Immuno- and enzyme-histochemical detection of phosphoprotein phosphatase in rat epidermis

A phosphoprotein phosphatase (PPase: EC 3.1.3.2) was recently purified from rat epidermis. The enzyme dephosphorylates phosphoprotein, and its properties, such as pH optimum, inhibitor spectrum, and Fe2+ activation, differ from those of other soluble phosphatases. We investigated in 2-day-old rat skin the distribution of immunologically detectable PPase and intracellular localization of PPase activity. The reaction of rabbit monospecific anti-PPase IgG was identified in granular and cornified cells by the avidin-biotin complex method. For activity staining, basic principles of the Gomori lead-salt method and azo dye technique with the substrates p-nitrophenylphosphate (p-NPP) and alpha-naphthyl phosphate (NP), respectively, were modified according to the biochemical properties of PPase activity which is resistant to formalin, Na tartrate, and NaF. Activity was detectable in granular cells including keratohyalin granules and the lower strata of cornified cells. The activity was inhibited by 1 mM CuSO4 and enhanced by a mixture of 0.5 mM FeSO4 and 1 mM ascorbic acid. We consider that PPase may be involved in dephosphorylation of histidine-rich proteins in granular and cornified cells and may play a key role in intracellular catabolism associated with epidermal cell differentiation.     

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)     

唾液富组氨酸蛋白

唾液富组氨酸蛋白（histidine-rich protein, HRPs）是由人类和高等灵长动物的腮腺、下颌下腺、舌下腺分泌到唾液中的碱性小分子多肽,分子量为4 kD左右,其氨基酸序列富含组氨酸,分子表面带有正电荷和稳定的α螺旋结构．唾液富组氨酸蛋白具有广谱抗菌性,对革兰氏阴性菌、革兰氏阳性菌及真菌都有杀伤作用,其抗菌机制是作用线粒体呼吸通路,诱发ATP的释放．此外,唾液富组氨酸蛋白具有促进伤口愈合的能力,其作用机制与细胞内信号调节激酶1/2通路相关．同时,它也具有金属离子结合能力、机体免疫调节能力,其作用机制与分子表面本身结构特点和阻滞下游信号通路相关．唾液富组氨酸蛋白是一种天然的功能性蛋白质,有望应用在相关疾病的治疗上,本文对唾液富组氨酸蛋白的结构特点、功能机制、应用前景作一简要论述,为后续功能蛋白质的研发和应用奠定了理论基础．     

Preferred heme binding sites of histidine-rich glycoprotein

The heme binding sites of rabbit histidine-rich glycoprotein (HRG), 94 kDa, were studied with rose bengal (RB), a fluorescein derivative that associates with histidine residues. Difference absorbance spectra indicate that HRG binds RB at two thermodynamically preferred sites (Kd approximately 2 microM) that are spectroscopically equivalent. Up to 18-22 equiv of RB can also be bound by a set of lower affinity sites. Mesoheme is capable of displacing RB from the two preferred sites (Kd = 0.6 microM) and provides evidence that the two sites are not identical. Two peptides isolated from plasmin-digested HRG, one 35-kDa peptide rich in histidine (approximately 30 mol %) and one 15-kDa peptide relatively poor in histidine (approximately 4 mol %), also bind RB and mesoheme. The two preferred RB binding sites of HRG are located on the 15-kDa histidine-poor peptide and the lower affinity "class" of sites on the 35-kDa histidine-rich peptide. Mesoheme or RB quenches the tryptophan fluorescence of HRG and the histidine-poor peptide with an apparent binding stoichiometry near 2. Fluorescence quenching also indicates that 1-2 equiv of Cu(II) binds to the 15-kDa peptide, and absorbance spectroscopy provides evidence that Cu(II) is capable of displacing heme from the peptide. The fluorescence lifetimes of RB, determined by phase-modulation fluorometry, indicate that the two preferred sites in the histidine-poor domain are more apolar than the more numerous sites located in the histidine-rich region of the protein.     

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.     

Effect of dietary histidine content on the change in content of skin urocanic acid isomers in hairless mice irradiated with ultraviolet B

Hairless mice were fed with a 10% amino acid mixture diet (control diet, 0.42% histidine content), the control diet without histidine (histidine-free diet), or the control diet rich in histidine (histidine-rich diet; histidine content, 4.2%) for 32 days. They were irradiated with UV light of 312 nm for 30 min, and skin samples were periodically taken for measuring the urocanic acid isomers. Total urocanic acid isomers were decreased by UV irradiation in all the three groups, the recovery being the fastest in the histidine-rich group. The percentage increase in cis-urocanic acid/total urocanic acid was quickly increased by UVB irradiation. The recovery of the ratio was slightly higher in the histidine-rich group, although the total urocanic acid level was higher in the histidine-rich group than in the others. Therefore, the absolute cis-urocanic acid content in the skin was almost the same among the three groups. These results indicate that the increased histidine intake strengthened UVB protection without any decrease in immune suppression.     

rLj-RGD3全RGD缺失基因重组突变体——rLj-112的分子克隆及其广谱抑真菌活性

Lj-112是日本七鳃鳗RGD毒素蛋白野生型Lj-RGD3的RGD全缺失基因突变体,其一级结构具有富组氨酸的特点．富组氨酸糖蛋白具有抑菌功能．为了研究Lj-112是否具有抑菌作用,使用基因合成和原核表达的方法,获得了纯化重组蛋白rLj-112,将其对不同菌种进行抑菌试验.结果表明,以野生型rLj-RGD3、RGD模体与组氨酸全缺失突变体rLj-26为对照,rLj-112有较广谱的抑菌活性,其中对白念球菌的最低抑菌浓度（MIC）为7 μmol/L,对稻瘟病菌的MIC值为7.5 μmol/L,对毛癣菌和禾谷病菌的MIC值分别为11.9 μmol/L和29.9 μmol/L．可见,rLj-112能作为抗真菌药物候选,为提高农作物的生产质量和减轻人类感染疾病的用药压力奠定了基础．     

Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.     

Cutting edge structural protein from the jaws of Nereis virens

The fang-like jaws of the marine polychaete Nereis virens possess remarkable mechanical properties considering their high protein content and lack of mineralization. Hardness and stiffness properties in the jaw tip are comparable to human dentin and are achieved by extensive coordination of Zn (2+) by a histidine-rich protein framework. In the present study, the predominant protein in the jaw tip, Nvjp-1, was purified and characterized by partial peptide mapping and molecular cloning of a partial cDNA from a jaw pulp library. The deduced amino acid sequence revealed an approximately 38 kDa histidine-rich protein rich in glycine and histidine (approximately 36 and 27%, respectively) with no well-defined repetitive motifs. The effects of pH and metal treatment on aggregation, secondary structure, and hydrodynamic properties of recombinant Nvjp-1 are described. Notably, Zn treatment induced the formation of amyloid-like fibers.     

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.     

Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in Ichthyosis vulgaris

Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo.     

Histidine-rich glycoprotein exerts antibacterial activity

Histidine-rich glycoprotein (HRGP), an abundant heparin-binding protein found in plasma and thrombocytes, exerts antibacterial effects against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fluorescence studies and electron microscopy to assess membrane permeation showed that HRGP induces lysis of E. faecalisbacteria in the presence of Zn2+ or at low pH. Heparin blocked binding of the protein to E. faecalis and abolished antibacterial activity. Furthermore, truncated HRGP, devoid of the heparin-binding and histidine-rich domain, was not antibacterial. It has previously been shown that peptides containing consensus heparin-binding sequences (Cardin and Weintraub motifs) are antibacterial. Thus, the peptide (GHHPH)4, derived from the histidine-rich region of HRGP and containing such a heparin-binding motif, was antibacterial for E. faecalis in the presence of Zn2+ or at low pH. The results show a previously undisclosed antibacterial activity of HRGP and suggest that the histidine-rich and heparin-binding domain of HRGP mediates the antibacterial activity of the protein.     

Studies on human kininogens. I. Isolation, characterization, and cleavage by plasma kallikrein of high molecular weight (HMW)-kininogen.

1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein.     

High affinity binding of serum histidine-rich glycoprotein to nickel-nitrilotriacetic acid: the application to microquantification

Histidine-rich glycoprotein (HRG) is a serum protein with possible pluripotent activities. In this study, a method for the quantification of rabbit histidine-rich glycoprotein (rHRG) was developed based upon the high affinity binding profile of rHRG to nickel-nitrilotriacetic acid (Ni-NTA), an improved chelation agent. When the binding profile of Ni-NTA for whole serum proteins was assessed by Western blotting, Ni-NTA exhibited the binding specificity only to rHRG even after washing with 20 mM imidazole, owing to the unusual amounts of histidine residues in rHRG. In the following experiments, the rHRG immobilized onto a microplate with specific antibody was determined spectrophotometrically with peroxidase-labeled Ni-NTA. This method permitted evaluation of rHRG concentrations ranging from 1.0 to 100 ng/ml, and was actually applicable to the monitoring of rHRG in Resource Q-fractionated serum preparations. Also, the co-addition of L-histidine into the incubation mixture significantly diminished the specific binding between rHRG and Ni-NTA. These findings indicate the potential usefulness of this method for the specific measurement of small amounts of rHRG and for understanding the roles of abundant histidine residues in rHRG-metal cation interaction.     

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.     

Plasmodium falciparum malaria: association of knobs on the surface of infected erythrocytes with a histidine-rich protein and the erythrocyte skeleton

Plasmodium falciparum-infected erythrocytes (RBC) develop surface protrusions (knobs) which consist of electron-dense submembrane cups and the overlying RBC plasma membrane. Knobs mediate cytoadherence to endothelial cells. Falciparum variants exist that lack knobs. Using knobby (K+) and knobless (K-) variants of two strains of P. falciparum, we confirmed Kilejian's original observation that a histidine-rich protein occurred in K+ parasites but not K- variants (Kilejian, A., 1979, Proc. Natl. Acad. Sci. USA, 76:4650-4653; and Kilejian, A., 1980, J. Exp. Med., 151:1534-1538). Two additional histidine-rich proteins of lower molecular weight were synthesized by K+ and K- variants of both strains. We used differential detergent extraction and thin-section electron microscopy to investigate the subcellular location of the histidine-rich protein unique to K+ parasites. Triton X-100, Zwittergent 314, cholic acid, CHAPS, and Triton X-100/0.6 M KCl failed to extract the unique histidine-rich protein. The residues insoluble in these detergents contained the unique histidine-rich protein and electron-dense cups. The protein was extracted by 1% SDS and by 1% Triton X-100/9 M urea. The electron-dense cups were missing from the insoluble residues of these detergents. The electron-dense cups and the unique histidine-rich protein appeared to be associated with the RBC skeleton, particularly RBC protein bands 1, 2, 4.1, and 5. We propose that the unique histidine-rich protein binds to the RBC skeleton to form the electron-dense cup. The electron-dense cup produces knobs by forming focal protrusions of the RBC membrane. These protrusions are the specific points of attachment between infected RBC and endothelium.     

Differentiation of snake epidermis, with emphasis on the shedding layer

Little is known about specific proteins involved in keratinization of the epidermis of snakes. The presence of histidine-rich molecules, sulfur, keratins, loricrin, transglutaminase, and isopeptide-bonds have been studied by ultrastructural autoradiography, X-ray microanalysis, and immunohistochemistry in the epidermis of snakes. Shedding takes place along a shedding complex, which is composed of two layers, the clear and the oberhautchen layers. The remaining epidermis comprises different layers, some of which contain beta-keratins and others alpha-keratins. Weak loricrin, transglutaminase, and sometimes also iso-peptide-bond immunoreactivities are seen in some cells, lacunar cells, of the alpha-layer. Tritiated histidine is mainly incorporated in the shedding complex, especially in dense beta-keratin filaments in cells of the oberhautchen layer and to a small amount in cells of the clear layer. This suggests the presence of histidine-rich, matrix proteins among beta-keratin bundles. The latter contain sulfur and are weakly immunolabeled for beta-keratin at the beginning of differentiation of oberhautchen cells. After merging with beta cells, the dense beta-keratin filaments of oberhautchen cells become immunopositive for beta-keratin. The uptake of histidine decreases in beta cells, where little dense matrix material is present, while pale beta-keratin filaments increase. During maturation, little histidine labeling remains in electron-dense areas of the beta layer and in those of oberhautchen spinulae. Some roundish dense granules of oberhautchen cells rich in sulfur are negative to antibodies for alpha-keratin, beta-keratin, and loricrin. The granules eventually merge with beta-keratin, and probably contribute to the formation of the resistant matrix of oberhautchen cells. In conclusion, beta-keratin, histidine-rich, and sulfur-rich proteins contribute to form snake microornamentations.     

The complete sequence of the gene for the knob-associated histidine-rich protein from Plasmodium falciparum.

'Knobs' at the surface of erythrocytes infected with mature stages of Plasmodium falciparum are believed to be important in adherence of these cells to capillary walls. They contain at least one parasite protein, designated the knob-associated histidine-rich protein (KAHRP). We present here the sequences of a cDNA and chromosomal clone that predict the complete sequence of KAHRP. The gene contains a single intervening sequence, located at the 3' boundary of the hydrophobic core of a putative signal sequence. Exon two encodes a short region that is rich in histidine as well as two separate regions of repetitive sequence, the 5' repeats (five copies related to SKKHKDNEDAESVK) and the 3' repeats (seven copies related to SKGATKEAST). These repeat blocks were both shown to bear epitopes recognized by the human immune system during natural infection by expressing them separately in Escherichia coli, and reacting human antibodies affinity-purified on lysates of the resulting clones with the corresponding synthetic oligopeptides. The 3' end of the molecule, presumably the repetitive region, is a site of size variation in KAHRP from different isolates.     

THE CHEMICAL NATURE OF KERATOHYALIN GRANULES OF THE EPIDERMIS

Keratohyalin granules were isolated in the native form from the epidermis of newborn rats by the use of citric acid and a detergent. The isolated granules revealed a fine granular substructure in the electron microscope similar to that seen in situ. Analyses of amino acids by automated column-chromatography showed that proline and cystine are present in large proportions whereas histidine is present in a small amount. Accordingly, it was concluded that keratohyalin represents a sulfur-rich amorphous precursor of the horny cell content, rather than a sulfur-poor side product of the keratinization process, or a unique histidine-rich protein as proposed by in situ histochemical and radioautographic studies.