Figure 1.
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.
Figure 2.
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.
Figure 3.
Spink9 mRNA expression in human skin and keratinocytes.
(A) Expression profile of Spink9 mRNA. Fragments were obtained after RT-PCR amplification on human multiple tissue cDNAs with primers specific to the human GAPDH and Spink9. Lanes are labelled according to the template tissue. The Spink9 fragments are of 175 bp in size. H2O (no cDNA) and RT-control (no RNA template) were used as negative controls. (B) Spink9 mRNA expression in cultured primary keratinocytes. Quantitative realtime PCR was conducted on RT-PCR products of total RNA samples collected from keratinocytes treated with 1.0 mM CaCl2 for the indicated time. Bar graphs represent the relative mRNA expression of Spink9 against GAPDH. Data are obtained from three independent experiments with different sources of keratinocytes and are indicated as the mean+/−SD. * indicates significant (p<0.05); ** indicates significant (p<0.01).
Figure 4.
Western blot analyses of LEKTI-2 antibodies.
Proteins were separated in a 16.5% SDS-tricine polyacrylamide gel containing 8 M urea and blotted from both, the anode (left panel) and the cathode (right panel) using protein transfer at pH 10.5. Recombinant and natural LEKTI-2 used here have been HPLC-purified, stored below −70°C and used immediately prior to experiments unless otherwise stated. Lanes 1 and 7: Precisions Plus Protein Dual Color standards Biorad (it includes peroxidase-active markers), each 3 µl; lane 2: stratum corneum extract, 20 µl; lane 3: rLEKTI-2 (200 ng); lane 4: stratum corneum extract, 40 µl; lane 5: natural LEKTI-2 from stratum corneum extract (off RP-8-HPLC, see Fig. 1A), 30 µl; lane 6: rLEKTI-2 (200 ng). WB analyses performed with LEKTI-2 antibodies, pretreated for 1 h with 20 µg r LEKTI-2, did not show any LEKTI-2 immunostaining, which further confirms specificity of the LEKTI-2 antibodies. Note that cathodal transfer seems to be more efficient than anodal protein transfer. A representative out of three independent experiments is shown.
Figure 5.
LEKTI-2 is expressed at palmar and plantar sites.
Immunohistochemical staining (Vector red) of LEKTI-2 of paraffin-embedded human skin samples using polyclonal antibodies. Only palmar and plantar localizations (A, n = 8) exhibited obvious LEKTI-2 immunoreactivity at the stratum granulosum and stratum corneum. Other localizations (e.g. trunk, B, n = 16) did not reveal LEKTI-2 immunoreactivity. Bars indicate 50 µm.
Figure 6.
Inhibition of KLK5 by rLEKTI-2.
(A) Enzyme kinetic of KLK5 (5 nM) and LEKTI-2 (0 to 2000 nM) in comparison to no inhibition of KLK14 (B), KLK7 (C), trypsin (D) and chymotrypsin (E). The substrate for KLK5 was MeO-Suc-Arg-Pro-Tyr-pNA (1 mM). Points represent mean of triplicate experiments and the solid line represents the fitted curve according to equation of Baici model [33].
Table 1.
Protease Inhibition by rLEKTI-2.
Figure 7.
LETKI-2 and KLK5 are expressed at the stratum granulosum of palmar skin.
Immunofluorescence localization of LEKTI-2 (red) and KLK5 (green) in human skin. Nuclei staining was done using Hoechst 33258 reagent. (B–E) shows the magnification of the white-square area of (A). LEKTI-2 staining showed granular structures inside the keratinocytes at the stratum granulosum (C) with a faint intercellular staining pattern and remaining immunoreactivity inside the stratum corneum. KLK5 staining (D) exhibited only intercellular staining at the stratum granulosum. Comparative localization of LEKTI-2 and KLK5 is shown in the merged image (D). (E) shows the control omitting the first antibody.
Figure 8.
LEKTI-2 is highly expressed at sites of hyperkeratosis.
Immunohistochemical staining (Vector red) of LEKTI-2 of a paraffin-embedded section of a clavus. Notice the enormously enlarged stratum corneum (hyperkeratosis) in the upper two third of the picture with a sweat gland sectioned at the upper first third. Clefts between the stratum corneum and the stratum granulosum are artefacts derived from histology processing. The upper part of the living epidermis, the stratum granulosum, exhibits a massive increase in LEKTI-2 immunoreactivity (red). A representative result out of three different samples of clavi is shown.