Identification of proteolytic cleavage sites in the conversion of profilaggrin to filaggrin in mammalian epidermis

Profilaggrin consists of multiple filaggrin domains joined by linker segments which are removed during proteolytic conversion to filaggrin. Analysis of tryptic peptides of filaggrin defined a 26-residue linker segment when aligned on the amino acid sequence of one repeat unit of mouse profilaggrin deduced from a cDNA sequence (Rothnagel, J. A., Mehrel, T., Idler, W. W., Roop, D. R., and Steinert, P. M. (1987) J. Biol. Chem. 262, 15643-15648). Two types of linker segments were distinguished by their different susceptibility to thermolysin and by the presence of a Phe-Tyr-Pro-Val sequence in only one type. These data led to a model of profilaggrin in which the two types of linker segments alternate along the length of profilaggrin. This model provides a structural basis for the two stages of proteolytic processing seen in vivo. In the first stage intermediates accumulate which have several filaggrin domains still joined by linker segments lacking Phe-Tyr-Pro-Val. In the second stage, the other linker segments are cleaved and mature filaggrin domains are released. Proteolytic activity with specificity consistent with first stage cleavage was partially purified from rat epidermis. Chymostatin inhibited both the in vitro enzymatic activity and the processing of profilaggrin in a cultured rat keratinocyte cell line. The products formed in vitro were 3-5 kDa larger than intermediates produced in vivo, suggesting that the linker segments are cleaved at one end only. This implies the existence of a third protease which completes the removal of the linker segments.     

Identification of a rapidly labelled 350K histidine-rich protein in neonatal mouse epidermis

The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6-9 h lag period. A number of putative precursor HRPs in the 70-300 K molecular weight range were initially identified using short pulse labeling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [3H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100 degrees C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides

The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 500 000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26 000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the filaggrin sequence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phosphorylated in profilaggrin.     

A comparative study on wound-healing in neonatal and adult mouse epidermis in vivo

Abstract. A cut made into the back skin of either newborn or adult mice evokes, at both ages, a hyperproliferative response in the epidermis. Differences in the reaction of neonatal as compared with adult epidermis are found in the spatial distribution of proliferative activity as well as in its time course. The response in adult mouse epidermis is inhibited by local application of indomethacin, whereas the response of the newborn epidermis is not.     

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.     

Identification of murine type I keratin 9 (73 kDa) and its immunolocalization in neonatal and adult mouse foot sole epidermis

The foot sole epidermis of the fore and hind feet of the adult mouse contains an acidic (type I) mRNA-encoded 73-kDa keratin polypeptide which cannot be detected in any other skin site of the mouse integument. Western blot analysis using an antibody specific for the 64-kDa keratin 9 of human and bovine callus-forming epidermis [A. C. Knapp et al. (1986) J. Cell Biol. 103, 657-667] demonstrates that the 73-kDa keratin represents the murine analog of keratin 9 of man and cow. Concomitant investigations in two related rodent species indicate that the size of this keratin varies more among species than that of any other orthologous keratin. Histological examination of adult mouse foot sole skin reveals an extremely thick and undulated epidermis covering the apical portion of the six footpads, whereas the epidermal-dermal junction of the lateral walls of these nodular protuberances as well as that of the remainder of the foot sole skin is essentially flat. If sections of adult foot sole skin are investigated by indirect immunofluorescence with the keratin 9-specific antibody, intense cytoplasmic staining is restricted to the apical rete pegs of the footpad epidermis in which virtually all suprabasal cells express keratin 9. However, we also observed keratin 9-negative cell columns ascending straight above the tips of the dermal papillae and separating the keratin 9-positive rete pegs from each other. At the transition from the strongly undulated apical epidermis to the flat epidermis of the lateral walls of the footpads, keratin 9-positive cells loose their coherence and gradually disappear toward the inter-footpad epidermis. This intimate relationship between the morphogenesis of epidermal ridges and inter-ridges and the expression of keratin 9 is also visible in foot sole epidermis of neonatal mice. Here we observed the appearance of keratin 9-positive suprabasal cells concomitant with the onset of pronounced folding of the apical footpad epidermis by about Day 3 after birth. Our findings confirm the view that the expression of keratin 9 is characteristic of a highly specialized pathway of epidermal differentiation. We propose a hypothesis for keratin expression in skin sites which are subject to pronounced mechanical wear and tear.     

Identification of two distinct hybrid state intermediates on the ribosome

High spatial and time resolution single-molecule fluorescence resonance energy transfer measurements have been used to probe the structural and kinetic parameters of transfer RNA (tRNA) movements within the aminoacyl (A) and peptidyl (P) sites of the ribosome. Our investigation of tRNA motions, quantified on wild-type, mutant, and L1-depleted ribosome complexes, reveals a dynamic exchange between three metastable tRNA configurations, one of which is a previously unidentified hybrid state in which only deacylated-tRNA adopts its hybrid (P/E) configuration. This new dynamic information suggests a framework in which the formation of intermediate states in the translocation process is achieved through global conformational rearrangements of the ribosome particle.     

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.     

DNA synthesis rate changes during the S phase in mouse epidermis

The in vivo DNA synthesis rate throughout the S phase of mouse epidermal cells was investigated. Epidermal basal cells were isolated at various times of the day from normal animals injected with [3H]TdR 30 min before sacrifice, and from pulse-labelled animals with regenerating and growth-inhibited epidermis. The cells were analysed by DNA flow cytometry combined with cell sorting. Cells from successive fractions of the S phase were sorted on glass slides and subjected to quantitative [3H]TdR autoradiography. The results confirmed the presence of unlabelled (slowly replicating) cells in the S phase, the proportion of which was circadian stage-dependent with minimum values at midnight and in the early morning. The DNA synthesis rate throughout the S phase showed a general trend with high values in the mid-fractions, a pattern which was similar in normal and in growth perturbed epidermis. In the early morning the DNA synthesis rate pattern was bimodal with maxima both in the first and second half of the S phase, with a corresponding trough in mid-S. At this time of day the cell progression rate through S is at its maximum, indicating a relationship between the overall DNA synthesis rate and the rate distribution pattern through S.     

Excess tyrosine stimulates eumelanin and pheomelanin synthesis in cultured slaty melanocytes from neonatal mouse epidermis

The mouse slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase (DCT). The reduced DCT activity inhibits melanosome maturation and reduces the melanin content in the skin, hair and eyes. It is not known whether eumelanin and pheomelanin synthesis in slaty melanocytes is modulated by melanogenic factors. In this study, to address this point, epidermal melanocytes derived from 0.5-, 3.5- and 7.5-day-old wild-type mice (Dct(+)/Dct(+) at the slaty locus) and from congenic mice mutant (Dct(slt) /Dct(slt) at that locus) were cultured in serum-free primary culture with or without additional L-tyrosine (Tyr). The content of melanin was measured by high-performance liquid chromatography in the cultured melanocytes as well as culture supernatants in serum-free primary culture. L-Tyr was found to increase the content of pheomelanin in addition to eumelanin in cultured slaty melanocytes and cuture supernatants at all ages tested. The eumelanin and pheomelanin contents in culture supernatants were greater than in cultured melanocytes. The eumelanin and pheomelanin contents in culture supernatants from 7.5-day-old slaty melanocytes in the presence of L-Tyr were greater than those from wild-type melanocytes. These results suggest that the inhibition of eumelanin synthesis by the slaty mutation can be partly restored by the addition of excess L-Tyr. Eumelanin and pheomelanin may accumulate with difficulty in slaty melanocytes and be easily released from them during skin development. L-Tyr may stimulate this release.     

Expression of cytoplasmic antigens linked to orthokeratosis during the development of parakeratosis in newborn mouse tail epidermis

An unusual example of two different types of epidermal differentiation occurs side by side in adult mouse tail. The neonate shows only orthokeratotic differentiation, but develops parakeratotic scales in precise patterns over the first two weeks of life, retaining orthokeratotic differentiation at the necks of hair follicles. Certain human IgG antibodies from patients receiving bone marrow transplants bind only to cytoplasmic components of orthokeratotic epithelium. As markers of orthokeratotic differentiation in indirect immunofluorescent studies, these antibodies allowed the timing and location of the change in epidermal differentiation. A specific loss of 'orthokertotic' antigens in the natural switch from orthokeratosis to parakeratosis was demonstrated. The sequence of the orthokeratotic antigens disappearance suggested that the switch to parakeratotic differentiation occurred at a supra-basal level.     

Targeted somatic mutagenesis in mouse epidermis

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.