Characterization of polypeptide chains of epidermal prekeratin and reconstituted filaments

Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0-9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HCl buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization.     

Prekeratin biosynthesis in human scalp epidermis.

Analysis of human scalp epidermal prekeratin polypeptides by two-dimensional gel electrophoresis revealed that each of the bands observed in one-dimensional electrophoresis consisted of three to five polypeptides of the same molecular weight but differing in isoelectric points. It was possible to divide the polypeptides into two families, with isoelectric points in the ranges pH 6.0-8.0 and pH 5.0-5.5 respectively. Incorporation of radiolabelled amino acids into freshly excised pieces of scalp epidermis showed that some of the polypeptides had relatively greater contents of glycine and serine than others. Radiolabelled methionine and leucine were, in contrast, incorporated more or less uniformly into all the polypeptides. After incubation with 32P-labelled orthophosphate, relatively more intense labelling by 32P was observed in the higher molecular weight bands of each family. The most basic of the isoelectric variants in each case did not take up phosphate, implying that at least some of the variation in charge was due to different degrees of phosphorylation. Polyadenylated RNA isolated from scalp epidermis was translated in an RNA-dependent reticulocyte haemolysate system followed by immunoprecipitation and electrophoresis. The polypeptides isolated by using anti-(human scalp prekeratin) immunoglobulin G had similar electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels to authentic prekeratin polypeptides, but had different isoelectric properties. This suggested that the products of keratin gene expression undergo post-translational modification.     

Keratin metabolism in the epidermis and hair of mice with experimental diabetes]

An animal trial was performed using mice with streptozotocine-induced diabetes, with investigation of velocity of prekeratin and keratin biosynthesis and degradation using 14C-glycine, and evaluation of the content of -SH and -S-S groups in epidermal prekeratin. It has been found out that velocity of epidermal prekeratin and keratin in diabetic animals is higher than that in healthy group. SS and SH groups ratio in prekeratin in diabetic animals is 10 times as high as that in the control group. In the hair of diabetic mice an increased keratin turnover was observed as compared with the norm. The data testify that experimental diabetes manifests itself in increased intensity of keratin metabolism in epidermis and hair. These results may be used as the criteria in elaboration of non-invasive methods for diabetes diagnosis.     

Modification of human prekeratin during epidermal differentiation.

The polypeptide-chain components of human epidermal prekeratin and keratin were analysed by high-resolution SDS (sodium dodecyl sulphate)/polyacrylamide-gradient-gel electrophoresis. Size heterogeneity existed amongst prekeratin components and at least ten polypeptides, in the molecular-weight range 46,000-70,000, were observed in 0.1 M-citric acid/sodium citrate buffer (pH 2.65) extracts of scale epidermis. Prekeratin from scalp pilosebaceous ducts was identical with that from the contiguous epidermis, and no prekeratin was found in extracts of scale dermis. Prekeratin from plantar epidermis contained additional polypeptide chains, but only slight anatomical variation existed between the non-callus sites examined. Keratin differed from prekeratin in at least two major respects: (a) many major components did not co-electrophorese on high-resolution SDS/polyacrylamide slab gels, and (b) keratin, but not prekeratin, required denaturing and reducing conditions for extraction. Keratin extracted from scale epidermis after complete removal of prekeratin was identical with forearm stratum-corneum keratin. Palmar and plantar keratin contained additional polypeptide chains and had a different size distribution compared with forearm and scalp keratin components. Modification of prekeratin components to produce the keratin polypeptide profile occurred during epidermal differentiation, and these changes appeared to take place in the granular-layer region of the epidermis.     

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.     

Characterization of cells of amniotic fluids by immunological identification of intermediate-sized filaments: Presence of cells of different tissue origin

Summary Antibodies against intermediate-sized filaments, of the prekeratin or vimentin type, were used to investigate the presence of these filaments by indirect immunofluorescence microscopy in cultured and non-cultured amniotic fluid cells, in frozen sections of the placenta and in isolated cells of the amniotic epithelium. Two major classes of cells can be cultured from amniotic fluids, namely cells of epithelial origin containing filaments of the prekeratin type and cells of different origin which contain filaments of the vimentin type but are negative when tested with antibodies to epidermal prekeratin. The presence of prekeratin type filaments correlates with the morphology of colonies of amniotic fluid cell cultures in vitro as classified by Hoehn et al. (1974). Cells of E-type colonies are shown to be of epithelial origin. In contrast our data indicate a different origin of almost all cells of F-type colonies and of the large majority of cells of AF-type colonies. Cells of epithelial origin and positively stained with antibodies to epidermal prekeratin are occasionally scattered in F-type colonies and in variable percentages (up to 30%) in AF-type colonies. Surprisingly, cryostat sections of the amniotic epithelium and isolated groups of amniotic cells showed positive reactions with both antibodies to vimentin and prekeratin. The possibility that amniotic cells may be different from other epithelial cells in that they contain both types of filaments simultaneously already in situ is presently under investigation.Part of this work is included in the doctoral thesis of Irmgard Treiss to be submitted to the Faculty of Medicine of the University of Heidelberg     

Role of microtubules and intermediate filaments in maintaining the shape of epithelial cells

The role of microtubules and intermediate filaments in control of cell shape of cultured cells of hepatomas McA-RH-7777 and 27 was investigated. Indirect immunofluorescence with specific polyclonal antibodies against tubulin and monoclonal antibodies against prekeratin with molecular weight 49 kD and vimentin was used. Incubation of cells in colcemid, resulting in specific distribution of microtubules did not change either prekeratin or vimentin distribution in cells of both the hepatomas, but reversed polarization of elongated McA-RH-7777 cells. These data suggest that the effect of disruption of microtubular system on the cell shape is not mediated by alterations of intermediate filaments.     

Isolation and primary cultures of human intrahepatic bile ductular epithelium

Summary A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments.     

Mallory bodies: Lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Summary Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

Simultaneous Expression of Two Different Types of Intermediate Sized Filaments in Mouse Keratinocytes Proliferating in vitro

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

The relationship between glandular kallikrein and growth factor-processing proteases of mouse submaxillary gland.

Three highly specific trypsin-like proteases from mouse submaxillary gland; nerve growth factor gamma subunit, beta nerve growth factor-endopeptidase, and epidermal growth factor-binding protein were tested for kallikrein activity. Low molecular weight kininogen was purified from mouse plasma and used as substrate for the three enzymes, and the kinin released by the enzymes was assayed by its ability to induce contraction of isolated rat uterus. All three enzymes were found to have significant kininogenase activity, and the most active enzyme, beta nerve growth factor-endopeptidase, has activity comparable to authentic kallikreins from other glandular sources. Essentially all of the kininogenase activity of submaxillary gland co-purifies with beta nerve growth factor-endopeptidase. Hence, beta nerve growth factor-endopeptidase appears to be identical with submaxillary gland kallikrein. Nerve growth factor gamma subunit, epidermal growth factor-binding protein, and beta nerve growth factor-endopeptidase have similar amino acid compositions and molecular weights, and are immunologically similar. Comparison of published partial primary sequence data confirms our conclusion that nerve growth factor gamma subunit, epidermal growth factor-binding protein, and kallikrein are very closely related enzymes. It is postulated that these three enzymes are members of a larger family of similar enzymes, all of which are involved in the processing of precursors to polypeptide hormones and growth factors.     

Simultaneous expression of two different types of intermediate sized filaments in mouse keratinocytes proliferating in vitro.

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

The extraction and characterization of bovine epidermal alpha-keratin.

1. The alpha-fibrous protein (alpha-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the 'keratinized' stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of alpha-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.     

Structures Indicative of Keratinization in Lactating Cells of Bovine Mammary Gland

Keratohyalin granule-like aggregates and tangles of filaments similar to epidermal prekeratin fibrils have been observed in lactating cells of bovine mammary epithelium. The concomitant occurrence of such structures, which are characteristic of early stages of keratinization, with typical secretory products such as casein micelles demonstrates that keratinization can take place in functional secretory cells. The observations are discussed in relation to current concepts of keratinization in epidermal and in non-epidermal epithelia.     

Epidermal proteins of cultured human and bovine keratinocytes.

Bovine and human epidermal cells were cultured on mitomycin C treated fibroblasts. The cells were carried through four passages and found to synthesize fibrous proteins and insoluble cell envelopes. Acid buffer soluble fibrous protein, prekeratin, and urea soluble fibrous protein were both identified and the latter was the major component in older cultures. Some of the prekeratin polypeptides of intact tissue were not found in cultured cells, but the ones that were present corresponded to those of whole tissue. X-ray diffraction, amino acid analysis and immunological techniques were used to establish that the polypeptides were keratins. The insoluble cell envelopes had a higher proline and 1/2 cystine content than the fibrous protein, similar to what is found in whole epidermis. Histidase, a characteristic enzyme marker of whole epidermis, was not observed in cultured cells. These studies indicate that differentiation occurs in cultured cells but it may not be as complete as in intact tissue.     

Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species.

Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue

Hepatocytes of mammalian liver are known to contain intermediate-sized filaments of tonofilament morphology. Unlike many other epithelial cells, including cultured hepatocytes and hepatoma cells, hepatocytes present in normal liver tissue have been reported not to react, in significant intensity, with various preparations of antibodies to human and bovine epidermal prekeratin [2,6]. We have therefore examined, by biochemical and immunological methods, the cytoskeletal composition of hepatocytes grown in the body.Cytoskeletal preparations from hepatocytes of mouse and rat liver tissue resistant to high salt buffer and Triton X-100 are enriched in tangles of intermediate filaments and contain, besides some residual microfilamentous actin, a characteristic set of polypeptides. One- and two-dimensional gel electrophoresis reveals the presence of two major cytokeratin components, which appear as ‘pairs’ of isoelectric variants (component A, M_r 55 000, apparent pI values, 6.40 and 6.45; component D, M_r 49000, apparent pI values 5.43 and 5.38), and five minor components (M_r range from 41000 to 53 000), most of them also as ‘pairs’ of polypeptides slightly different in isoelectric pH value. These polypeptide patterns are very similar in mouse and rat liver although some minor but significant differences have been noted between the two species. The polypeptide patterns of liver cytoskeletons are also similar to—but clearly not identical with—the cytoskeletal protein patterns observed in other epithelial tissues and cells, including various lines of cultured rat hepatocytes and hepatoma cells.Guinea pig antibodies raised against individual cytokeratin proteins of mouse liver and against certain prekeratin polypeptides present in desmosome-attached tonofilaments of bovine muzzle are described which differ from previously described prekeratin antibodies. These prekeratin antibodies not only react with filament bundles of the prekeratin type present in many cultured epithelial cells (e.g. murine HEL, human HeLa, rat kangaroo PtK2) and various epithelial tissues, but also allow the detection of the cytokeratin components present in parenchymal cells of liver and pancreas of various species, man included. Immunofluorescence microscopy on frozen sections of liver using these antibodies reveals a novel structure, i.e. a three-dimensional filament meshwork extending throughout the whole cytoplasm of the hepatocyte, with higher intensity of staining in pericanalicular regions.The results show that parenchymal cells of normal liver and pancreas contain filaments of the cytokeratin type that are related to but not identical with epidermal prekeratin. The hepatocyte filaments appear to be different from prekeratin-type filaments present in epidermis and several other epithelial cells, both in some antigenic determinants exposed and in polypeptide composition. Our findings support the concept of the existence of a family of intermediate filament proteins, cytokeratins, containing many different polypeptides that are expressed in different epithelial cells in certain characteristic subsets in a cell type-specific mode.     

Cloning and characterization of the yeast nuclear gene for subunit 5 of cytochrome oxidase

The nuclear gene COX5 coding for subunit 5 of cytochrome oxidase has been cloned by transformation of the cox5-1 mutant aE4-238/AL1 with a library of yeast genomic DNA. The recombinant plasmid pG46/ST2 bearing a nuclear DNA insert of 1.17 kilobase pairs restores the ability of cox5 mutants to respire and to synthesize a wild type subunit 5. The COX5 gene has been sequenced and determined to code for a 153-amino acid long protein with a molecular weight of 17,121. The amino-terminal 20 residues comprise the signal peptide. The sequence starting from residue 21 matches the partial sequence reported for the mature subunit 5. The sequence of the subunit 5 gene indicates that the mature protein has a molecular weight of 14,858 which agrees with previous size estimates based on electrophoretic migration. The primary sequence and polarity profile of yeast subunit 5 establishes that it is homologous to subunit 4 of bovine cytochrome oxidase.