Cytokeratins of the bovine hoof: classification and studies on expression

The bovine hoof has been examined as a model for the study of keratinized skin appendages. We characterized the keratin polypeptides of hoof bed and matrix and compared them to epidermis using two-dimensional electrophoresis and immunoblot techniques. Both hoof tissues express keratins 6 and 16 (as described by Franke et al. (1981) J. Mol. Biol. 153, 933-959) and b2 and a1-4 which are previously undescribed proteins unique to the bovine hoof. Keratins of hoof matrix and bed share one or more common antigenic components as defined by immunoblot analysis. Hoof matrix expresses keratins 7 and 14, which are absent in hoof bed, and also expresses a greater number of isoelectric variants of keratin 6. Biopsies of hoof bed and matrix transplanted onto athymic mice both made hard hoof and underwent active keratin synthesis as evidenced by incorporation of [3H]leucine. Indirect immunofluorescence studies of the grafts showed that they had the histology and immunoreactivity previously noted for hoof bed and matrix. The two-dimensional gel electrophoretic patterns of both grafts were similar and expressed keratins b2 and a1-4. We conclude that a unique group of keratins exists in hoof. Furthermore, while hoof matrix is the major contributor to hard hoof, hoof bed epidermis maintains the capacity to make hard hoof and may contribute to the synthesis of the hoof plate in vivo. The ability to graft hoofs onto athymic mice provides an opportunity for the study of a number of aspects of hoof formation.     

Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system

The translation products synthesized in a rabbit reticulocyte lysate system, from total cellular mRNA from the human epithelial cell-line ME-180, have been examined. Keratin proteins are prominent among these translation products, and they precisely coelectrophorese in sodium dodecyl sulfate-polyacrylamide gels with keratins purified from the cells. Type-I, acidic, keratins which are acetylated in vivo, are also acetylated by the reticulocyte lysate. Examination by two-dimensional electrophoresis, of two acidic keratins known to be phosphorylated in vivo reveals that only one of these proteins is phosphorylated in the lysate system. Phosphorylation of this protein occurs after release of the completed polypeptide chain from the ribosome. The protein phosphorylated by the lysate is known to be the only ME-180 phosphokeratin modulated by cyclic AMP, reflecting in vitro the differential modification of ME-180 keratins in vivo.     

Multiple keratins of cultured human epidermal cells are translated from different mRNA molecules.

The keratins of human epidermis consist of several distinct proteins of different molecular weight that can be separated by gel electrophoresis in the presence of sodium dodecylsulfate. These proteins are very similar in structure, as determined by amino acid composition, polypeptide mapping and immunological reactivity. At least five such keratins are found in cultured human epidermal cells. We have examined the mode of synthesis of these keratins by isolating the poly(A)⁺ mRNA from the cultured cells and translating it in a reticulocyte system. All the keratins characteristic of the cultured cells were synthesized in vitro from the mRNA; they were identified by their molecular weight and by polypeptide mapping. No evidence was found for any precursor of substantially greater molecular weight. A study of the kinetics of synthesis showed that all the keratins were labeled within 2 min after the addition of ³⁵S-methionine to a translation system preincubated with epidermal mRNA, and the relative intensities of labeling did not change upon further incubation. It was therefore improbable that one keratin could be the precursor of another. The mRNAs of the large keratins could be completely separated from those of the small keratins by gel electrophoresis under either native or denaturing conditions. Within the group of small mRNAs, each had a different mobility although resolution was incomplete. Upon translation, the mRNA fractions yielded different groups of keratins corresponding in molecular weight to their counterparts in the cells. Consequently, most if not all keratins of different size are translated from different messages. The approximate sizes of the mRNA molecules for different keratins were determined from their mobility under denaturing conditions. The size of the mRNA was not always proportional to the size of the encoded keratin, demonstrating the existence of noncoding segments of different length in the different mRNA molecules.     

The cDNA sequence of a Type II cytoskeletal keratin reveals constant and variable structural domains among keratins

We present the cDNA and amino acid sequences of a cytoskeletal keratin from human epidermis (Mr = 56K) that belongs to one of the two classes of keratins (Type I and Type II) present in all vertebrates. In these two types of keratins the central approximately 300 residue long regions share approximately 30% homology both with one another and with the sequences of other IF proteins. Within this region, all IF proteins are predicted to contain four helical domains demarcated from one another by three regions of beta-turns. The amino and carboxy termini of the Type II keratin are very different from those of microfibrillar keratins and other nonkeratin IF proteins. However, they contain unusual glycine-rich tandem repeats similar to the amino terminus of the Type I keratin. Thus the size heterogeneity among keratins appears to be a result of differences in the length of the terminal ends rather than the structurally conserved central region.     

Different polypeptides form the intermediate filaments in bovine hoof and esophageal epithelium and in aortic endothelium

Polypeptides that form 10-nm filaments in vitro were isolated from three different bovine tissues: the viable portion of the hoof epithelium, the epithelium of the esophagus, and cultured endothelial cells derived from aorta. The seven polypeptides from hoof, the two from esophagus, and the one from endothelial cells were different with respect to mobility in SDS polyacrylamide gels and/or limited proteolytic digestion. Peptide maps of the different filament-forming polypeptides (FFP's) showed that none of the smaller FFP's was a fragment of any of the larger FFP's. Several isomobile fragments were found in the peptide maps of different FFP's, suggesting that they might contain regions of amino acid sequence homology. We present a hypothesis that suggests how the different 10-nm filament-forming proteins may be related.     

The molecular heterogeneity and diversity of reptilian keratins

Summary Reptile keratins produce complex electrophoretic patterns, contain a number of size classes, and contain protein fractions analogous to the fractions found in other keratins. Thus, reptile keratins are similar to the heterogenous keratins of birds and mammals, and quite different from amphibian epidermal keratins. This heterogeneity may be related to the multiple functions performed by the epidermis of these organisms.The chemical diversity of reptile keratins seems to depend on the morphological differences between the tissues in which they occur. This situation is also found among these proteins in mammals and birds suggesting that keratin diversity is related to the morphological and presumably functional differentiation of epidermal tissues. The distribution of the keratin fractions in each tissue contributes to this diversity but the significance of these fractional differences is uncertain.A comparison of the half-cystine and glycine content of vertebrate andØ keratins suggests that the andØ proteins of reptiles may be related to the soft keratins of mammals and amphibians. Mammalian hard keratins probably represent a uniquely derived group of proteins which are unlike the other vertebrate keratins. The presence of a high sulphur matrix component in both hard mammalian and reptilian Ø keratins may represent some form of molecular convergence which provides these distantly related proteins with similar physical or organizational properties.     

The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.     

Interactions of intermediate filament proteins from wool.

Filaments of wool are heteropolymers formed by interaction of type I and type II intermediate filament (IF) proteins. There are four proteins in each of these two classes. Interaction of the reduced wool IF proteins was studied by two-dimensional electrophoresis which showed that complexes between type I and type II proteins were formed in solution at urea concentrations below 6 M. Complex formation between the carboxymethyl derivatives of wool IF proteins was studied using a filter binding assay in which radio-labelled individual components were allowed to react under various conditions with SDS-PAGE separated components after transfer to nitrocellulose. The results suggested that (i) absolute type specificity of interaction was maintained, (ii) fine specificity, i.e. preferential reaction between specific components is observed, (iii) wool IF proteins (hard keratins) also react, with the same type specificity, with soft keratins isolated from cow snout, (iv) the initial step in the polymerization sequence that leads to filament formation yields heterodimers.     

Proteins of the hard keratins of echidna, hedgehog, rabbit, ox and man

In the accompanying paper it has been shown that two major groups of proteins (low-sulphur and high-sulphur) of ovine wool, horn, and hoof contain similar components although the overall proportions of the groups of proteins and the relative proportions of components within the groups may show significant differences. In the present paper it has been shown for five other species (echidna, hedgehog, rabbit, ox and man) that the hard keratins produced by one animal contain the same groups of protein components but in different relative proportions. The wide apparent differences in the type and relative proportions of the the low-sulphur components which comprise the major constituent proteins of the microfibrils suggest that microfibrils can tolerate a considerable variation in the constituent proteins and still produce functional structures. The low-sulphur protein components are sufficiently well resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis to make this procedure potentially useful for animal identification and classification.     

Immunofluorescence confocal laser scanning microscopy and immuno-electron microscopic identification of keratins in human materno-foetal interaction zone

We show here that at least 5 keratin proteins are present in villous trophoblast and the same 5 in extravillous trophoblast. A further 14 tested were undetectable in these tissues. In contrast, 10 of the 19 keratins tested were present in amniotic epithelium. The marking of amniotic epithelium on the one hand, as distinct from villous and extravillous trophoblast on the other, can be achieved using 5 keratins (K4, 6, 13, 14 and 17) with a mixture of positive and negative discrimination that is expected, in combination, to be highly sensitive. All the specific keratins identified in trophoblast were apparently up-regulated on the pathway to extravillous trophoblast. Co-ordinated differentiation at the molecular expression level is indicated by this finding. The relevant keratins are K5, 7, 8, 18 and 19. Specific keratins have been identified that are down-regulated in villous trophoblast in pre-eclamptic pregnancy. This difference between healthy and pre-eclamptic chorionic villous trophoblast keratin expression was statistically significant in 4 out of the 5 keratins. This was not the case for the extravillous trophoblast at the immunofluorescence confocal level but significant differences were obtained using immunogold electron microscopy. We suggest that the villous trophoblast in pre-eclamptic placentae is cytoskeletally weaker with respect to the filaments made from these specific proteins and that this is one reason why, in pre-eclampsia, trophoblast is deported in greater quantity than in healthy placentae.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation.

Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope.     

The quest for the function of simple epithelial keratins

Simple epithelial keratins K8 and K18 are components of the intracellular cytoskeleton in the cells of the single-layered sheet tissues inside the body. As members of the intermediate filament family of proteins, their function has been a matter for debate since they were first discovered. Whilst there is an indisputable case for a structural cell-reinforcing function for keratins in the mutilayered squamous epithelia of external barrier tissues, some very different stress-protective features now seem to be emerging for the simple epithelial keratins. Even the emerging evidence of pathological mutations in K8/K18 looks very different from mutations in stratified epithelial keratins. K8/K18-like keratins were probably the first to evolve and, whilst stratified epithelial (keratinocyte) keratins have diversified into a large group of keratins highly specialised for providing mechanical stability, the simple epithelial keratins have retained early features that may protect the internal epithelia from a broader range of stresses, including osmotic stress and chemical toxicity.     

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.     

The expression of keratin genes in epidermis and cultured epidermal cells

Cultured human epidermal cells and human stratum corneum (callus) contain a number of keratins of different molecular size, but the size distribution is not the same in the two cases. To characterize these keratins in more detail, we compared them by amino acid analysis, immunological reactivity and one-dimensional peptide mapping (Cleveland et al., 1977). No differences in amino acid composition could be detected among keratins of stratum corneum differing in molecular size by as much as 50%, suggesting that some repeating structure may be present in these molecules. Examination of polypeptide fragments produced by partial enzymatic hydrolysis showed strong similarities among all the keratins of stratum corneum and of cultured epidermal cells, even extending to the keratins of rodents; but the keratins of similar size, whether of stratum corneum or cultured cells, were more closely related than keratins of different size. This conclusion was supported by studies of the immunological reactivity of the keratins.How the epidermal cell generates a family of keratins is a problem of considerable interest. The differences in size and structure between the keratins of stratum corneum and cultured epidermal cells suggest that the epidermal cell can modify the expression of its keratin genes.     

Characterization of the keratin filament subunits unique to bovine snout epidermis.

Steinert [Biochem. J. (1975) 149, 39-48] reported that the alpha-keratin polypeptides (the subunits of the intracellular keratin filaments) of bovine hoof and snout epidermis are the same. We now demonstrate that this is not so: in addition to the seven polypeptides previously identified in hoof epidermis, snout epidermis also contains at least three other polypeptides of higher molecular weight. These unique polypeptides were isolated, purified and characterized. They are chemically and structurally very similar to the other polypeptides of bovine epidermis and readily polymerize in vitro with them to form native-type epidermal keratin filaments.     

Translational products of mRNAs coding for non-epidermal cytokeratins

Total RNA and poly(A)+ RNA were isolated from tissues and cultured cells of various mammalian species (bovine muzzle epidermis and bladder urothelium; rat hepatoma cells; human cell lines HeLa, MCF-7 and A-431) and examined by translation in vitro using the reticulocyte lysate system. Polypeptides were separated and identified by two-dimensional electrophoresis and cytokeratins were selectively enriched from the translation assays by co-polymerization with added heterologous cytokeratins. In all three species, non-epidermal cytokeratins A, D and mol. wt. 40,000 (corresponding to numbers 8, 18 and 19 of the human cytokeratin catalog of Moll et al., 1982) were identified as translation products capable of co-polymerization with epidermal keratins. Several other basic and other acidic cytokeratins were also identified as translational products. In addition, two unidentified polypeptides (mol. wt. 52,000 and 43,000) which were minor polypeptides in cytoskeletons and translation assays were found to be specifically enriched in co-polymers with bovine epidermal keratins. The results indicate that many, perhaps all, non-epidermal cytokeratins characteristic of simple epithelia are genuine products of translation and that their diversity is not due to post-translational modification or processing. These findings, taken together with observations of in vitro translation of epidermal mRNAs, suggest that the diversity of cell type-specific expression of the different members of the cytokeratin polypeptide family is largely due to the cell type-specific synthesis of diverse mRNAs.     

Expression of new proteins of the intermediate filament protein family in differentiating F9 embryonal carcinoma cell cytoskeleton

Differentiation of F9 embryonal carcinoma cells by retinoic acid treatment results in extraembryonic endoderm-like cells. The effects of this process on the protein composition of the intermediate filaments were studied by two-dimensional gel electrophoresis and by immunoblotting. By this approach, two new proteins induced in differentiating cells, p57 and p54, were identified in cytoskeletal preparations enriched in intermediate filaments. The 57-kDa protein could be resolved into at least three components (pI 5.6-5.9), and the 54-kDa protein into at least two components (pI approximately 5.6). Both proteins reacted with a monoclonal antibody which recognizes an antigenic determinant common to all intermediate filaments. Based on these results, the two proteins were identified as members of the intermediate filament protein family. Partial digestion with V8 protease showed that p57 was different from vimentin, another intermediate filament protein present in these cells. p57 and p54 were also immunodetected by a polyclonal anti-keratin anti-serum, which suggests that these proteins share some homology with the keratins. These two proteins are different from the endodermal cytoskeletal protein A and B (endo A and endo B) keratins, which are known to be present in extraembryonic endoderm-like cells. They were also more abundant than endo A and endo B in differentiating F9 embryonal carcinoma cells, but almost undetectable in terminally differentiated extraembryonic endoderm-like cells, where endo A and endo B are readily detectable. This suggests that p57 and p54 have a different pattern of expression than endo A and endo B.     

Novel insights into intermediate-filament function from studies of transgenic and knockout mice

Summary The function of intermediate-filament (IF) proteins has been a matter of speculation for a long time. Now, the analysis of genetically altered mice is contributing to the understanding of their function. While the initial analysis of knockout mice supports the global view that keratins in epidermis and desmin in muscle serve an important structural function by protecting these tissues against mechanical stress, the detailed examination of these and other mice suggests that IF are more than passive cytoskeletal proteins. This is highlighted by mice with deficiencies for keratins in internal epithelia, vimentin, GFAP, or neurofilament proteins. These lack overt phenotypes expected as a result of cytoskeletal deficiency but show defects compatible with a role of IF in protecting tissues against toxic and other forms of stress. Moreover, the first round of gene replacement experiments suggests that keratins from internal epithelia are unable to take the place of their epidermal counterparts. The development of mice with point mutations, paralleled by the mutation analysis of human diseases and the characterization of IF-associated proteins will be instrumental to understand why evolution has produced such a diverse gene family to encode simple 10 nm diameter filaments.     

The mesonephric (wolffian) and paramesonephric (müllerian) ducts of golden hamsters express different intermediate-filament proteins during development

We analysed the expression of intermediate-filament proteins in the developing mesonephric duct (the precursor of the male genital ducts) and the paramesonephric duct (the precursor of the female genital ducts) of golden-hamster embryos using immunohistochemical methods. Embryos were investigated from the early stages of duct development, i.e. at 9.5 days post conceptionem (dpc), through sexual differentiation, until birth (15.5 dpc). Monospecific antibodies to vimentin or keratins 7, 8, 18 or 19 as well as two keratin antibodies that are pan-epithelial in human tissues were tested. Both ducts expressed vimentin to some degree from their early stages (mesonephric duct from 9.5 dpc onwards; paramesonephric duct from 10.5 dpc onwards) until birth. No keratins were detectable at these earliest stages. In the mesonephric duct, keratins 7, 18 and 19 appeared simultaneously at 10.5 dpc and persisted until birth. In the paramesonephric duct, only keratin 18 was detectable at first (at 12.0 dpc), with the expression of keratins 7 and 19 being delayed until 14.5 dpc. This feature was irrespective of sexual differentiation, which begins at 11.0 dpc, so that, in males, these keratins appeared on cue, even though the paramesonephric duct was regressing at this time. The expression of keratin 8 could not be demonstrated in either duct using the antibodies tested in our study. By 14.5 dpc, the differentiated male mesonephric duct and the differentiated female paramesonephric duct exhibited the same intermediate-filament protein pattern (weak vimentin expression and strong expression of keratins 7, 18 and 19), in spite of differences in the intermediate-filament protein patterns exhibited by the two ducts during early development. These different programmes of intermediate-filament protein regulation do not support the concept that the mesonephric duct makes a cellular contribution to the paramesonephric duct during the development of the latter.