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Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

Meyer-Hoffert U, Wu Z, Schröder JM - PLoS ONE (2009)

Bottom Line: Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin.LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5.At sites of plantar hyperkeratosis, LEKTI-2 expression was increased.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

ABSTRACT
Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

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Molecular identification of the Spink9 gene.(A) Schematic physical map of human SPINK genes locus (5q33.1). Genes are ordered from centromere (left hand side) to telomere (right hand side). (B) Schematic diagram of the Spink9 gene, based on its cDNA isolated from foreskin-derived keratinocyte identified by RT-PCR. It consists of four exons and three introns. The positions of the exons (boxes) and introns (curve lines) of Spink9 are deduced by comparing its full-length cDNA sequence with the corresponding genomic DNA. 5′/3′-UTRs and coding sequences are indicated by gray- and green-filled boxes, respectively. (C) The full-length cDNA sequence of Spink9 and its predicted protein sequence. The N-terminal signal peptide (residues 1–16; underlined) and the Kazal domain (residues 32–86; double-underlined) were detected with the SMART algorithm. The poly(A) signal site was coloured green. (D) Common characteristics of Lekti2 and Lekti. The alignment of the Kazal domains of Lekti-2 and Lekti domains 2 and 15 were generated by using M-COFFEE, displayed by using GeneDoc and shown in the down panel. The middle panel shows a schematic pattern of the typical Kazal domain including conserved tyrosine residue (Y) and disulfide bonds [14]. # represents the residue at the P1 site. The residue numbers spacing the cysteine residues are indicated on the top panel for the Kazal domain, LEKTI-2 and the LEKTI domains 2 and 15, respectively.
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pone-0004372-g002: Molecular identification of the Spink9 gene.(A) Schematic physical map of human SPINK genes locus (5q33.1). Genes are ordered from centromere (left hand side) to telomere (right hand side). (B) Schematic diagram of the Spink9 gene, based on its cDNA isolated from foreskin-derived keratinocyte identified by RT-PCR. It consists of four exons and three introns. The positions of the exons (boxes) and introns (curve lines) of Spink9 are deduced by comparing its full-length cDNA sequence with the corresponding genomic DNA. 5′/3′-UTRs and coding sequences are indicated by gray- and green-filled boxes, respectively. (C) The full-length cDNA sequence of Spink9 and its predicted protein sequence. The N-terminal signal peptide (residues 1–16; underlined) and the Kazal domain (residues 32–86; double-underlined) were detected with the SMART algorithm. The poly(A) signal site was coloured green. (D) Common characteristics of Lekti2 and Lekti. The alignment of the Kazal domains of Lekti-2 and Lekti domains 2 and 15 were generated by using M-COFFEE, displayed by using GeneDoc and shown in the down panel. The middle panel shows a schematic pattern of the typical Kazal domain including conserved tyrosine residue (Y) and disulfide bonds [14]. # represents the residue at the P1 site. The residue numbers spacing the cysteine residues are indicated on the top panel for the Kazal domain, LEKTI-2 and the LEKTI domains 2 and 15, respectively.

Mentions: To identify the gene corresponding to the amino acid sequence, a BLAT search with the N-terminal 25-residue sequence of the novel peptide (where×was replaced by the cysteine residue) against the April 2003 human genome assembly localized this sequence to a chromosome 5 clone RP11-373N22 on 5p33.1 (Fig. 2A). Subsequent analysis of the retrieved RP11-373N22 DNA sequence identified two putative exons exactly encoding the isolated peptide (Fig. 1). Based on the generated theoretical partial DNA sequence, gene-specific primers were designed to perform 3′- and 5′-RACE. The full-length cDNA sequence (453 bp) was completed by combining the overlapping sequences from each PCR product and then confirmed by a long distance PCR (Fig. 2C; GenBank accession No. AY396740). Alignment of the mRNA sequence against human genome sequences clearly indicated that each unique mRNA segment represents an individual exon and that all introns are flanked by the consensus donor and acceptor splice sites conforming to the GT/AG rule (Fig. 2B; data not shown). This gene contains an open reading frame of 261 nucleotides encoding a protein of 86 amino acids; a polyadenylation signal (AAUAAA) is situated 13 nucleotides 5′ of the polyadenine tail (Fig. 2C). By SMART analysis, the 16 residues from the first Met are a leader sequence containing a signal peptide while the last 55 residues correspond to a typical Kazal domain. A BLAST search revealed that this Kazal domain is about 33% identical (50% similar) and 32% identical (40% similar) to domains 2 and 15 of human Lekti, respectively, (Fig. 2D) that is encoded by Spink5, the defective gene in Netherton syndrome [12]. These three Kazal domains possess similar domain patterns, including a conserved tyrosine residue, disulfide bonds and the residue numbers spacing the cysteine residues. Only the P1 residue of the putative active site is different, suggesting they might have different substrate binding modes. Therefore, we designated this novel gene as serine protease inhibitor Kazal-type 9 with the gene symbol Spink9, which was approved later by the HUGO gene nomenclature committee, while its protein product was named lympho-epithelial Kazal-type inhibitor 2 (LEKTI-2).


Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

Meyer-Hoffert U, Wu Z, Schröder JM - PLoS ONE (2009)

Molecular identification of the Spink9 gene.(A) Schematic physical map of human SPINK genes locus (5q33.1). Genes are ordered from centromere (left hand side) to telomere (right hand side). (B) Schematic diagram of the Spink9 gene, based on its cDNA isolated from foreskin-derived keratinocyte identified by RT-PCR. It consists of four exons and three introns. The positions of the exons (boxes) and introns (curve lines) of Spink9 are deduced by comparing its full-length cDNA sequence with the corresponding genomic DNA. 5′/3′-UTRs and coding sequences are indicated by gray- and green-filled boxes, respectively. (C) The full-length cDNA sequence of Spink9 and its predicted protein sequence. The N-terminal signal peptide (residues 1–16; underlined) and the Kazal domain (residues 32–86; double-underlined) were detected with the SMART algorithm. The poly(A) signal site was coloured green. (D) Common characteristics of Lekti2 and Lekti. The alignment of the Kazal domains of Lekti-2 and Lekti domains 2 and 15 were generated by using M-COFFEE, displayed by using GeneDoc and shown in the down panel. The middle panel shows a schematic pattern of the typical Kazal domain including conserved tyrosine residue (Y) and disulfide bonds [14]. # represents the residue at the P1 site. The residue numbers spacing the cysteine residues are indicated on the top panel for the Kazal domain, LEKTI-2 and the LEKTI domains 2 and 15, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2631147&req=5

pone-0004372-g002: Molecular identification of the Spink9 gene.(A) Schematic physical map of human SPINK genes locus (5q33.1). Genes are ordered from centromere (left hand side) to telomere (right hand side). (B) Schematic diagram of the Spink9 gene, based on its cDNA isolated from foreskin-derived keratinocyte identified by RT-PCR. It consists of four exons and three introns. The positions of the exons (boxes) and introns (curve lines) of Spink9 are deduced by comparing its full-length cDNA sequence with the corresponding genomic DNA. 5′/3′-UTRs and coding sequences are indicated by gray- and green-filled boxes, respectively. (C) The full-length cDNA sequence of Spink9 and its predicted protein sequence. The N-terminal signal peptide (residues 1–16; underlined) and the Kazal domain (residues 32–86; double-underlined) were detected with the SMART algorithm. The poly(A) signal site was coloured green. (D) Common characteristics of Lekti2 and Lekti. The alignment of the Kazal domains of Lekti-2 and Lekti domains 2 and 15 were generated by using M-COFFEE, displayed by using GeneDoc and shown in the down panel. The middle panel shows a schematic pattern of the typical Kazal domain including conserved tyrosine residue (Y) and disulfide bonds [14]. # represents the residue at the P1 site. The residue numbers spacing the cysteine residues are indicated on the top panel for the Kazal domain, LEKTI-2 and the LEKTI domains 2 and 15, respectively.
Mentions: To identify the gene corresponding to the amino acid sequence, a BLAT search with the N-terminal 25-residue sequence of the novel peptide (where×was replaced by the cysteine residue) against the April 2003 human genome assembly localized this sequence to a chromosome 5 clone RP11-373N22 on 5p33.1 (Fig. 2A). Subsequent analysis of the retrieved RP11-373N22 DNA sequence identified two putative exons exactly encoding the isolated peptide (Fig. 1). Based on the generated theoretical partial DNA sequence, gene-specific primers were designed to perform 3′- and 5′-RACE. The full-length cDNA sequence (453 bp) was completed by combining the overlapping sequences from each PCR product and then confirmed by a long distance PCR (Fig. 2C; GenBank accession No. AY396740). Alignment of the mRNA sequence against human genome sequences clearly indicated that each unique mRNA segment represents an individual exon and that all introns are flanked by the consensus donor and acceptor splice sites conforming to the GT/AG rule (Fig. 2B; data not shown). This gene contains an open reading frame of 261 nucleotides encoding a protein of 86 amino acids; a polyadenylation signal (AAUAAA) is situated 13 nucleotides 5′ of the polyadenine tail (Fig. 2C). By SMART analysis, the 16 residues from the first Met are a leader sequence containing a signal peptide while the last 55 residues correspond to a typical Kazal domain. A BLAST search revealed that this Kazal domain is about 33% identical (50% similar) and 32% identical (40% similar) to domains 2 and 15 of human Lekti, respectively, (Fig. 2D) that is encoded by Spink5, the defective gene in Netherton syndrome [12]. These three Kazal domains possess similar domain patterns, including a conserved tyrosine residue, disulfide bonds and the residue numbers spacing the cysteine residues. Only the P1 residue of the putative active site is different, suggesting they might have different substrate binding modes. Therefore, we designated this novel gene as serine protease inhibitor Kazal-type 9 with the gene symbol Spink9, which was approved later by the HUGO gene nomenclature committee, while its protein product was named lympho-epithelial Kazal-type inhibitor 2 (LEKTI-2).

Bottom Line: Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin.LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5.At sites of plantar hyperkeratosis, LEKTI-2 expression was increased.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

ABSTRACT
Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

Show MeSH
Related in: MedlinePlus