Two-dimensional analysis of proteins of rat oral epithelia and epidermis.
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Animals↗
Biomedical subjects
B A Dale
Publications and source records attributed to B A Dale.
Distribution of histidine-rich basic protein, a possible keratin matrix protein, in rat oral epithelium.
The indirect fluorescent-antibody technique was used to locate histidine-rich basic protein, filaggrin. In the newborn, immunofluorescence was seen in the cornified layers and in keratohyalin granules throughout the mouth using antibody specific for epidermal filaggrin, a distribution similar to that in epidermis where it is thought that filaggrin functions as the keratin matrix protein. In the adult immunofluorescence was in keratohyalin granules of palate, buccal and tongue epithelium but in the stratum corneum was limited to the soft palate with weak, patchy areas in the densely keratinized epithelium of the hard palate and tongue. Immunofluorescence was delineated at the boundary between the soft and hard palates. A protein apparently identical to epidermal filaggrin was identified in extracts of newborn palate by its mobility on sodium dodecyl sulphate-polyacrylamide gels and subsequent reaction with the antibody used for immunofluorescent studies. This protein was not detected in extracts of adult oral epithelia. Both newborn and adult tissues contained high mol. wt cross-reactive protein, suggestive of the filaggrin-precursor protein extractable from keratohyalin granules. The distribution of filaggrin was consistent with its function as a keratin matrix protein in the newborn oral epithelium and some less densely keratinized regions of the adult. However, in the adult mouth, filaggrin is not detectable in the stratum corneum of the most densely keratinized regions. Thus, the protein must be lost or its antigenic sites altered with maturation of the animal, depending on the type and extent of keratinization.
Aging↗
Abnormal epidermal keratinization in the repeated epilation mutant mouse.
Repeated epilation (Er) is a radiation-induced, autosomal, incomplete dominant mutation in mice which is expressed in heterozygotes but is lethal in the homozygous condition. Many effects of the mutation occur in skin: the epidermis in Er/Er mice is adhesive (oral and nasal orifices fuse, limbs adhere to the body wall), hyperplastic, and fails to undergo terminal differentiation. Skin from fetal +/+, Er/+ and Er/Er mice at ages pre- and postkeratinization examined by light, scanning, and transmission electron microscopy showed marked abnormalities in tissue architecture, differentiation, and cell structure; light and dark basal epidermal cells were separated by wide intercellular spaces, joined by few desmosomes, and contained phagolysomes. The numbers of spinous, granular, and superficial layers were highly variable within any given region and among various regions of the body. In some areas, 2-8 layers of granular cells, containing large or diminutive keratohyalin granules, extended to the epidermal surface; in others, the granular layers were covered by several layers of partially keratinized or nonkeratinized cells. In rare instances, a single or small group of cornified cells was present among the granular layers but was not associated with the epidermal surface. Both the granular and nonkeratinized/partially keratinized upper epidermal layers Er/Er skin gave positive immunofluorescence with antiserum to the histidine-rich, basic protein, filaggrin. Proteins in epidermal extracts from +/+, Er/+ and Er/Er mice were separated and identified by radio- and immunolabeling techniques. The Er/Er extract was missing a 26.5- kdalton protein and had an altered ratio of bands in the keratin region. The 26.5-kdalton band was histidine-rich and cross-reacted with the antiserum to rat filaggrin. Several high molecular weight bands present in both Er/Er and +/+ extracts also reacted with the antiserum. These are presumed to be the precursors of filaggrin and to account for the immunofluorescence om Er/Er epidermis even though the product protein is absent. The morphologic and biochemical data indicated that the genetic defect has a general and profound influence on epidermal differentiation, including alteration of two proteins (filaggrin and keratin) important in normal terminal differentiation, tissue architecture, and cytology. Identification of epidermal abnormalities at early stages of development (prekeratinization) and defective structure of other tissues and gross anatomy suggest that the mutation is responsible for a defect in same regulatory step important in many processes of differentiation and development.
Animals↗
Repeated epilation (ER): a semidominant autosomal gene reducing synthesis of skin filaggrin in mice.
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Abnormalities, Multiple↗
Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels. Diagonal peptide mapping of proteins from epidermis.
A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.
Animals↗
Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis.
The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.
Animals↗
Characterization of a class of cationic proteins that specifically interact with intermediate filaments.
We describe a class of cationic structural proteins that associate specifically with intermediate filaments (IF) but not with other types of cytoskeletal proteins. These proteins, for which the term filaggrin is introduced, are isolated from the stratum corneum of mammalian epidermis. They are species-distinct proteins; for example, rat and mouse filaggrin have different molecular weights and amino acid compositions, but are nevertheless chemically and functionally ver similar. They interact in vitro with the IF several different types of cells to form large fibers or macrofibrils in which many IF are highly aligned in parallel arrays. Stoichiometric analyses suggest that two molecules of filaggrin bind to each three-chain building block of the IF, possibly by ionic interactions with the coiled-coil alpha-helical regions of the IF.
Amino Acids↗
Induced hypotension with a mixture of sodium nitroprusside and trimetaphan camsylate.
Thirty patients undergoing induced hypotension were given a 10:1 mixture of trimetaphan camsylate and sodium nitroprusside by intravenous infusion. A readily controllable hypotension was produced with considerable reduction in the dosage of these drugs compared to their used individually. There were no cases of prolonged hypotension.
Adolescent↗
Congenital abnormalities of the skin: future directions.
The inherited abnormalities of the skin are easily recognizable and readily accessible. They involve structures of different embryonic origins, are amenable to study in in vitro systems, are available for study in laboratory and domestic animal models, and are eminently suited to the type of interdisciplinary research presented at this workshop. Biochemists, geneticists, and clinicians in various disciplines all profit by this exchange of information. Ultimately, the gain is for the patient in the treatment and prevention of inherited dermatologic disorders.
Cell Differentiation↗
A phosphorylated keratohyalin-derived precursor of epidermal stratum corneum basic protein.
The precursor of the cationic protein called stratum corneum basic protein (SCBP) has been purified from extracts of epidermal keratohyalin granules. The precursor and SCBP have virtually identical amino acid compositions and similar reactivities to antibody to SCBP. Peptide mapping studies using elastase in sodium dodecyl sulfate (SDS)-polyacrylamide gels suggest that the primary amino acid sequences of SCBP and the precursor are similar. The proteins differ in their mobilities on SDS-polyacrylamide gel electrophoresis and in their net charges. The lower pI of the precursor (6.9) appears to be due to 15 to 20 mol of covalently bound phosphate per mol of protein; SCBP contains no phosphate.
Amino Acids↗
Stratum corneum basic protein: an interfilamentous matrix protein of epidermal keratin.
The stratum corneum basic protein (SCBP) aggregates specifically with keratin filaments, forming macrofibrils in which the filaments are highly aligned. The appearance of the macrofibrils formed in vitro is similar to the keratin pattern in the intact stratum corneum, suggesting that SCBP is an interfilamentous matrix substance of epidermal keratin (Dale et all., Nature, 276: 729, 1978, see Ref. 13). Electron microscope and biochemical analyses were performed to determine the optimum molar ratio of SCBP and keratin filaments (average subunit molecular weight, 62,000). The optimum mixtures, in which all of the proteins were aggregated into macrofibrils, contained 1-2 moles of SCBP per 3 moles filament subunits. At ratios below this value, free filaments were observed by electron microscopy; above this value, excess SCBP was observed in the supernate by immunologic assay. The precursor of SCBP has been purified after extraction from keratohyalin granules in 1 M potassium phosphate. The precursor is immunologically similar to SCBP. Their amino acid compositions are essentially identical. However, in contrast to SCBP, the precursor is an insoluble, neutral protein which contains approximately 10 moles of phosphate per mole protein. This phosphate is covalently bound to the precursor and is not due to the presence of nucleic acid. During the conversion of a granular cell to a cornified cell, the precursor, present in keratohyalin granules, must be converted by proteolysis and/or dephosphorylation to active SCBP which aggregates with keratin filaments and forms the complex of filaments embedded in a matrix.
Amino Acids↗
Evidence of a precursor form of stratum corneum basic protein in rat epidermis.
The fully differentiated anucleate cells of the stratum corneum of newborn rat epidermis contain a cationic protein called stratum corneum basic protein (SCBP). This protein has a molecular weight (49 000) and an amino acid composition similar to a protein extracted from the less differentiated cell layers of the epidermis. Pulse--chase experiments with radiolabeled histidine were undertaken to test the possiblity that SCBP is derived from a preexisting protein. A protein of 52 000 daltons is rapidly but transiently labeled in extracts of the less differentiated cell layers. As the amount of label in the 52 000-dalton protein decreases, an increase in radiolabel is observed in extracts of the fully differentiated cells. This label is found in SCBP, a protein of lower molecular weight (49 000) than that initially labeled. These proteins are immunologically related and both are resistant to cyanogen bromide cleavage. They differ in apparent molecular weight on sodium dodecyl sulfate--polyacrylamide gels and in their net charge. The results are consistent with the conversion of a precursor protein into SCBP.
Amino Acids↗
Immunologic cross-reaction of stratum corneum basic protein and a keratohyalin granule protein.
Antiserum to the stratum corneum basic protein (SCBP) of newborn rat epidermis was used to test for cross-reactive proteins in extracts of skin and in tissue sections. The antibody reacts strongly with epidermal extracts but very poor with dermal extracts. Buffer extracts of epidermis give a reaction of partial identity or identity with the antigen, SCBP. Urea extracts of isolated stratum corneum give a reaction of identity with SCBP. When the proteins of these extracts are separated by SDS polyacrylamide gel electrophoresis, the immunoreactive peak in the 4 M urea extract corresponds to the 49,000 MW SCBP. The immunoreactive peak in the 1 M potassium phosphate extract corresponds to a 52,000 MW protein. This protein is rapidly and transiently labeled after injection of 3H-histidine into newborn rats, in contrast to the SCBP which is labeled after a 5-hr lag. The 52,000 MW protein appears to be an immunologically related precursor of the SCBP. Immunoreactive proteins were localized in tissue sections by the indirect immunoperoxidase method. A strong positive reaction was seen in keratohyalin granules and in the stratum corneum. The reaction of keratohyalin granules corroborates the extraction of a cross-reactive protein by 1 M potassium phosphate, a method for extraction of keratohyalin granules from epidermis. These results are consistent with the hypothesis that a protein of 52,000 MW is present in keratohyalin and is converted to the SCBP (49,000 MW) concomitantly with the conconversion of a granular cell to a stratum corneum cell.
Animals↗
Assembly of stratum corneum basic protein and keratin filaments in macrofibrils.
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Animals↗
Purification and characterization of a basic protein from the stratum corneum of mammalian epidermis.
A basic protein has been isolated and purified from the stratum corneum of newborn rat epidermis. This protein is referred to as stratum corneum basic protein. It was purified by ion-exchange chromatography on DE-52 and CM-52 cellulose. The protein has a molecular weight of 50 000 on sodium dodecyl sulfate-polyacrylamide gels. It is composed of one polypeptide chain and contains no detectable carbohydrate. The protein has an isoelectric point in the range of pH 9-10, but decomposes during isoelectric focusing giving rise to a polypeptide of less than 10 000 daltons. Amino acid analysis reveals high quantities of glutamic acid, glycine, serine, arginine and relatively high levels of histidine, with these five amino acids composing 74% of the total residues. The amino acid analysis is very similar to histidine-containing keratohyalin proteins isolated from the granular layer of epidermis by several investigators. The stratum corneum basic protein differs from fibrous proteins isolated from the same cell layer with respect to net charge, amino acid composition, and molecular weight. The protein does not react with antibody to the fibrous protein. The basic protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein.
Amino Acids↗
Initial characterization of the proteins of keratinized epithelium of rat oral mucosa.
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Amino Acids↗
The identification of fibrous proteins in fetal rat epidermis by electrophoretic and immunologic techniques.
Two proteins have been identified in extracts of fetal rat skin which are related to the two major fibrous proteins of newborn rat stratum corneum. The relative amount of these proteins increases daily from the 16th to the 20th day (d) of gestation when judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoelectrophoresis using antibody to the purified fibrous protein. Two-dimensional analysis by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis demonstrates that these two proteins are the only cross-reactive species in the fetal skin from 16d to 19d development. Some additional lower-molecular-weight components can be detected at 20d and 21d. In double-diffusion analysis, cross-reactive proteins in 19d fetal extracts show partial identity but have fewer antigenic sites than proteins in 20d extracts. The 20d protein shows a reaction of identity with purified newborn fibrous protein. Immunofluorescence studies on fetal skin support the prescence of cross-reacting components at 16d development related to the newborn fibrous protein. Intensity of fluorescence increases at 18d and 20d in the spinous and granular cell cytoplasm and in the keratohyaline granules. The stratum corneum, first seen at 20d, is intensely fluorescent. The cellular localization and time of appearance of the cross-reactive proteins suggest that they may be associated with tonofilaments.
Animals↗
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins of newborn rat skin. I. Cell strata and nuclear proteins.
The proteins obtained from separated cells of neonatal rat dermis, four cell populations of epidermis, and an epidermal nuclear preparation were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Comparison of the results of the insoluble proteins of the dermis and epidermis show no similarity of the major protein bands, indicating the effective separation of the dermis and epidermis and absence of cross-contamination. The gels of the soluble proteins of the basal, spinous, and granular layers of the epidermid are very similar. Only the pattern of bands of the cornified cells differs in that some of these bands are absent and at least three new bands are present. The insoluble proteins have specific differences in the protein content related to the cell structure. An example is the nuclear protein bands which correspond with the most prominent bands in the gels of basal and spinous layer proteins, but are absent, with the possible exception of one band, from gels of cornified cell proteins.
Animals↗