Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns.

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland.

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two-dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue-specific patterns of their protein subunits.

Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells.

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH1C1) were used to isolate cytokeratin subunit complexes by solubilization in low salt buffer containing 4 M-urea. Alternatively, the complexes were prepared by solubilization of total cytoskeletal proteins in 9.5 M-urea or 6 M-guanidinium hydrochloride (Gu . HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 M-urea or 6 M-Gu . HCl and then against buffers containing either 4 M-urea or 2 M-Gu . HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1 : 1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A (Mr 55,000; isoelectric point in 9.5 M-urea, pH 6.4) and D (Mr 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 M. The complex had a sedimentation coefficient S25,w of 4.96 S in 2 M-Gu . HCl. Sedimentation equilibrium analysis gave an average Mr value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 M-urea or 2 M-Gu . HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional ("diagonal") gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when cytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I.

Cytoskeletal filaments of the alpha-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology. We compare two polypeptides of the acidic cytokeratin subfamily, VIb (Mr 54,000) and VII (Mr 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3' ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the alpha-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its alpha-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of Mr 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycine-rich and contain a canonic sequence GGGSGYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine Mr 59,000 keratin but is almost identical to the human cytokeratin number 14 of Mr 50,000, both in the alpha-helical and in the non-alpha-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3' non-coding ends. The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the alpha-helical and the non-helical regions as well as in the 3' non-coding portions of their mRNAs.(ABSTRACT TRUNCATED AT 400 WORDS)

Patterns of expression and organization of cytokeratin intermediate filaments.

Cytokeratins are a large multigene family comprising two polypeptide types, i.e. acidic (type I) and basic (type II) ones, which are distinguished on the basis of immunological, peptide mapping, mRNA hybridization, and primary amino acid sequence data. The acidic (type I) cytokeratins can be subdivided into at least two different subtypes on the basis of their carboxy-terminal sequences. Considerable interspecies conservation of sequences exists, even extending to the 3'-non-coding mRNA regions. Different pairs of type I and II cytokeratins show different resistance to dissociation in urea. Sequence differences of the type I cytokeratins containing functional domains may be an explanation of the observed preference of co-expression with certain type II cytokeratins. The distribution of the different type I and II cytokeratins in normal epithelia and in carcinomas is differentiation related and can be used for cell typing and identification. The cell type-specific expression of cytokeratin polypeptides is recognized at both the protein and the mRNA level. The building block of cytokeratin IFs is a heterotypic tetramer, consisting of two type I and two type II polypeptides arranged in pairs of laterally aligned coiled coils. This principle of tetrameric organization is thought to be generally applicable to IFs.

Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins.

Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation.

A subfamily of relatively large and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells.

Epithelial cells contain a class of intermediate-sized filaments formed by proteins related to epidermal alpha-keratins ('cytokeratins'). Different epithelia can express different combinations of cytokeratin polypeptides widely varying in apparent mol. wt. (40 000-68 000) and isoelectric pH (5.0-8.5). We have separated, by two-dimensional gel electrophoresis, cytokeratin polypeptides from various tissues and cultured cells of man, cow, and rodents and examined their relatedness by tryptic peptide mapping. By this method, a subfamily of closely related cytokeratin polypeptides has been identified which comprises the relatively large (greater than or equal to mol. wt. 52 500 in human cells) and basic (pH greater than or equal to 6.0) polypeptides but not the smaller and acidic cytokeratins. In all species examined, the smallest polypeptide of this subfamily is cytokeratin A, which is widespread in many simple epithelia and is the first cytokeratin expressed during embryogenesis. This cytokeratin polypeptide subfamily is represented by at least one member in all epithelial and carcinoma cells examined, indicating that polypeptides of this subfamily serve an important role as tonofilament constitutents . Diverse stratified epithelia and tumours derived therefrom contain two or more polypeptides of this subfamily, and the patterns of expression in different cell types suggest that some polypeptides of this subfamily are specific for certain routes of epithelial differentiation.

Change of cytokeratin organization during development of Mallory bodies as revealed by a monoclonal antibody.

A monoclonal murine antibody (KM 54-5) was produced against Mallory body (MB) material isolated from liver tissue of griseofulvin treated mice. The antigen was identified by positive immunofluroescence microscopy of MBs and by the immunoblotting technique on polypeptides separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In immunoblotting experiments, antibody KM 54-5 reacted with cytokeratins A (human no. 8) and D (human no. 18) of murine, bovine, and human hepatocytes as well as with cytokeratin A (no. 8) and its degradation products present in isolated murine MB. In immunofluorescence microscopy the antibody did not react with cytokeratin filaments of normal liver but showed a positive reaction with MBs after a certain stage in MB development had been reached. In a dot blot assay, using individual cytokeratin polypeptides isolated from murine liver and purified by ion exchange chromatography in pH 8 buffer containing 8 M urea, the antibody reacted with the individual polypeptides A (no. 8) and D (no. 18) but not with the heterotypic tetramer (A2D2) reconstituted from these polypeptides in 4 M urea. These findings confirm the cytokeratin nature of MB filaments. In addition, they show that the pathologic process of MB formation involves changes in cytokeratin organization and conformation, resulting in the accessibility of a specific antigenic determinant which is inaccessible ("masked") in the heterotypic tetramer subunit and in the cytokeratin filaments of normal cells. Hence this study presents an example of a pathological change of cytokeratin filaments and illustrates the value of monoclonal antibodies in the detection of such changes.

Stimulation of phospholipase C by a mutationally activated G protein alpha 16 subunit.

Alpha 16, a member of the alpha q subfamily of G protein alpha subunits, was recently identified in human hematopoietic cells. In order to elucidate the function of this novel alpha subunit, we cloned and mutagenized its cDNA to obtain a constitutively active protein. COS-1 cells were transfected with both wild-type and mutant cDNAs. Expression was confirmed by immunoblotting using a rabbit antiserum raised against the C-terminal decapeptide of alpha 16. The constitutively activated mutant alpha 16-R186C caused a two-fold increase in the formation of inositol trisphosphate in intact COS-1 cells, while the wild-type alpha 16 subunit had no effect. We conclude that alpha 16 is involved in coupling cell surface receptors of human hematopoietic cells to stimulation of phospholipase C.

Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics.

Subunit complexes of cytokeratin polypeptides from intermediate-sized filaments (IF) of various tissues and cultured cells from rat, cow, and man were solubilized in low-salt buffer containing 4 M urea and exposed to increasing concentrations of urea, followed by urea gradient electrophoresis or two-dimensional gel electrophoresis at different urea concentrations. Correspondingly, cytokeratin polypeptides dissociated in 9.5 or 10 M urea were dialyzed into lower concentrations of urea and allowed to reassociate into specific complexes. It was found that the polypeptide constituents of a given cytokeratin complex dissociate in the form of a rather sharp "melting curve" and that dissociated polypeptides reassociate in the same mode of dependence on urea concentration. The midpoint of melting in urea (Um) is a characteristic property of a given complex of cytokeratin polypeptides. Um values differ markedly between different cytokeratin complexes, ranging from 5.9 to 9.0 M urea. The results also show that cytokeratins do not form complexes with vimentin, another type of IF protein. The data suggest that certain cytokeratin polypeptides are complementary and contain sequences that direct their association into specific complexes forming IF subunits.

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays.

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.

Cessation of cytokeratin expression in a rat hepatoma cell line lacking differentiated functions.

Intermediate-sized filaments (IF) of diameter 7-11 nm occur in the cytoplasm of most cells of vertebrates and their constituent proteins are abundant in most cell types. Expression of IF proteins depends on the route of cell differentiation and five major subclasses of IF proteins have been distinguished: of these, cytokeratins are typical of epithelial cells whereas vimentin occurs in mesenchymally derived cells and some other non-epithelial cells. When epithelial cells are grown in culture this restriction of IF expression is often lost and they begin to synthesize vimentin in addition to cytokeratin, although examples of maintenance of the cell-type-specific expression of only cytokeratin have also been reported. No IFs have been detected in mammalian germ cells or in pre-morula stages of mouse embryogenesis, and the first IF proteins identified in murine blastocysts are cytokeratins of trophectodermal cells. We report here that a dedifferentiated rat hepatoma cell clone, which has become resistant to the action of the glucocorticoid hormone analogue dexamethasone and has lost various liver-specific functions, also stops all synthesis of IF proteins, without obvious consequences for growth and proliferation. The existence of such cells devoid of IF supports the notion that such filaments are not involved in basic cellular functions necessary for growth and proliferation but are related to special functions of the differentiated cell.

Desmoplakins of epithelial and myocardial desmosomes are immunologically and biochemically related.

Guinea pig antibodies against desmoplakins from bovine muzzle epidermis showed specific reaction in several epithelial tissues with desmoplakin I (Mr 250,000) and desmoplakin II (Mr 215,000). By immunofluorescence microscopy, prominent punctate staining was observed in various lines of cultured epithelial cells, revealing desmosomal junctions at sites of established cell-to-cell contacts as well as hemidesmosomes and internalized desmosome-derived membrane domains. On frozen tissue sections punctate staining was observed along plasma membranes of epithelial cells, and electron microscopy using the immunoperoxidase technique revealed that the antibodies were specifically localized at the plaques associated with desmosomes and hemidesmosomes. Of a large number of non-epithelial cells examined positive staining was only observed on desmosome-like junctions of myocardial cells and Purkinje fiber cells. In both epithelial and myocardial tissues the antibodies showed a broad range of cross-reactivity between diverse vertebrate species such as man, cow, rodent, and chicken, indicating that desmoplakins contain determinants strongly conserved during evolution. When binding of these antibodies to cytoskeletal polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper sheets was examined, specific reaction was noted with desmoplakin I and, to a variable degree, also desmoplakin II from various epithelial cells. Reaction was also observed with a myocardial polypeptide from bovine and human hearts which had a similar Mr value (250,000) and isoelectric pH range as desmoplakin I. We conclude that desmoplakins are the major proteins present in the desmosomal plaques of both epithelial and myocardial cells and that the desmoplakin polypeptides present in these two different cell types are very similar, if not identical.

Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein.

Plakoglobin is a major cytoplasmic protein that occurs in a soluble and a membrane-associated form and is the only known constituent common to the submembranous plaques of both kinds of adhering junctions, the desmosomes and the intermediate junctions. Using a partial cDNA clone for bovine plakoglobin, we isolated cDNAs encoding human plakoglobin, determined its nucleotide sequence, and deduced the complete amino acid sequence. The polypeptide encoded by the cDNA was synthesized by in vitro transcription and translation and identified by its comigration with authentic plakoglobin in two-dimensional gel electrophoresis. The identity was further confirmed by comparison of the deduced sequence with the directly determined amino acid sequence of two fragments from bovine plakoglobin. Analysis of the plakoglobin sequence showed the protein (744 amino acids; 81,750 Da) to be unrelated to any other known proteins, highly conserved between human and bovine tissues, and characterized by numerous changes between hydrophilic and hydrophobic sections. Only one kind of plakoglobin mRNA (3.4 kilobases) was found in most tissues, but an additional mRNA (3.7 kilobases) was detected in certain human tumor cell lines. This longer mRNA may be represented by a second type of plakoglobin cDNA, which contains an insertion of 297 nucleotides in the 3' non-coding region.

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.