonuclei in regulation of alternative splicings occur in a single muscle fiber.     

Human fibronectin: cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats

The nucleotide sequence of five independent cDNA clones, which cover 4843 nucleotides from the poly(A) addition site of human fibronectin (FN) mRNA was determined. The deduced amino acid sequence (1383 residues) covers the COOH-terminal 60% of human FN, spanning the C-terminus, fibrin-, heparin- and cell-binding domains, and shows the exact location of the only two free sulphydryl groups present in each subunit chain. We have recently reported two different FN mRNA species; one of them containing an additional 270 nucleotide insert (ED) that encodes exactly one of the homology type III repeats of the protein. The two mRNAs arise by alternative splicing of a common precursor. S1 nuclease mapping of cDNA/RNA hybrids shows that the expression of the two mRNAs is cell specific. Liver only produces the mRNA without the ED, whereas hepatoma cells, breast tumor cells and normal fibroblasts produce both forms of mRNA. Another area of alternative splicing generating three different FN mRNAs in rat liver has been reported by Schwarzbauer et al (16). We here provide evidence for the existence in human cells of a fourth mRNA species different from the three described in rat liver.     

Aberrant and alternative splicing in skeletal system disease

The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

Altered fibronectin mRNA splicing in skin fibroblasts from Ehlers-Danlos syndrome patients: in situ hybridization analysis.

The expression of fibronectin (FN) mRNA isoforms generated by alternative splicing of the EDA region was studied by dot-blot and in situ hybridization, using specific FN cDNA probes, in skin fibroblasts from controls and Ehlers-Danlos Syndrome (EDS) patients (types III, IV, VII and non classified). An Image Analysis program was used for the quantitative evaluation and comparison of FN mRNAs levels in the different cell strains. The in situ hybridization analysis showed that FN mRNAs are homogeneously expressed in all cells of each fibroblast strain analyzed. While in control fibroblasts about 70% of FN mRNA isoforms contain the EDA region (EDA+ FN mRNAs), in EDS fibroblasts this fraction is reduced up to about 30%. This indicates that in the EDS fibroblasts analyzed a deregulation of the alternative splicing processes acting at the EDA region takes place.     

Identification of an RNA binding specificity for the potential splicing factor TLS

The TLS/FUS gene is involved in a recurrent chromosomal translocation in human myxoid liposarcomas. We previously reported that TLS is a potential splicing regulator able to modulate the 5'-splice site selection in an E1A pre-mRNA. Using an in vitro selection procedure, we investigated whether TLS exhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this motif abolishes the interaction of TLS with the selected RNAs. We showed that TLS can bind GGUG-containing RNAs with a 250 nm affinity. By UV cross-linking/competition and immunoprecipitation experiments, we demonstrated that TLS recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contributes to the specificity of the TLS.RNA interaction, whereas only RRM and RGG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS.RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the beta-tropomyosin pre-mRNA. Moreover, we determined that RNA binding specificities of TLS and high nuclear ribonucleoprotein A1 were different. Hence, our results help define the role of the specific interaction of TLS with RNA during the splicing process of a pre-mRNA.     

Variation in alternative splicing across human tissues

Background  

Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues.     

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.