1. Human Peptidylarginine Deiminase Type III: Molecular Cloning and Nucleotide Sequence of the cDNA, Properties of the Recombinant Enzyme, and Immunohistochemical Localization in Human Skin 
Takuya Kanno, Masakazu Shiraiwa, Hidenari Takahara 
Journal of Investigative Dermatology 
Volume 115, Issue 5, Pages (November 2000)
DOI: /j x
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2. Figure 1
Cloning strategy of human PAD type III cDNA by reverse transcription–PCR, 5′-RACE and 3′-RACE. Products A, B, and C showed cDNA fragments produced by PCR amplification. The clones phPAD3–1, phPAD3–2, and phPAD3–3 were obtained by reverse transcription–PCR. phPAD3–4 and phPAD3–5 were cloned by 5′-RACE and 3′-RACE, respectively. Horizontal arrows denote primers used for reverse transcription–PCR, 5′-RACE, and 3′-RACE amplification. These sequences were combined to construct the composite cDNA in which the open box and lines indicate the coding and noncoding sequences, respectively. Only the relevant restriction site (KpnI) is shown.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
Nucleotide sequence and deduced amino acid sequence of human PAD type III. The DNA sequence is numbered from the first nucleotide of the cDNA. The numbers are on the right of each line. The deduced amino acid sequence is displayed below the nucleotide sequence in one letter code starting from the putative initiation methionine. A putative polyadenylation signal is underlined. The amino acid sequence indicated by wave line is that of a synthetic peptide for producing anti-hPAD3 antibody.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
Sequential fractionation of PAD activity from an extract of E. coli harboring pKKhPAD3. (A) DEAE-Sepharose Fast Flow column after an ammonium sulfate fractionation step. (B) Fractionation of the PAD peak from (A) on an aminohexyl-Bio Gel A15m column. PAD activity of each fraction was measured with Bz-L-Arg-O-Et as a substrate. The fractions indicated by horizontal bar in (B) were pooled and stored at 4°C until use.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Amino acid sequence comparison of PAD type III of different species. The sequences are aligned by using the Malign program of GENETYX-MAC. The identical residues that conserved in at least three gene products are boxed. The Abbreviations used in the figure are: mPAD3, mouse PAD type III; rPAD3, rat PAD type III; sPAD, sheep PAD type III; hPAD, human PAD type III. The accession numbers of the sequence data cited for the comparison are as follows: mouse PAD type III, AB013849; rat PAD type III, D88034; sheep PAD type III, U85264; human PAD type III, AB
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6. Figure 5
SDS–PAGE of fractions in each purification step of the recombinant human PAD type III. Aliquots of samples were subjected to SDS–PAGE and stained with Coomassie Brilliant Blue. Lane M, molecular weight markers; lane 1, crude extract (25 μg of protein); lane 2, ammonium sulfate fractionation step (25 μg); lane 3, DEAE-Sepharose Fast Flow fractionation step (25 μg); lane 4, aminohexyl-Bio Gel A15m fractionation step (2 μg). Purified recombinant PAD type III was indicated by arrowhead.
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7. Figure 6
Time course of deimination of filaggrin repeat unit and THH-8 by human PAD type III. Each protein was deiminated by the enzyme as described in Materials and Methods. At the indicated times, the reaction was terminated by ethylenediamine tetraacetic acid, and the citrulline residues in each preparations were determined by amino acid analysis. Conversion rate of arginine residues to citrulline residues of each protein (○, filaggrin repeat unit; •, THH-8) was shown.
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8. Figure 7
PAGE analyses of deimination of filaggrin repeat unit and THH-8 by human PAD type III. Each protein was deiminated by the enzyme as described in Materials and Methods. After the appropriate incubation period, an aliquot of each reaction mixture was subjected to SDS–PAGE (A, C) and native PAGE (B, D) and stained with Coomassie Brilliant Blue. (A, B) PAGE patterns of filaggrin repeat unit incubated with human PAD type III. (C, D) PAGE patterns of THH-8 incubated with human PAD type III. Lane M, molecular weight markers; lane 1, 0 min; lane 2, 10 min; lane 3, 20 min; lane 4, 40 min; lane 5, 80 min; lane 6, 360 min; lane 7, 720 min; lane 8, 1440 min. –, cathode; +, anode.
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9. Figure 8
Monospecific recognition of anti-hPAD3 antibody toward PAD type III isoform. Four different recombinant mouse PAD isoforms and recombinant human PAD type III (0.3 μg of each protein), extracts from cultured human epidermal keratinocyte and hair follicle (10 μg of each protein) were resolved on SDS–PAGE and blotted on to a polyvinylidene difluoride membrane. The membrane was stained with Ponceau S (A) for protein and immunoblotted with anti-hPAD3 antibody (B). Lane 1, mouse PAD type I, lane 2, mouse PAD type II; lane 3, mouse PAD type III; lane 4, mouse PAD type IV; lane 5, human PAD type III; lane 6, extract from cultured human epidermis; lane 7, extract from human hair follicle. Arrowhead shows the immunoreactive band of human PAD type III. The positions of protein standards run in the same experiment are indicated on the left: myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa).
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10. Figure 9
Immunofluorescence staining of human hair follicle with anti-hPAD3, anti-trichohyalin, and anti-keratin 14 antibodies. (A–C, H–J) Longitudinal sections of hair follicles. (D–G, K–N) Transverse sections that are roughly corresponded to the area stated by numerals in C and J, respectively. The correspondences of each transverse section with the longitudinal sections are as follows: 1–D, 2–E, 3–F, 4–G in (C); 1–K, 2–L, 3–M, 4–N in (J). (A) Immunofluorescence staining with anti-hPAD3 antibody. (B) Immunofluorescence staining with anti-trichohyalin antibody. (C–G) Double immunofluorescence stainings with anti-hPAD3 and anti-trichohyalin antibodies. (H) Control staining with the preimmune serum. (I) Immunofluorescence staining with anti-keratin 14 antibody. (J–N) Double immunofluorescence stainings with anti-hPAD3 and anti-keratin 14 antibodies. Notes: PAD type III was coexpressed with trichohyalin in the IRS between supramatrix and keratogenous zone and in medulla. The type III enzyme was also localized to the ORS and cuticle of the hair. irs, inner root sheath; ors, outer root sheath; m, medulla; ch, cuticle of the hair; hs, hair shaft. Scale bar: 50 μm.
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11. Figure 10
Immunofluorescence staining of human epidermis and ORS at the infundibulum region with anti-hPAD3, anti-keratin 14, and anti-filaggrin antibodies. (A) Immunofluorescence staining with anti-hPAD3 antibody. (B) Double immunofluorescence stainings with anti-hPAD3 and anti-keratin 14 antibodies. The yellow color in the basal layer of the epidermis indicates that the expression of the PAD type III starts at the early stage of epidermal proliferation. (C) Double immunofluorescence stainings with anti-hPAD3 and anti-filaggrin antibodies. Most of the granular and cornified cells containing filaggrin show red color. A part of the granular cells facing out on the spinous cell layer turned yellow color, suggesting that the PAD type III enzyme partly colocalizes with filaggrin in these cells. epi, epidermis; ors, outer root sheath, ifb, infundibulum region; b, basal cell layer; g, granular cell layer; c, cornified cell layer. Scale bar, 200 μm.
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