LEKTI Is Localized in Lamellar Granules, Separated from KLK5 and KLK7, and Is Secreted in the Extracellular Spaces of the Superficial Stratum Granulosum

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LEKTI Is Localized in Lamellar Granules, Separated from KLK5 and KLK7, and Is Secreted in the Extracellular Spaces of the Superficial Stratum Granulosum Akemi Ishida-Yamamoto, Céline Deraison, Chrystelle Bonnart, Emmanuelle Bitoun, Ross Robinson, Timothy J. O'Brien, Kotaro Wakamatsu, Sawa Ohtsubo, Hidetoshi Takahashi, Yoshio Hashimoto, Patricia J.C. Dopping-Hepenstal, John A. McGrath, Hajime Iizuka, Gabriele Richard, Alain Hovnanian Journal of Investigative Dermatology Volume 124, Issue 2, Pages 360-366 (February 2005) DOI: 10.1111/j.0022-202X.2004.23583.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Mutations of the serine protease inhibitor Kazal-type 5 gene identified in case 1. Sequence chromatograms illustrate a heterozygous transition (2368C/T) in exon 25 leading to amino acid replacement R790X (A, left) compared with the wild-type sequence (A, right). The mutation results in loss of a TaqI site and prevents digestion of the mutant sequence. TaqI restriction fragment analysis of a PCR amplicon of exon 25 from the affected patient (P) results in two bands, which represent the undigested fragment (367 bp) of the mutant allele in addition to the cleaved fragments (181 and 186 bp, both appear as one band) of the wild-type allele. In contrast, amplicons of six normal controls (C1–C6) show complete digestion. M: DNA 100 bp ladder (Promega, Madison, Wisconsin). (B) Illustrates a heterozygous 2 base pair deletion in exon 5 (377delAT) resulting in frameshift and a premature termination codon (B, top left). The mutant sequence of exon 5 is two nucleotides shorter than the wild-type sequence (B, bottom right) as evident from the second peak of the HPLC profile (B, top right) compared with normal (B, bottom right). Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is expressed earlier than kallikrein (KLK)7 (A) and KLK5 (B) in the epidermis. LEKTI staining (mouse monoclonal antibody, seen in red) is expressed from the upper spinous layer, whereas KLK7 and KLK5 are detected in the most superficial granular cells (green). A double-positive zone is seen in between (orange arrows). Note the apical localization of LEKTI (white arrows). Stratum corneum is positive to both KLK, but negative to LEKTI. Confocal laser scanning microscopy. Differential interference microscopy images are superimposed upon immunofluorescent images. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is deficient and cornified cells are less adhesive in Netherton syndrome (NS). Immunohistochemical staining using a polyclonal antibody against the D1–D6 domains of LEKTI revealed the presence of LEKTI protein in the apical parts of the spinous and granular cells of normal skin (A), but not in the NS skin (case 1) (B). Using hematoxylin–eosin staining of paraffin-embedded sections, the stratum corneum was found to be thickened in NS (case 1) (C) than in normal skin (D). In NS, the outlines of the cells were undulated and extensive clefts were noted starting in the deeper layer, as indicated by the arrow, in contrast to the smooth outlines of normal stratum corneum. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is transported through the lamellar granule (LG) system. Post-embedding immunoelectron microscopy using Lowicryl HM20 resin (A, D) and a cryo-ultramicrotomy method (B, C, E). (A, B) LEKTI signals (black arrows) are detected in the LG using polyclonal antibodies against D8–D11 in (A) and D1–D6 in (B). Note that LG are seen as isolated granules in the post-embedding method (A), but appear as beaded tubular structures in the cryo-ultramicrotomy (B). Lamellar internal structures are seen in both methods (white arrows). (C) LEKTI is secreted from the apical side of upper granular cells (arrows). d, desmosomes. Polyclonal antibody to D8–D11 and desmoglein 1 monoclonal antibody were used and were labeled with 5 and 10 nm immunogold, respectively. (D) LEKTI antibody to D8–D11 labels are closely associated with those of the TGN46-positive trans-Golgi network. (E) LEKTI (monoclonal antibody, 5 nm gold labels) and glucosylceramides (GlcCer, 10 nm gold labels) are localized within the same continuous beaded tubular structure of the LG system. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is secreted earlier than kallikrein (KLK)7. (A) Shows a Lower magnification view of the stratum corneum (SC), the most superficial stratum granulosum (SG1), the second (SG2), and the third granular layer (SG3). The rectangular area marked as “B” is shown at higher magnification in (B), which shows LEKTI secreted into the extracellular spaces (ECS) between SG2 and SG3. (C) Shows a higher magnification view of the boxed area marked “C” in (B) showing KLK7-containing lamellar granules in SG2. d, desmosomes. Cryo-ultramicrotomy method. A monoclonal antibody to LEKTI D1–D6 and a polyclonal KLK7 antibody were used and were labeled with 10 and 5 nm immunogold, respectively. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) and kallikrein (KLK)7 are transported separately in the lamellar granule system. (A) Lower magnification view of the most superficial stratum granulosum (SG1), the second (SG2), the third (SG3), and the most superficial stratum spinosum (SS). The marked rectangular area in SG3 is shown at higher magnification in (B). Cryo-ultramicrotomy method. A monoclonal LEKTI antibody to D1–D6 and a polyclonal KLK7 antibody were used and were labeled with 10 and 5 nm immunogold, respectively. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) and kallikrein (KLK)7 are co-localized in the extracellular spaces (ECS). In the ECS of the stratum granulosum (SG1)–stratum corneum (SC) interface, LEKTI (5 nm gold labels, small arrows) and KLK7 (10 nm gold labels, large arrows) can be seen separated in some places, but are apparently co-localized in others. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Kallikrein (KLK)5 is localized in the lamellar granule (LG) system, but separated from Lympho-epithelial Kazal-type-related inhibitor (LEKTI). (A) Note beaded tubular structure with partial lamellar internal structures (white arrows) of the LG system containing KLK5 as aggregates (black arrows). (B) Shows LEKTI (10 nm gold) and KLK5 (5 nm gold) double labeling. Cryo-ultramicrotomy method. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Early desmosome split in Netherton syndrome (NS). By conventional transmission electron microscopy, a desmosomal split is seen in the superficial granular cells (A, B). The rectangular area marked in (A) is shown at higher magnification in (B). Note that the midline structures of the desmoglea remain on the basal side of split desmosomes (d) (arrows in B). Using desmoglein 1 immunoelectron microscopy, immunolabeling is detected in the desmoglea in the basal part of the split desmosome in NS (arrow, C), and intact desmosomes in normal control skin (D) (post-embedding method). Asterisks indicate lamellar granule contents secreted into the extracellular spaces. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Desmosome disintegration coincides with kallikrein (KLK)7 secretion. (A) In the lower stratum corneum where KLK7 labels (5 nm gold) are detected in the intercellular spaces, desmoglein 1 (DSG1) (10 nm gold) labels are associated with intact desmosomes in the normal skin. (B) In Netherton syndrome (NS) skin, the early sign of desmosomal split is detected (right-hand side). Note the many KLK7 labels around DSG1 labels. KLK7 and DSG1 double immunolabeling. (C) A KLK7-positive lamellar granule found in NS skin. Post-embedding immunoelectron microscopy. Journal of Investigative Dermatology 2005 124, 360-366DOI: (10.1111/j.0022-202X.2004.23583.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions