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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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Identification of a new KLK5-inhibiting peptide in human stratum corneum.(A) RP-HPLC separation of extract stratum corneum extracts from human plantar callus. Top, components eluting between 0% and 100% acetonitrile. Bottom, KLK5-inhibiting-activity of the fractions. One out of three similar experiments is shown. Arrow indicates fractions with high activity to absorbance ratio at 20–21 min eluting time. (B) SDS page analyses of fractions eluting between 18 and 22 minutes. Since the fractions were not collected by time but by peaks, distance of fractions is not linear. A dominant peak of around 7 kDa is visible at 20–21 min eluting time. (C) ESI-MS analyses revealed a mass of 7058.18 kDa for the KLK5-inhibiting fraction. (D) Edman degradation resulted in the indicated N-terminal sequence.
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pone-0004372-g001: Identification of a new KLK5-inhibiting peptide in human stratum corneum.(A) RP-HPLC separation of extract stratum corneum extracts from human plantar callus. Top, components eluting between 0% and 100% acetonitrile. Bottom, KLK5-inhibiting-activity of the fractions. One out of three similar experiments is shown. Arrow indicates fractions with high activity to absorbance ratio at 20–21 min eluting time. (B) SDS page analyses of fractions eluting between 18 and 22 minutes. Since the fractions were not collected by time but by peaks, distance of fractions is not linear. A dominant peak of around 7 kDa is visible at 20–21 min eluting time. (C) ESI-MS analyses revealed a mass of 7058.18 kDa for the KLK5-inhibiting fraction. (D) Edman degradation resulted in the indicated N-terminal sequence.

Mentions: To follow the hypothesis that specific inhibitors for KLK5 exist in human skin, extracts from healthy persons' SC were analyzed for KLK5-inhibiting activity. Preparative reverse-phase HPLC (RP-HPLC) was used to separate heparin-bound cationic peptides. Results from KLK5-inhibiting activity revealed a fraction (Fig. 1A), which was further purified by analytical RP-HPLC using a C2C18-column (data not shown). SDS-PAGE analysis (Fig. 1B) of these HPLC fractions, eluting at low acetonitrile concentration and containing KLK5-inhibitory activity showed the presence of a 7-kDa peptide. Electrospray-ionisation mass spectrometry (ESI-MS) analysis (Fig. 1C) resulted in a principal ion corresponding to a mass of 7058.19 Da. Da. N-terminal sequencing of the dominant fraction yielded a sequence of 25 residues (TKQMVDXSHYKKLPPGQQRFXHHMY; Fig. 1D). A blast search using the 25-residue sequence retrieved no matches in any protein/gene/EST databases, suggesting a novel human gene may encode this sequence.


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)

Identification of a new KLK5-inhibiting peptide in human stratum corneum.(A) RP-HPLC separation of extract stratum corneum extracts from human plantar callus. Top, components eluting between 0% and 100% acetonitrile. Bottom, KLK5-inhibiting-activity of the fractions. One out of three similar experiments is shown. Arrow indicates fractions with high activity to absorbance ratio at 20–21 min eluting time. (B) SDS page analyses of fractions eluting between 18 and 22 minutes. Since the fractions were not collected by time but by peaks, distance of fractions is not linear. A dominant peak of around 7 kDa is visible at 20–21 min eluting time. (C) ESI-MS analyses revealed a mass of 7058.18 kDa for the KLK5-inhibiting fraction. (D) Edman degradation resulted in the indicated N-terminal sequence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2631147&req=5

pone-0004372-g001: Identification of a new KLK5-inhibiting peptide in human stratum corneum.(A) RP-HPLC separation of extract stratum corneum extracts from human plantar callus. Top, components eluting between 0% and 100% acetonitrile. Bottom, KLK5-inhibiting-activity of the fractions. One out of three similar experiments is shown. Arrow indicates fractions with high activity to absorbance ratio at 20–21 min eluting time. (B) SDS page analyses of fractions eluting between 18 and 22 minutes. Since the fractions were not collected by time but by peaks, distance of fractions is not linear. A dominant peak of around 7 kDa is visible at 20–21 min eluting time. (C) ESI-MS analyses revealed a mass of 7058.18 kDa for the KLK5-inhibiting fraction. (D) Edman degradation resulted in the indicated N-terminal sequence.
Mentions: To follow the hypothesis that specific inhibitors for KLK5 exist in human skin, extracts from healthy persons' SC were analyzed for KLK5-inhibiting activity. Preparative reverse-phase HPLC (RP-HPLC) was used to separate heparin-bound cationic peptides. Results from KLK5-inhibiting activity revealed a fraction (Fig. 1A), which was further purified by analytical RP-HPLC using a C2C18-column (data not shown). SDS-PAGE analysis (Fig. 1B) of these HPLC fractions, eluting at low acetonitrile concentration and containing KLK5-inhibitory activity showed the presence of a 7-kDa peptide. Electrospray-ionisation mass spectrometry (ESI-MS) analysis (Fig. 1C) resulted in a principal ion corresponding to a mass of 7058.19 Da. Da. N-terminal sequencing of the dominant fraction yielded a sequence of 25 residues (TKQMVDXSHYKKLPPGQQRFXHHMY; Fig. 1D). A blast search using the 25-residue sequence retrieved no matches in any protein/gene/EST databases, suggesting a novel human gene may encode this sequence.

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