Tenascin-W: An extracellular matrix protein associated with osteogenesis and cancer

Tenascin-W was the last member of a family of four large extracellular matrix glycoproteins to be discovered. The original member of the tenascin family, tenascin-C, has been widely studied due to its association with asthma, fibrosis, infection, inflammation and cancer. Recent studies report multiple common features between tenascin-W and tenascin-C in terms of structure, expression and function, especially in the context of tumorigenesis. Furthermore, specific functions for tenascin-W in osteogenesis have been revealed. This review presents an update on our current knowledge concerning tenascin-W and discusses potential medical applications of this cancer-enriched extracellular matrix protein.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)⁺ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH₂ and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type genomic clone is a functional protein exhibiting RNA N-glycosidase activity.     

Identification of a wound-induced inhibitor of a nuclear factor that binds the carrot extensin gene

Following wounding of carrot (Daucus carota L.) roots, the activity of a nuclear factor (EGBF-1) that binds a 5′-region of the carrot extensin gene declines to undetectable levels within 48 h. Mixing of nuclear extracts from wounded roots with nuclear extracts from unwounded roots has demonstrated the existence of a wound-induced inhibitor of EGBF-1. Inhibition of EGBF-1 DNA-binding activity by nuclear extracts from wounded roots is shown to be specific for EGBF-1, and to be destroyed by heat treatment. In addition, inhibition is saturable and occurs rapidly. Active EGBF-1 can be reconstituted from its inhibited state by renaturation of proteins from mixed extracts following denaturation by boiling in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoretic separation, indicating that inhibition is dependent upon the reversible interaction of EGBF-1 with a titratable factor. However, EGBF-1 activity could not be detected in nuclear extracts from wounded roots following denaturation and electrophoretic separation. Inhibitory activity was not detectable in nuclear extracts from roots that had been trated with ethylene. The action of the inhibitor indicates one possible mechanism for the control of EGBF-1 activity in carrot roots following wounding.     

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.     

Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin

Recently, a novel type of calcium-dependent protein kinase (CDPK) that requires neither calmodulin nor phospholipids for activation, has been described in plants. We have isolated a cDNA clone for carrot CDPK by probing a library of somatic embryo cDNAs with oligonucleotides corresponding to highly conserved regions of protein kinases. The product of this gene overexpressed in Escherichia coli reacted strongly with monoclonal antibodies to soybean CDPK. The deduced amino acid sequence of carrot CDPK reveals two major functional domains. An N-terminal catalytic domain with greatest homology to calcium/calmodulin-dependent protein kinase type II from rat brain is coupled to a C-terminal calcium-binding domain resembling calmodulin. These features of the primary sequence explain how CDPK binds calcium and suggest a model for CDPK regulation based on similarities to animal calcium/calmodulin-dependent protein kinases.     

The extracellular matrix protein fibronectin is a substrate for kallikrein 7

Kallikrein 7 (hK7), a chymostatin-like serine protease, is overexpressed in pancreatic adenocarcinomas as well as other human cancers. Although it has been demonstrated to participate in normal desquamation by facilitating cell shedding at the skin surface, its role in human malignancies remains unclear. To investigate the ability of hK7 to degrade components of the extracellular matrix (ECM), recombinant hK7 was expressed and purified from cultured mammalian cells. Using a three-step chromatographic purification procedure, recombinant hK7 was obtained that displayed robust proteolytic activity against a fluorogenic peptide substrate following activation by thermolysin. We demonstrate that the active protease is able to cleave fibronectin in a time-dependent manner, but not laminin, using an in vitro degradation assay. These findings indicate that the aberrant expression and secretion of hK7 in human tumors may facilitate metastasis by directly degrading components of the extracellular matrix and may thus play an important role in tumorigenesis.     

Characterization of a binding protein for leukemia inhibitory factor localized in extracellular matrix

Leukemia Inhibitory Factor (LIF) interacts with two classes of high affinity binding sites on rat UMR cells cultured in monolayer. One class of binding sites was found to be localized in the extracellular matrix (ECM) after removal of cells from the culture dish. The interaction of LIF with ECM-localized binding sites is not dependent upon either glycosylation of LIF or the presence of extracellular glycosyaminoglycans. Chemical cross-linking studies demonstrate that LIF interacts with a 200-kD cell-associated protein and a 140-kD ECM- localized protein. A 140-kD protein could also be specifically precipitated from solubilised metabolically radiolabeled UMR ECM by antibodies directed against LIF by virtue of its ability to form a stable complex with unlabeled LIF. In addition, soluble LIF associated with this ECM-localized protein is biologically active in terms of inhibition of ES cell differentiation. The properties of ECM-localized 140-kD species are very similar to those of the secreted form of the LIF receptor suggesting that the ECM localization of LIF and LIF signal transduction may be closely coupled.     

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.     

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.     

Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH₂-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5 signal sequence, an open reading frame of 718 nucleotides, a 3 AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.     

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.     

Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.     

Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato

Differential screening of a cDNA bank constructed from ripe tomato fruit mRNA allowed the isolation of cDNA clone 2A11 which is entirely fruit-specific, is expressed at steadily increasing levels from anthesis to breaker, and accounts for approximately 1% of the messenger RNA in mature tomato fruit. A genomic clone corresponding to the 2A11 cDNA was isolated from a tomato genomic library. Sequence comparison of the cDNA clone with the genomic clone shows they are identical over the shared region with the genomic clone possessing a single large intron near the 5 end of the message.The open reading frame of 2A11 would encode a sulfur-rich polypeptide 96 amino acids in length. The identity of the putative protein is unknown. In situ hybridization shows that the 2A11 message is found throughout the pericarp cells in a tomato fruit. In contrast, in situ hybridization of early ripening stages with a polygalacturonase probe shows higher mRNA levels in cells of the outer pericarp and cells surrounding the vascular regions of the pericarp.     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.