Studies on the synthesis and degradation of a high molecular weight, histidine-rich phosphoprotein from mammalian epidermis

The synthesis and subsequent fate of the histidine-rich proteins, which form a major component of keratohyalin granules in mammalian epidermis, have been studied in the guinea-pig and new-born rat. In both species the protein first synthesised is of very high molecular weight, approximately 340 000. It is short-lived and breaks down to lower molecular weight proteins 1-2 days after its synthesis. These smaller proteins differ in the two species. In the guinea-pig, the high molecular weight protein breaks down to proteins of molecular weight 250 000 and 200 000, which are themselves unstable and break down to low molecular weight species, probably amino acids. The initial breakdown of the high molecular weight protein coincides with the dispersion of the keratohyalin granules and the transition of the granular cell into the stratum corneum. This high molecular weight histidine-rich protein has been purified to homogeneity, despite its instability to several treatments during purification. The protein is highly phosphorylated, containing 6 mol% of phosphoserine, but is otherwise very basic. The possibility that dephosphorylation of the protein produces highly basic matrix proteins in the stratum corneum is discussed.     

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.     

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.     

Identification of a Rapidly Labelled 350K Histidine-Rich Protein in Neonatal Mouse Epidermis

Abstract. 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 labelling 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°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.     

Endogenous lectin from terminally differentiated epidermal cells

We isolated a concanavalin A (Con-A)-binding glycoprotein from human stratum corneum by nonionic detergent extraction, lectin affinity chromatography, and preparative gel electrophoresis. This glycoprotein migrates as a single band at 40 kilodaltons at sodium-dodecyl-sulfate gel electrophoresis with or without the presence of 2-mercaptoethanol. It was shown to have a heterogeneous distribution between pH 5.6 and 7.6 by isoelectric focusing. The glycoprotein is histidine rich (10.4%) but is distinct from other histidine-rich proteins (epidermal filaggrin and the histidine-rich glycoprotein from serum). It does not bind to lectins specific for L-fucose or alpha-D-galactose. We prepared a monospecific polyclonal antibody to the 40-kilodalton glycoprotein; at the ultrastructural level, it cytoimmunolocalizes exclusively to the membranes of the stratum corneum. A unique feature of the glycoprotein is that it is an endogenous lectin: it hemagglutinates trypsinized and gluteraldehyde-fixed rabbit erythrocytes. The inhibition of its hemagglutination was found to be greatest with amino sugars, down to a saccharide concentration of 10(-5) mM. Such a high affinity of binding at the cell surface suggests that this glycoprotein is a major carbohydrate-binding, cross-linking molecule that holds adjacent corneocytes together in the stratum corneum. We hypothesize that this lectin plays a role in the adhesion and desquamation of the stratum corneum.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

Stratum corneum lipid phase transitions and water barrier properties

In mammals, the outer skin layer, the stratum corneum, is the ultimate barrier to water loss. In order to relate barrier function to stratum corneum structure, samples from porcine skin were investigated by using differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and water permeability techniques. Results of DSC and IR studies show that stratum corneum lipids undergo thermal transitions between 60 and 80 degrees C similar to lipid thermotropic transitions seen in a variety of synthetic and biological membranes. Results of water flux experiments performed under conditions similar to those of the DSC and IR studies show an abrupt change in permeability at about 70 degrees C. At low temperatures, water flux values are similar to those obtained for human skin in vivo, yielding an activation energy of 17 kcal/mol, in excellent agreement with values obtained for water flux through a variety of lipid biomembranes. In contrast, at temperatures above about 70 degrees C, water flux is characterized by an activation energy only slightly higher than that of free diffusion, suggesting that the stratum corneum offers little diffusional resistance under these conditions. These combined results suggest that increased disorder in stratum corneum lipid structure, brought about by thermotropic transitions, results in dramatically altered diffusional resistance of this tissue to water flux. Thus, as found for numerous biological membranes, water flux and lipid order in porcine stratum corneum are inversely related.     

The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein

A small proportion of the protein of stratum corneum of human epidermal callus is insoluble even when boiled in solutions containing sodium dodecylsulfate and a reducing agent. This protein is present in the cornified envelope, a structure located beneath the plasma membrane. When cornified envelopes were dissolved by exhaustive proteolytic digestion and the products analyzed by chromatography, approximately 18% of the total lysine residues were found as the cross-linking dipeptide ?-(γ-glutamyl) lysine.Labeled cornified envelope protein was synthesized by human epidermal keratinocytes allowed to differentiate terminally in culture. The extent of cross-linking, determined from the proportion of radioactive lysine in ?-(γ-glutamyl) lysine after exhaustive proteolysis, was similar to that in stratum corneum. The properties of the cornified envelopes (insolubility in detergent and reducing agents, and solubility following proteolytic digestion) are readily explained by a structure consisting of a cross-linked protein lattice.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

The acyl-CoA binding protein is required for normal epidermal barrier function in mice

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ～50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.     

Two polypeptide chain constituents of the major protein of the cornified layer of newborn rat epidermis.

The insoluble component of stratum corneum of rat epidermis yields two major bands after extraction with 8 M urea-mercaptoethanol-dithiothreitol. The ratio of these two bands is about 1:1 in terms of protein stain intensity and S-[14C]carboxymethyl label. Both polypeptides were purified to homogeneity by DE-52-cellulose, sodium dodecyl sulfate hydroxylapatite C column chromatography, and preparative DodSO4-polyacrylamide gel electrophoresis. The heavier polypeptide contains 30% alpha helix and the lighter contains 27% alpha helix as determined by circular dichroism studies. Both are sensitive to Pronase and resistant to trypsin, collagenase, and elastase. The lighter chain is stable to pepsin but the heavier can be partially degraded to a smaller polypeptide with a molecular weight similar to that of light chain. Amino acid analysis shows that the light chain contains 12 more tyrosine residues than does the heavy chain, suggesting that the light chain is not generated from the heavy chain. However, the two chains may have a common peptide region. Antiserum prepared against the heavier polypeptide can be completely absorbed by purified lighter polypeptide and vice versa indicating that both chains have some common antigenic determinants. Antibody against either chain can cross-react with the stratum corneum and keratohyalin granules in the epidermis of newborn rat as indicated by fluorescent microscopic observation. Similarly, this antibody also cross-reacts with the cell surface or the contents of spinous and granular cells, and very weakly with basal cells, indicating that the two proteins may be present in the lower strata as well as the stratum corneum.     

Histidine-rich peptide selection and quantification in targeted proteomics

Agarose based immobilized copper (II) affinity chromatography (Cu(II)-IMAC) in tandem with reversed-phase chromatography was applied to a yeast protein extract. Histidine-rich peptides were selected and, in the process, samples were substantially simplified prior to mass spectral analysis. Samples of proteins from the yeast extract at fermentation time periods of 2.5 and 10 h were compared quantitatively used the GIST protocol. Acylation of the N-terminus of tryptic peptides with N-acetoxysuccinamide was used to globally label and quantify relative protein concentration changes. Together with N-terminal acylation, an imidazole elution procedure allowed histidine-rich peptides to be preferentially selected by Cu(II)-IMAC. An inverse labeling strategy was applied to increase reliability in determinations of up- and down-regulation. It was found that the concentration of some histidine-rich proteins changed in excess of 4-fold during fermentation. These proteins covered a wide range of molecular weight and pI values.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.     

Sphingolipid activator proteins are required for epidermal permeability barrier formation

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.     

Genomic organization, structure and possible function of histidine-rich proteins of malaria parasites

The current status of histidine-rich proteins in malaria parasites with regard to their genomic organization, protein structure and function is discussed, one of such protein present in an avian malaria parasite Plasmodium lophurae contains about 73% histidine and called as HRP (histidine-rich protein). Among human malaria parasites, in Plasmodium falciparum, only three such proteins have been described, namely knob protein also known as knob associated histidine-rich protein (KP or KAHRP), soluble histidine-alanine rich protein (soluble HARP or PfHRP II) and small histidine-alanine rich protein (SHARP) containing 8, 35 and 30% histidine contents respectively. With rapid emergence of powerful tools in molecular biology the genes of all these histidine-rich proteins have been cloned and sequenced within a short period of time. The genomic organizations of all these proteins are very much similar to each other, in each case the gene contains a signal peptide coding sequence (exon 1) followed by an intron. This intron is followed by the main coding region (exon 2) which has no further intervening sequences. In the main coding region of each gene, the histidine-rich sequences start after 25-30 amino acids from N-terminal end (75-90 nucleotides from 5' in exon 2). All the three histidine-rich proteins of P. falciparum share some homology with the HRP of P. lophurae; they all cross react with anti HRP and incorporate higher amount of exogenous histidine. The relationship between KP and HRP resides in the repeated polyhistidine sequences, (His) 6-9, from the core of the multiple tandem repeats of HRP, whereas, the peptide Ala-His-His is commonly shared by HRP and two other proteins of P. falciparum (soluble HARP and SHARP).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus.

Klebsiella aerogenes UreE, one of four accessory proteins involved in urease metallocenter assembly, contains a histidine-rich C terminus (10 of the last 15 residues) that is likely to participate in metal ion coordination by this nickel-binding protein. To study the function of the histidine-rich region in urease activation, ureE in the urease gene cluster was mutated to result in synthesis of a truncated peptide, H144* UreE, lacking the final 15 residues. Urease activity in cells containing H144* UreE approached the activities for cells possessing the wild-type protein at nickel ion concentrations ranging from 0 to 1 mM in both nutrient-rich and minimal media. In contrast, clear reductions in urease activities were observed when two ureE deletion mutant strains were examined, especially at lower nickel ion concentrations. Surprisingly, the H144* UreE, like the wild-type protein, was readily purified with a nickel-nitrilotriacetic acid resin. Denaturing polyacrylamide gel electrophoretic analysis and N-terminal sequencing confirmed that the protein was a truncated UreE. Size exclusion chromatography indicated that the H144* UreE peptide associated into a homodimer, as known for the wild-type protein. The truncated protein was shown to cooperatively bind 1.9 +/- 0.2 Ni(II) ions as assessed by equilibrium dialysis measurements, compared with the 6.05 +/- 0.25 Ni ions per dimer reported previously for the native protein. These results demonstrate that the histidine-rich motif is not essential to UreE function and is not solely responsible for UreE nickel-binding ability. Rather, we propose that internal nickel binding sites of UreE participate in urease metallocenter assembly.