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-165- Keratinocyte(1) 9/5 Room-D
PARTIAL PURIFICATION AND CHARACTERIZATION OF EPIDERMAL ICE-LIKE ENZYMES.
Tadahito Takahashi, Masashi Ogo, Tsutomu Soma, Toshihiko Hibino.
Shiseido Life Science Research Laboratories, Yokohama, Japan.
ICE and its homlogs play essential roles in apoptosis. Here we report a partial purification and characterization of epidermal ICE-like enzymes from human epidermis. Two distinct types of ICE-like proteases, which are reffered to as F-I and F-II respectively, were obtained from the extract of 30g of human cornified cells scraped from heels of healthy individuals. F-I effectively cleaved the synthetic substrate for CPP32, Ac-DEVD-MCA, but had no effect on the substrate for ICE, Ac-YVAD-MCA. The molecular weight was estimated to be 30 kDa by gel filtration. Kinetic parameters for the substrates and inhibitors suggested that is similar to CPP32. In contrast, F-II hydrolyzed both substrates and the molecular weight was estimated as 80 kDa, which is much higher than the other ICE-like proteases, suggesting that F-II is a hetero-oligomer. Both fractions cleaved poly(ADP-ribose) polymerase (PARP), a substrate for CPP32, and psoriastatin which belongs to ovserpin family. RT-PCR analysis showed that ICE, CPP32, Mch3, ICErelII and Ich-1 were expressed in cultured keratinocytes among ICE/ced-3 family members. These results indicate that several kinds of ICE-like enzymes are involved in terminal differentiation of keratinocytes.
-166- Keratinocyte(1) 9/5 Room-D
GENOMIC ANALYSIS OF HUMAN CYSTATIN A.
Masashi Yamazaki1, Kazumi Ishidoh2, Ryoji Tsuboi1, Eiki Kominami2, Hideoki Ogawa1.
1Department of Dermatology, 2Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
Cystatin A is a cysteine proteinase inhibitor with a molecular mass of 11 kDa, and is located mainly in the keratohyaline granules of the stratum granulosum and the cornified envelope of the stratum corneum in the epidermis. In this study, we analyzed the genomic structure of this proteinase inhibitor in which there were three exons of 111 bp, 102 bp and 226 bp in length, while the lengths of the 1st and 2nd intron were approximately 14 Kbp and 4 Kbp, respectively. There were binding sites for SP-1 and AP-2 in the promoter region and an AP-1 binding site in the 1st intron. The successful amplification of each exon of cystatin A may possibly contribute to the detection of the genomic abnormality of some skin disorders e.g. keratinization disorder, chronic bacterial infection.
-167- Keratinocyte(1) 9/5 Room-D
PROSAPOSIN IN HUMAN SKIN.
Chang-Yi Cui, Tokuji Seguchi, Masae Takahashi, Tadashi Tezuka.
Department of Dermatology, Kinki University School of Medicine, Osaka, Japan.
Saposin D is the one of the sphingolipid activator proteins. A polyclonal antibody to saposin D, elicitated by immunizing rabbits with the synthetic polypeptide from cDNA of saposin D, cross-reacted with a single, 65 kDa, pI 5.6 epidermal protein in a two-dimensional immunoblot study, suggesting it to be the precursor protein of saposin D, prosaposin, from its molecular weight and isoelectric point. Immuno-histochemical study showed the 65 kDa protein localized in the innermost cell layers of the stratum corneum. The Immunoelectron microscopy showed the antigenic material in the cytoplasm of the granular cells and the intercellular spaces either between the stratum granulosum and the stratum corneum or on the stratum corneum cell envelope. By ELISA, the amount of the 65 kDa protein in the inner surface skin of the upper arm of atopic dermatitis patients (non-lesional skin) was found to be significantly decreased than normal control. The suppression of prosaposin synthesis may be related to the abnormal stratum corneum formation in atopic skin through lower activation of sphingomyelinase or glucosylcerebrosidase.
-168- Keratinocyte(1) 9/5 Room-D
THE BIOLOGICAL ROLES OF PEPTIDYLARGININE DEIMINASE IN VITRO.
Masayuki Mizoguchi1, Yasushi Kawamura1, Motomu Motomu1, Tatsuo Senshu2, Hideoki Ogawa1.
1Department of Dermatology, Juntendo University School of Medicine, 2Department of Cell Chemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
The outer layers of mammalian epidermis have citrulline-containing proteins formed in situ by the activity of peptidylarginine deiminases on arginine residues. We investigated the role of protein deimination in epidermal differentiation by studying citrulline-containing proteins in cultured epidermal keratinocytes. After ionomycin treatment, major deiminated proteins were found to be associated with the nuclear lamina preceding the processes of apoptosis. We propose that deimination of a specific nuclear lamina protein is an important step in the removal of the nuclear lamina in the final stages of terminal differentiation of epidermal cells.
-169- Keratinocyte(1) 9/5 Room-D
PREFERRED SITES OF DEIMINATION IN KERATIN K1.
Tatsuo Senshu, Kyoichi Akiyama.
Department of Cell Chemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
We have reported recently that major deiminated proteins in the cornified layers of mammalian epidermis are derived from keratin K1. They are partially degraded/disulfide-cross-linked and deiminated by peptidylarginine deiminase at some arginine residues. In order to study the functional role of keratin deimination during the cornification of epidermis, we attempted to identify preferred acting sites of peptidylarginine deiminase in keratin K1. Deiminated keratin K1 was obtained from newborn mouse epidermis by differential extraction using urea/2-mercaptoethanol and anion exchange chromatography. It was subjected to limited proteolytic cleavages, and deiminated peptides released were fractionated by SDS-PAGE or HPLC. N-terminal sequencing showed deiminated arginine residues in V1 and V2 subdomains. Deimination of these regions may promote organization of epidermal proteins facilitating formation and maintenance of the outermost protective barrier of mammalian skin.
-170- Keratinocyte(1) 9/5 Room-D
CHARACTERIZATION OF LACTOFERRICIN B LIKE PROTEIN IN NEWBORN RAT EPIDERMIS.
Masae Takahashi, Tadashi Tezuka.
Department of Dermatology, Kinki University School of Medicine, Osaka, Japan.
We haveinvestigated the properties of proteins with epidermal barrier function such as a phosphorylatead cystatin a in newborn rat epidermis. In this experiment, properties of an epidermal protein related with lactoferricin B which is an antimicrobial peptide derived from the N-terminal region of bovine lactoferrin, is examined. A peptide, whose amino acid sequence was -L-Q-K-E-L-K-R-I-K-I-P-D-Y-S-D-S-F-K-I-K-H-L-G-K-G- (lactoferricin B peptide), was synthesized and conjugated with KLH as a carrier protein by MBS method. The conjugated peptide was injected to rabbits to rise an antibody. Because this produced antibody reacted with lactoferricine B peptide, the specific antibody agaist the peptide was obtained. Tris-HCl buffer (pH 8.0) or 8 M alkaline urea extract of newborn rat epidermis was subjected to SDS-PAGE and immunoblot analysis using anti-lactoferricin B/KLH antibody was performed. The positive protein band appeared in the Tris-HCl buffer extract. Its molecular weight and isoelectric point were 40 kDa and pI 5.5, respectively. Therefore, this protein may have a role as antimicrobial substance in the epidermis.