- Email: [email protected]
172 Targeted disruption of the transglutaminase 1 gene
131
JSID Abstracts
169
172
PREFERRED SITES OF DEIMINATION IN KERATIN Kl. L Senshu’ Department of Cell Chemistry, Tokyo Metropolitan lnstllute of Gerontology, Tokyo, Japan We have reported recently that major deiminated proteins in the cornified layers of mammalian epidermis are derived from keratin Ki. 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 Kl. Deiminated keratin Kl 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 delminated peptides released were fractionated by SDS-PAGE or HPLC. N-terminal sequencing showed deiminated arginine residues in Vl and V2 subdomains. Deimination of these regions may promote organization of epidermal proteins facilitating formation and maintenance of the outermost protective b;rrrier of mammalian skin.
TARGETED
170
OF THE TRANSGLUTAMINASE
1 GENE
‘Dcpment of Dmnatology, Kyoto Prefectural University of Medicine, Kyoto, ‘Department of Science for Laboratory Animal Experimentation and ’ Irnmunoregulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan. Transglutaminase 1 (TGase 1) is expressed during the process of keratinizatlon and is believed to produce cell envelope by catalyzing protein cross-linking reaction. ‘l%e gene for TGase 1 is a disease gne of autosomal recessive lamellar ichtbyosis. In thii study, to elucidate in viva function of TGasc 1 gene, we generated mice lacking TGase 1 gene by targeted disruption of the Z-kb mouse TGase 1 gene containing exons From 1 to 3. ‘Ihe heterozygous mutant mice were normal in phenotype, but the homozygous mutant mice showed neonatal lethality with severe morphological changes of the stratum curneum. Thus, the TGase 1 gene is essential to development of the stratum comeurn and for survival of mice.
173
CHARACTERlZATION
OF LACKFERRICINB
LIKE PROTEIN
IN
NEWBORNRATEPIDBRMIS.
M.mmmtof Damatology, Kinki University School of McdicIne, Osaka, Japan. We havcinv&igatai the pmpatics of proteins with epidennal barrier timction such asa pho@myIatcad cysta!jn u. in newborn rat epidermis. In thisexpaiment, paopatiesofane@dezmaIpmteinre4a~edwithlactofezrlcIn B which is an antimic?ubiaI peptide derived tirn the N-terminal
region of bovine lauofariu,
is ex&ned.
A peptide, whose amino acid
sequence was -LQ-K-E-L-K-R-I-K-I-P-D-Y-S-D-S-F’-K-I-K-H-LG-K-G - (laUofarl& B pepi&), was synthesized and conjugated with KLH as a car&r pmtcin by MBS method. ‘I% conjugated peptide was injected
to rabbits tn rise an antibody. Because this pmdoced antibody reacted with laUof6ricIne B peptide, the speclflc antibody agais2 the peptide was oWned. This-I-El buffa @II 8.0) or 8 M allmliae urea extract of newborn rat @damIs was subja2cd to SDS-PAGE and Immunoblot analysis using anti-WcIn BKLH andtmdy was paformed. The pusitive pmteln band appear& in the Ttls-IKl buffer extract. Its molecular weight and iso-
electric point were 40 kDa and ~15.5, re.specCvrJy. Therefore, this protein may have a role as antimIcmbiaI substance in the epidermis.
BCLXLACI-IVATES IN KERAlTNOC~.K.*‘. -1 y F
HUMAN
TRANSGLUTAMINASE 1 PROMOTER . .I 1. U&l, K. Yamanlshl. su)lmatps, ‘Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, and ‘Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Suita, Japan. Transglutaminase 1 (TGasel) is expressed at the late stage of terminal differentiation of keratinocytes to form cell envelope by pmtein crosslinking reaction. Bcl-XL, a member of the bcl-2 family regulating programmed cell death, is expressed in the upper spinous and granular layers of the epidermis. The localization of bcl-xL is closely related to that of TGasel mRNA, suggesting its regulatory role in TGasel gene expression. When the 5’ flanking region of human TGasel gene linked to a h&erase reporter gene was co-tmnsfected with a chicken bcl-xL expression plasmid, luciferasc activity was increased in keratinocytes, but not in non-keratinocytic HTlO80 ceils. ‘Thus, bcl-xL may be involved in the activation of the human TGase 1 promoter in keratinocytes.
174
171 EXPRESSION OF ENTEROPEPTIDASE IN HUMAN KERATINOCYTES. y Shiseido Research Center, Yokohama, Japan.
We have previously
shown
EPIDERMAL
that two types of lrypsinogens
are
expressed in human epidermal keralinocytes and may play an important role in desquamation by cntalyzing the degradation of intercelluler cohesive s~ruclure.
DISRUPTION
In this study, we examined the expression
d enteropeplidase,
which speciFIcally cleaves trypsinogen IO yield aclive trypsin, in human keratinocyres by RT-PCR and in silu hybridization. Enteropeptidaae was not expressed in cul(ured human keratinocytes al growth phase and at contluency, even though in high calcium (1SmM) condition, while the expression was observed in the cells exposed IO the air. Furthermore, in silu hybridization study revealed that enleropeptidase was expressed only in the uppermost granular cells in human epidermis. ‘llwse results auggesl that trypsinogens expressed in suprabasal cells are activated to trypsin by enleropeplidase expressed in uppermost cells and that the aclive lrypsin may play imponant roles in terminal diEEerenlialion and desquamation.
CORNIPIED CELL ENVELOPE (CCE) PRECURSOR PROTEINS AND TRANSGLUTAMINASE (TGase) 1 ARE COORDINATELY EXPRESSED IN THE SlTES OF HAIR CANAL MORPHOGENESIS DURING HUMAN HAIR FOLLICLE DEVELOPMENT. M.‘. mSmirh’. M.‘. K.A.‘. mShimizu’. ‘Div. of Dermatology. Kitasaro lnsdlule Hospifnl.
Tokyo, Japan.2Dcpts. of Biological Structure and Medicine/Dermatology. Univ. of Washington School of Medicine, Seattle,WA, U.S.A. ‘Dept. of Dermatology, Kyoto Univ. Faculty of Medicine, Kyoto. Japan.‘Dept.%of Anatomy and Cell Biology and MedicinelDemwology. Univ. of Florida. Gainesville. FL. U.S.A. ‘Dept. of Dermatology. Keio Univ. School of Medicine. Tokyo, Japan. CCE formsdon is a key step of the final stage of keradnirarion. CCS precursor proteins are crosslinked by keratin&e T&se I Lothe’inner &face oiplasma membraneof co&ied cells in CCE formation and its outer surface is coated with content of lamellar rranulss (LG). To clarify the time and the site of expression of these CCE arsocialed moiecules in hair follicledwelopmeat. skin samples t?omm human fetuses of a series of estimated gestational aaes (ECiAs) were examined. Laricrin was seen in cells of the neck of hair oee @5-105 d E?3Aj aad?Gasc 1 and LG coatcat wac observed in the inner cells of hair &,. lmmunonaclivities of loricrin. SPRPs I & 2, involucrin. TGase I and LG cement were de~fxtedin inner rw sheathcells of the bulbous hair peg (105-135 d EGA) and in primitive hair canals in inlraepidermal portion of hair follicle +I35 d EGA). TGase I was also positive in outer roof sheath ti sebaceous gland. These data indicated that CCE pro&s. TGase I and lamellar granulelipid con&t are coordinately expressed in Ihe safes defined 1oform hair canalduring human hair follicledevelopment.
includinn loricrin. small omline-rich Drareins (SPRPs) I and 2 and involucrin
JSID Abstracts
169
172
PREFERRED SITES OF DEIMINATION IN KERATIN Kl. L Senshu’ Department of Cell Chemistry, Tokyo Metropolitan lnstllute of Gerontology, Tokyo, Japan We have reported recently that major deiminated proteins in the cornified layers of mammalian epidermis are derived from keratin Ki. 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 Kl. Deiminated keratin Kl 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 delminated peptides released were fractionated by SDS-PAGE or HPLC. N-terminal sequencing showed deiminated arginine residues in Vl and V2 subdomains. Deimination of these regions may promote organization of epidermal proteins facilitating formation and maintenance of the outermost protective b;rrrier of mammalian skin.
TARGETED
170
OF THE TRANSGLUTAMINASE
1 GENE
‘Dcpment of Dmnatology, Kyoto Prefectural University of Medicine, Kyoto, ‘Department of Science for Laboratory Animal Experimentation and ’ Irnmunoregulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan. Transglutaminase 1 (TGase 1) is expressed during the process of keratinizatlon and is believed to produce cell envelope by catalyzing protein cross-linking reaction. ‘l%e gene for TGase 1 is a disease gne of autosomal recessive lamellar ichtbyosis. In thii study, to elucidate in viva function of TGasc 1 gene, we generated mice lacking TGase 1 gene by targeted disruption of the Z-kb mouse TGase 1 gene containing exons From 1 to 3. ‘Ihe heterozygous mutant mice were normal in phenotype, but the homozygous mutant mice showed neonatal lethality with severe morphological changes of the stratum curneum. Thus, the TGase 1 gene is essential to development of the stratum comeurn and for survival of mice.
173
CHARACTERlZATION
OF LACKFERRICINB
LIKE PROTEIN
IN
NEWBORNRATEPIDBRMIS.
M.mmmtof Damatology, Kinki University School of McdicIne, Osaka, Japan. We havcinv&igatai the pmpatics of proteins with epidennal barrier timction such asa pho@myIatcad cysta!jn u. in newborn rat epidermis. In thisexpaiment, paopatiesofane@dezmaIpmteinre4a~edwithlactofezrlcIn B which is an antimic?ubiaI peptide derived tirn the N-terminal
region of bovine lauofariu,
is ex&ned.
A peptide, whose amino acid
sequence was -LQ-K-E-L-K-R-I-K-I-P-D-Y-S-D-S-F’-K-I-K-H-LG-K-G - (laUofarl& B pepi&), was synthesized and conjugated with KLH as a car&r pmtcin by MBS method. ‘I% conjugated peptide was injected
to rabbits tn rise an antibody. Because this pmdoced antibody reacted with laUof6ricIne B peptide, the speclflc antibody agais2 the peptide was oWned. This-I-El buffa @II 8.0) or 8 M allmliae urea extract of newborn rat @damIs was subja2cd to SDS-PAGE and Immunoblot analysis using anti-WcIn BKLH andtmdy was paformed. The pusitive pmteln band appear& in the Ttls-IKl buffer extract. Its molecular weight and iso-
electric point were 40 kDa and ~15.5, re.specCvrJy. Therefore, this protein may have a role as antimIcmbiaI substance in the epidermis.
BCLXLACI-IVATES IN KERAlTNOC~.K.*‘. -1 y F
HUMAN
TRANSGLUTAMINASE 1 PROMOTER . .I 1. U&l, K. Yamanlshl. su)lmatps, ‘Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, and ‘Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Suita, Japan. Transglutaminase 1 (TGasel) is expressed at the late stage of terminal differentiation of keratinocytes to form cell envelope by pmtein crosslinking reaction. Bcl-XL, a member of the bcl-2 family regulating programmed cell death, is expressed in the upper spinous and granular layers of the epidermis. The localization of bcl-xL is closely related to that of TGasel mRNA, suggesting its regulatory role in TGasel gene expression. When the 5’ flanking region of human TGasel gene linked to a h&erase reporter gene was co-tmnsfected with a chicken bcl-xL expression plasmid, luciferasc activity was increased in keratinocytes, but not in non-keratinocytic HTlO80 ceils. ‘Thus, bcl-xL may be involved in the activation of the human TGase 1 promoter in keratinocytes.
174
171 EXPRESSION OF ENTEROPEPTIDASE IN HUMAN KERATINOCYTES. y Shiseido Research Center, Yokohama, Japan.
We have previously
shown
EPIDERMAL
that two types of lrypsinogens
are
expressed in human epidermal keralinocytes and may play an important role in desquamation by cntalyzing the degradation of intercelluler cohesive s~ruclure.
DISRUPTION
In this study, we examined the expression
d enteropeplidase,
which speciFIcally cleaves trypsinogen IO yield aclive trypsin, in human keratinocyres by RT-PCR and in silu hybridization. Enteropeptidaae was not expressed in cul(ured human keratinocytes al growth phase and at contluency, even though in high calcium (1SmM) condition, while the expression was observed in the cells exposed IO the air. Furthermore, in silu hybridization study revealed that enleropeptidase was expressed only in the uppermost granular cells in human epidermis. ‘llwse results auggesl that trypsinogens expressed in suprabasal cells are activated to trypsin by enleropeplidase expressed in uppermost cells and that the aclive lrypsin may play imponant roles in terminal diEEerenlialion and desquamation.
CORNIPIED CELL ENVELOPE (CCE) PRECURSOR PROTEINS AND TRANSGLUTAMINASE (TGase) 1 ARE COORDINATELY EXPRESSED IN THE SlTES OF HAIR CANAL MORPHOGENESIS DURING HUMAN HAIR FOLLICLE DEVELOPMENT. M.‘. mSmirh’. M.‘. K.A.‘. mShimizu’. ‘Div. of Dermatology. Kitasaro lnsdlule Hospifnl.
Tokyo, Japan.2Dcpts. of Biological Structure and Medicine/Dermatology. Univ. of Washington School of Medicine, Seattle,WA, U.S.A. ‘Dept. of Dermatology, Kyoto Univ. Faculty of Medicine, Kyoto. Japan.‘Dept.%of Anatomy and Cell Biology and MedicinelDemwology. Univ. of Florida. Gainesville. FL. U.S.A. ‘Dept. of Dermatology. Keio Univ. School of Medicine. Tokyo, Japan. CCE formsdon is a key step of the final stage of keradnirarion. CCS precursor proteins are crosslinked by keratin&e T&se I Lothe’inner &face oiplasma membraneof co&ied cells in CCE formation and its outer surface is coated with content of lamellar rranulss (LG). To clarify the time and the site of expression of these CCE arsocialed moiecules in hair follicledwelopmeat. skin samples t?omm human fetuses of a series of estimated gestational aaes (ECiAs) were examined. Laricrin was seen in cells of the neck of hair oee @5-105 d E?3Aj aad?Gasc 1 and LG coatcat wac observed in the inner cells of hair &,. lmmunonaclivities of loricrin. SPRPs I & 2, involucrin. TGase I and LG cement were de~fxtedin inner rw sheathcells of the bulbous hair peg (105-135 d EGA) and in primitive hair canals in inlraepidermal portion of hair follicle +I35 d EGA). TGase I was also positive in outer roof sheath ti sebaceous gland. These data indicated that CCE pro&s. TGase I and lamellar granulelipid con&t are coordinately expressed in Ihe safes defined 1oform hair canalduring human hair follicledevelopment.
includinn loricrin. small omline-rich Drareins (SPRPs) I and 2 and involucrin