Smoothelin and intermediate filament proteins in human aortocoronary saphenous vein by-pass grafts

The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as smoothelin in the native great saphenous vein. When desmin and smoothelin were present in the neointima, smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.     

Smoothelin and Intermediate Filament Proteins in Humanaortocoronary Saphenous Vein By-pass Grafts

The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as smoothelin in the native great saphenous vein. When desmin and smoothelin were present in the neointima, smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.     

Smoothelin in adult and developing human arteries and myocardium

The aim of this investigation was to study, with immunohistochemical methods, the distribution of the novel cytoskeletal protein smoothelin in human cardiovascular tissues, the possible changes during the development of the cardiovascular system and its correlation to the intermediate filament proteins desmin and vimentin. Smoothelin was detected in smooth muscle cells of the fetal coronary arteries. In very young subjects (up to 3 months of age), only a few cells in the media of the elastic arteries contained smoothelin, whereas it was present in most smooth muscle cells in the muscular arteries. In individuals older than 1 year, most smooth muscle cells in the media of all blood vessels contained smoothelin. In vessels with a developed intima, smoothelin was present in a variable proportion of the smooth muscle cells. With few exceptions, smoothelin was more frequently detected than desmin in medial smooth muscle cells. Smoothelin and vimentin were codistributed in the smooth muscle cells of the media in most vessels. In the cardiomyocytes (fetal to adult age), the smoothelin antibody detected epitopes located at the Z-disc level but not in the intercalated discs. In conclusion, smoothelin is more widely distributed in the muscular arteries than in the elastic arteries early in life, and thus exhibits a variable distribution during postnatal development of vascular tissues. In the adult, smoothelin is detected in the media of most vascular smooth muscle cells, both in muscular and elastic arteries, and is not necessarily codistributed with either desmin or vimentin. Evidence that smoothelin is present in human striated cardiomyocytes is also presented. Accepted: 16 July 1999     

Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle [published erratum appears in J Cell Biol 1994 Feb;124(3):395]

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. A novel integrin alpha subunit partial cDNA isolated from TGF- beta stimulated guinea pig airway epithelial cells has previously been reported (Erle, D.J., D. Sheppard, J. Bruess, C. Ruegg, and R. Pytela. 1991. Am. J. Respir. Cell Mol. Biol. 5:170-177). We have now determined cDNA and amino acid sequence for the human homolog of this subunit, named alpha 9, from a human lung cDNA library, a human small intestine cDNA library, and cDNA from the cell lines U937, HL-60 and Tera-2. This sequence is predicted to encode a 1006-amino acid mature protein that shares 39% identity with the previously identified integrin subunit alpha 4. By Northern blot analysis, alpha 9 mRNA was detected in the human carcinoma cell lines Tera-2 and Caco-2. Anti-peptide antibodies against the predicted COOH-terminal sequence of alpha 9 immunoprecipitated a heterodimer (140 kD/115 kD nonreduced; 150 kD/130 kD reduced) from Tera-2 lysates. Immunodepletion of beta 1-containing integrins with Tera-2 lysates removed alpha 9 immunoreactivity, suggesting that beta 1 is the principal beta subunit partner for alpha 9 in these cells. alpha 9 was detected by immunohistochemistry in airway epithelium, in the basal layer of squamous epithelium, and in smooth muscle, skeletal muscle, and hepatocytes.     

The cDNA of the two isoforms of bovine cGMP-dependent protein kinase

cDNAs encoding the isoform I alpha of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in lambda gt10. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 4207-4218]. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform I beta of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb.     

Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization

Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors. Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies. The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells. In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA. The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084. According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines. The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences. Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27). Our results show that cytovillin is representative of a novel class of microvillar proteins.     

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.     

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Apparent up-regulation of stimulatory G-protein alpha subunits in the pregnant human myometrium is mimicked by elevated smoothelin expression.

Sensitization of adenylyl cyclase (AC) by increased expression of large isoforms of the stimulatory G-protein Galpha(s) has been suggested as a mechanism that governs uterine quiescence during pregnancy. We quantified several components of the AC pathway in pregnant (P, n=21) and nonpregnant human myometria (NP, n=10). AC activity was approximately sevenfold higher in P than in NP under basal and stimulated conditions (MnCl(2)/GTP/GTP + isoproterenol). In addition, relative stimulation (% of basal) by 5'-guanosine-betagamma-iminotriphosphate and forskolin was twofold higher in P. beta-Adrenoceptor density was low and unaltered in P. Galpha(s) mRNA splice variants did not differ in P. Using antisera against different epitopes of Galpha(s) (carboxyl-/more amino-terminal), we found unchanged expression of Galpha(s) short and long (45, 47 kDa) in P. Two additional proteins in P (51, 59 kDa) were detectable only by the carboxyl-terminal antiserum and lacked GTP binding properties. The 59 kDa protein could be identified as a recently discovered cytoskeletal protein, smoothelin, which was 10-fold increased in P. These data indicate that the apparent up-regulation of large Galpha(s) species in P is mimicked by elevated smoothelin. Therefore, the increase in AC cannot be attributed to changes in Galpha(s)- or beta-adrenoreceptors. Epitope sharing between Galpha(s) and smoothelin should be considered in experiments on smooth muscle tissues.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.     

ESDN, a novel neuropilin-like membrane protein cloned from vascular cells with the longest secretory signal sequence among eukaryotes, is up-regulated after vascular injury.

A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins.     

Complete primary structure and tissue expression of chicken pectoralis M-protein.

M-Protein (165 kDa) is a structural constituent of myofibrillar M-band in striated muscle. We generated a monoclonal antibody which recognized a 165-kDa protein from chicken pectoralis muscle in immunoblot analysis and stained the M-band under immunofluorescence microscopy. By screening a lambda gt11 cDNA library from chicken embryonic pectoralis muscle with this antibody, we isolated a cDNA clone encoding the M-protein. Northern blot analysis showed that M-protein mRNA is expressed in pectoralis and cardiac muscle but not in gizzard smooth muscle or non-muscle tissues. Moreover, the anterior latissimus dorsi muscle, which consists almost exclusively of slow fiber types, contains no detectable levels of the mRNA. The full-length cDNA sequence predicted a 1,450-amino acid polypeptide with a calculated molecular weight of 163 x 10(3). The encoded protein contains several copies of two different repetitive motifs: five copies of fibronectin type III repeats are in the middle part of the predicted molecule, and two and four copies of the immunoglobulin C2-type repeats are located toward the NH2-terminal and COOH-terminal regions, respectively. This indicates that M-protein, along with other thick filament-associated proteins such as C-protein, twichin, and titin, belongs to the superfamily of cytoskeletal proteins with immunoglobulin/fibronectin repeats.     

Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor.

To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.     

Partial amino acid sequence of human thrombospondin as determined by analysis of cDNA clones: homology to malarial circumsporozoite proteins

A lambda gt 11 library prepared from human umbilical vein endothelial cell RNA was screened for cDNAs encoding thrombospondin. Reagents included a monospecific antibody to human thrombospondin and a mixture of four synthetic oligodeoxyribonucleotides derived from an amino acid sequence near the NH2 terminus of mature human thrombospondin. Two series of cDNA clones coding for sequences at the 5' and 3' ends of thrombospondin mRNA, respectively, were isolated. The nucleotide sequence of a 1.3-kilobase (kb) 5' clone (lambda TS-33) coded for 99 bases of 5' untranslated RNA, a signal peptide of 18 amino acids, and the first 379 amino acids of thrombospondin. Northern blot analysis with lambda TS-33 detected a single mRNA species of approximately 6.0 kb in rat aortic smooth muscle cell RNA. Thrombospondin mRNA levels increased rapidly, but transiently, in quiescent smooth muscle cells treated with platelet-derived growth factor. The kinetics of this response were very similar to those of the thrombospondin protein to this growth factor. There was significant homology in amino acid sequence between thrombospondin and a conserved region in the circumsporozoite protein of two malarial sporozoites. This region of thrombospondin may therefore represent a potential recognition site for a cell surface thrombospondin receptor.