Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 958-963
March 31, 1992
MOLECULAR CLONING AND CHARACTERIZATION OF TYPE VII COLLAGEN cDNA Toshihiro Tanaka, Kenzo Takahashi, Fukumi Furukawa and Sadao imamura Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
Received February 12, 1992
SUMMARy; Type VII collagen, located in human epidermal basement membrane, is the primary pathogenic target molecule in epidermolysis bullosa acquisita and epidermolysis bullosa dystrophica. Using a monoctonal antibody against the non-collagenous domain of type VII collagen, ~lKb cDNA was isolated from human keratinocyte library. The deduced primary structure of this clone thus reflects the non-collagenous domain of type VII collagen that may be involved in cell attachment. This region shows a weak homology (~23%) to the cell attachment domain of fibronectin. Northern blot revealed ~9.5 Kb single band. ®1992 A c a d e m i c P ..... I n c .
The major protein of anchoring fibril, that may play an important role in the binding of keratinocytes to the underlying dermis, is composed of type VII collagen (1). This protein contains a collagenous domain, which makes a triple helical structure, and a non-collagenous domain that may be involved in cell attachment (2). Type VII collagen is of particular interest as they have been implicated in the pathogenesis of acquired epidermolysis bullosa and congenital epidermolysis bullosa, two clinical diseases that affect subbasement membrane region and cause skin blistering (3,4). The noncollagenous domain of type VII collagen is primarily targeted by pathogenic autoantibodies that bind to anchoring fibril in acquired epidermolysis bullosa (3). Moreover, type VII collagen is not detected by indirect immunofluorescence in the basement membrane zone (BMZ) of patients with dystrophic type epidermolysis bullosa (4). Therefore, hereditary defect of type VII collagen or dysfunction of this molecule by circulating autoantibody causes similar blistering disorder in human skin. Because such biochemical and immunological studies have highlighted the importance of type VII collagen, as a step toward examining the genetic bases of the structure of type VII collagen, characterizing the acquired epidermolysis bullosa antigen, and further investigation of congenital epidermolysis bullosa, the present study was undertaken to clone the cDNA encoding human type VII collagen. 0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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EXPERIMENTAL PROCEDURES Reagents: All the reagents for library construction, RNA blot, polymerase chain reaction (PCR) and DNA sequence were purchased from Takara Shuzo Co. (Kyoto Japan). Reagents for immunoscreening were purchased from BRL (MD, U.S.A). Construction of oDNA library and immunoscreening Random primer library was constructed with poly(A) rich RNA from cultured human keratinocytes (5,6) and resultant cDNAs were inserted in Eco RI site of expression vector Xgt11. Library was immunoscreened with the monoclonal antibody (mAb) which specifically reacts with type VII collagen (Chemicon International Inc, CA, U.S.A.). This mAb recognizes ~300KDa type VII collagen by Western blot and is now used as a tool for the diagnosis and prenatal diagnosis of dystrophic type epidermolysis bullosa (EB) (7.8). Polvmerase Chain Reaction: The following oligonucleotides were synthesized according to the nucleotide sequence of our clone (primer A) and Parente's clone (primer B and C) (9): primer A: 5'-GGAGCCGGAAACTCCAC TTG-3', primer B; 5'-GAGTGGCCGCCAGGATAGGAT-3' and primer C; 5'-CCC AAGTGCCAACACCAGACG-3'. ~lpg of poly(A) rich RNA from keratinocytes was used as a template by using primer C as a reverse transcriptase primer. Primer C was removed from the reaction mixture by membrane filter (Takara Shuzo Co, Kyoto Japan) and aliquot of the purified cDNA was amplified by primer A and B under the PCR condition described by supplier. The resultant fragment was gel electrophoresed. This fragment was subcloned into pGEM3Z vector (Promega Wl U.S.A.) and nucleotide sequenced. RNA blot. nucleotide seauence and comouter analysis: RNA was extracted from primary cultured keratinocytes. Agarose gel electrophoresis of RNA, transfer to nitrocellulose filter and hybridization were performed as described (10). Double stranded cDNA inserted in pGEM3Z was sequenced by dideoxy chain termination method (5) and followed with computer analysis on EMBL and Genbank data base.
RESULTS AND DISCUSSION cDNA isolation and PCR A human primary cultured keratinocyte cDNA library was constructed in Xgtl 1 and was screened with a mAb against type VII collagen. ~10 6 plaques were screened and one clone, named R1 with an insert cDNA of ~lKb, was isolated from this library. We tentatively assign this clone corresponding to the non-collagenous domain because the mAb used in this screening reacts with the non-collagenous domain of type VII collagen. The positive signal of R1 clone was observed only when 13-thiogalactopyranoside (IPTG) was added to this screening system. This result indicates that synthesis of the fusion protein, which reacts to the mAb, is regulated by 13-galactosidase gene operon of ;Lgt11. Positive signals were observed only when R1 clone was incubated with this mAb, and normal mouse serum IgG does not bind to this clone. When reactivities of randomly picked up clone and R1 clone against this screening mAb were tested, there was marked difference between these two clones; R1 959
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clone showed clear positive signal and the clone randomly picked up showed clear negative. Therefore, R1 clone is specifically recognized by this mAb, and these observations thus indicate that R1 clone encodes type VII collagen. Very recently, Parente et aL reported the molecular cloning of type VII collagen cDNA ( named as K131) by screening an expression library with epidermolysis bullosa acquisita patient serum (9). They identified that their clone encodes the central portion of the type VII collagen molecule. The amino acid sequences of our clone and Parente's one are different from each other, reflecting the difference of recognition sites of these two screening antibodies. To confirm that the two cDNAs, R1 and K131, are derived from same mRNA species, analysis by PCR was performed. As shown in figure 1, primers A and B, corresponding to K131 and R1 respectively, gave rise to a 1.15Kb single band. Furthermore, the sequence analysis showed that this DNA fragment contains both R1 and K131 sequences ( data not shown ). These results clearly demonstrate that R1 clone encodes a part of type VII collagen. Northern blot of this clone The size of this mRNA was determined by Northern blot analysis with ~lKb fragment of R1 clone as a probe. ~9.5Kb single band was detected as shown in figure 2. This size is large enough to code type VII collagen of M.W. ~300KDa (11). A single polyadenylation signal may be utilized during transcription of this gene. This is at variance with type I collagen where several different sites are utilized leading to a length polymorphism of the mRNA products (12). Nucleotide sequence The nucleotide sequence and deduced amino acid sequence of this clone, representing approximately one-tenth of the ~9.5Kb mRNA, is presented in figure 3. There is a single open reading frame of 1020 bp encoding for 340
3
m
2
,----
9,5
- -
7.5
1.6 1 0.5
- -
m
4.4
'----"
2.4 m
Q
®
1.35
Fig. 1. PCR product by primer A and B. Left lane shows molecular weight in Kb. Fig. 2. RNA blot of total RNA from cultured keratinocytes. Left lane shows molecular weight in Kb. 960
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i
GAATTCCGAGCCCACAGCCTCCTGGTGGCCTGGCGGAGTGTGCCAGGTGCCACTGGCTAC GluPheArgAlaHisSerLeuLeuValAlaTrpArgSerValProGlyAlaThrGlyTyr
61
CGTGTGACATGGCGGGTCCTCAGTGGTGGGCCCACACAGCAGCAGGAGCTGGGCCCTGGG ArgValThrTrpArgValLeuSerGlyGlyProThrGlnGlnGlnGluLeuGlyProGly
121 CAGGGTTCAGTGTTGCTGCGTGACTTGGAGCCTGGCACGGACTATGAGGTGACCGTGAGC GlnGlySerValLeuLeuArgAspLeuGluProGlyThrAspTyrGluValTyrValSer 181 ACCCTATTTGGCCGCAGTGTGGGGCCCGCCACTTCCCTGATGGCTCGCACTGACGCTTCT TyrLeuPheGlyArgSerValGlyProAlaThrSerLeuMetAlaArgThrAspAlaSer 241 GTTGAGCAGACCCTGCGCCCGGTCATCCTGGGCCCCACATCCATCCTCCTTTCCTGGAAC ValGluGlnThrLeuArgProValIleLeuGlyProThrSerIleLeuLeuSerTrpAsn 301 TTGGTGCCTGAGGCCCGTGGCTACCGGTTGGAATGGCGGCGTGAGACTGGCTTGGAGCCA LeuValProGluAlaArgGlyTyrArgLeuGluTrpArgArgGluThrGlyLeuGluPro 361 CCGCAGAAGGTGGTACTGCCCTCTGATGTGACCCGCTACCAGTTGGATGGGCTGCAGCGG ProGlnLysValValLeuProSerAspValThrArgTyrGlnLeuAspGlyLeuGlnArg 421 GCACTGAGTACCGCCTCACACTCTACACTCTGCTGGAGGGCCACGAGGTGGCACCCCTGC AlaLeuSerThrAlaSerHisSerThrLeuCysTrpArgAlaThrArgTrpHisProCys 481 AACCGTGGTTCCCACTGGACCAGAGCTGCCTGTGAGCCCTGTAACAGACCTGCAAGCCAC AsnArgGlySerHisTrpThrArgAlaAlaCysGluProCysAsnArgProAlaSerHis 541 CGAGCTGCCCGGGCAGGGGTGCGAGTGTCCTGGTGCCCAGTCCCTGGTGCCACCCAGTAC ArgAlaAlaArgAlaGlyValArgValSerTrpCysProValProGlyAlaThrGlnTyr 601 CGCATCATTTGCGCAGACACCCAGGGGGTTGAGCGGACCCTGGTGCTTCCTGGGAGTCAG ArgIleIleCysAlaAspThrGlnGlyValGluArgThrLeuValLeuProGlySerGln 661 ACAGCATTCGACTTGGATGACGTTCAGGCTGGGCTTAGCTACACTGTGCGGGTGTCTGCT ThrAlaPheAspLeuAspAspValGlnAlaGlyLeuSerTyrThrValArgValSerAla 721 CGAGTGGGTCCCCGTGAGGGCAGTGCCAGTGTCCTCACTGTCCGCCGGGAGCCGGAAACT ArgValGlyProArgGluGlySerAlaSerValLeuThrValArgArgGluProGluThr 781 CCACTTGCTGTTCCAGGGCTGCGGGTTGTGGTGTCAGATGCAACGCGAGTGAGGGTGGCC ProLeuAlaValproGlyLeuArgValValValSerAspAlaThrArgValArgValAla 841 TGGGGACCCGTCCCTGGAGCCAGTGGATTTCGGATTAGCTGGAGCACAGGCAGTGGTCCG TrpGlyProValProGlyAlaSerGlyPheArgIleSerTrpSerThrGlySerGlyPro 901 GAGTCCAGCCAGACACTGCCCCCAGACTCTACTGCCACAGACATCACAGGGCTGCAGCCT GluSerSerGlnThrLeuProProAspSerThrAlaThrAspIleThrGlyLeuGlnPro 961 GGAACCACCTACCAGGTGGCTGTGTCGGTACTGCGAGGCAGAGAGGAGGGCCCGGAATTC GlyThrThrTyrGlnValAlaValSerValLeuArgGlyArgGluGluGlyProGluPhe
Fig. 3. Nucleotideand predi~ed amino acid sequences.
amino acids of expected MW of ~36.8 KDa, about one-fourth of the noncollagenous domain of type VII collagen. This sequence is also in frame to ~ galactosidase gene of kgt11.
The deduced structure does not contain a
repetitive sequence of Gly-X-Y unit that is a characteristic amino acid 961
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HFNT Col
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EFRAHSLLVAWRSVPGA-TGYRVTWRVLS-GGPTQQQELGPGQGSVLLRDLE : : : : :::: : DITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLT
VII
P-EARGYRLEWRRETGLEPPQKVVLPSDVTRYQLDGLQRALS-TASHSTLCW
VII
:
:
:
::
:
:
:
:
:
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•
::
RGDSPASSKPISINYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVT VII
CADTQ--GVERTLVLPGSQTAFDLDDVQAGLSYTVRVSARVGPREGSASVLT
TTPKNG PT KT GPD EMTZEG TV VOS YIQNPSG SQP Q
HFNT VII
HFNT
HFNT
:
RATRWHPCNRGSHWTRAACEPCNRPASHRAARAGVRVSWCPVPGA-TQYRII :
Col
::
AVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTG
HFNT
Col
:
PGTDYEVTVSTLFGRSVGPATSLMARTDASVEQTLRPVILGPTSILLSWNLV
•
Col
:
PGTEYVVSIVA{NGREESPLLIGQQSTVSDVPRD£EVVAATPTSL£ISWDAP
HFNT Col
:
VRREPETPLAVPGLRVVVSDATRVRVAWGPVPGASG-FRISWST--GSGPES : :: : :: : : :
:
AVTNIDRP---KGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHEL VII
SQTLPPDSTATDITGLQPGTTYQVAVSVLRGREEGPEF FPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPL
Fig. 4. Comparison of amino acid sequence of type VII collagen (Col VII) and fibronectin (HFNT) that corresponds to amino acid number 1334 to 1683 (13). Identical amino acids are indicated by :. RGD sequence of fibronectin is underlined.
sequence of a collagenous domain. Because the mAb used in this screening recognizes the peripheral end of a non-collagenous domain as demonstrated by the electron microscopic study (8), it is likely that the amino acid sequence encoded by R1 clone corresponds to the non-collagenous domain of type VII collagen. Computer analysis was performed to find a molecule which has a homology to our R1 clone encoded protein. As shown in figure 4, this part of type VII collagen has a weak homology (~23%) to the cell attachment domain of fibronectin, (13). Although the amino acid sequence of R1 clone does not contain RGD sequence, it is interesting that type VII collagen and fibronectin, both of which have similar function in aspect of cell binding to the underlying layer, have similar amino acid sequences.
The screening antibody reactive
with R1 clone is now used as a standard probe for the diagnosis as well as the prenatal diagnosis for the dystrophic type of epidermolysis bullosa because, in this disease, type VII collagen is not detected at BMZ with this mAb (4,7). Therefore, the isolation of this cDNA that contains coding region of the epitope for diagnostic mAb will provide a valuable tool for our understanding of normal and diseased skin. 962
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ACKNOWLEDGMENTS This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan. We thank Dr. Ryoichiro Kageyama for the technical advices and criticisms.
REFERENCES 1. Sakai, L.Y., Keene, D.R., Morris, N.P., and Burgeson, R.E. (1986) J. Cell. Biol. 103, 1577-1586. 2. Seltzer, J.L., Eisen, A.Z., Bauer, E.A., Morris, N.P., Glanville, R.W., and Burgeson, R.E. (1987)J. Biol. Chem. 264, 3822-3826. 3. Woodley, D.T., Burgeson, R.E., Lunstrum, G., Bruckner-Tuderman, L., Reese, M. and Briggaman, R.A. (1988) J. Clin. Invest. 81,683-687. 4. Heagertry, A,H,M., Kennedy, A,R., Leigh, I., Purkis, P.E.,and Eady, R.A.J. (1986) Br. J. Dermatol. 115, 125-131. 5. Stanley, J.R., Tanaka, T., Mueller, S., Klaus-Kovtum, V., and Roop, D. (1988) J. Clin. invest. 82, 1864-1870. 6. Tanaka, T., Parry, D.A.D., Klaus-Kovtum, V., Steinert, P.M., and Stanley, J.R. (1991) J. Biol. Chem. 266, 12555-12559. 7. Heagerty, A.H.M., Kennedy, A.R., Gunner, D.B., and Eady, R.A.J. (1986) J. Invest. Dermatol. 86, 603-605. 8, Shimizu, H., McDonald, J.N., Gunner, D.B., Black, M.M., Bhogal, B., Leigh, I.M., Whitehead, P.C., and Eady, R.A.J. (1990) Br. J. Dermatol. 122, 577585. 9. Parente, M,G., Chung, L.C., Ryynaenen, J., Woodley, D.T., Wynn, K.C., Bauer, E.A., Mattei, M-G., Chu, N-L., and Uitto, J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6931-6935. 10. Davis, LG., Dibner, M.D., and Battey, J.F. (1986) Basic Methods in Molecular Biology. Elsevier, New York 388pp 11. Lunstrum, G.P., Sakai, L.Y., Keene, D.R., Morris, N.P. and Burgeson, R.E. (1986) J. Biol. Chem. 261, 9042-9048. 12. Myers, J.C., Dickson, L.A., de Wet, W.J., Bernard, M.P., Chu, M., Di Liberto, M., Pepe, G., Sangiorgi, F.O., and Ramirez, F. (1983) J. Biol. Chem. 258, 10128-10135. 13. Kornblihtt, A.R., Umezawa, K., Vibe-Pedersen, K., and Baralle, F.E. (1985) EMBO J. 4, 1755-1759.
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MOLECULAR CLONING AND CHARACTERIZATION OF TYPE VII COLLAGEN cDNA Toshihiro Tanaka, Kenzo Takahashi, Fukumi Furukawa and Sadao imamura Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
Received February 12, 1992
SUMMARy; Type VII collagen, located in human epidermal basement membrane, is the primary pathogenic target molecule in epidermolysis bullosa acquisita and epidermolysis bullosa dystrophica. Using a monoctonal antibody against the non-collagenous domain of type VII collagen, ~lKb cDNA was isolated from human keratinocyte library. The deduced primary structure of this clone thus reflects the non-collagenous domain of type VII collagen that may be involved in cell attachment. This region shows a weak homology (~23%) to the cell attachment domain of fibronectin. Northern blot revealed ~9.5 Kb single band. ®1992 A c a d e m i c P ..... I n c .
The major protein of anchoring fibril, that may play an important role in the binding of keratinocytes to the underlying dermis, is composed of type VII collagen (1). This protein contains a collagenous domain, which makes a triple helical structure, and a non-collagenous domain that may be involved in cell attachment (2). Type VII collagen is of particular interest as they have been implicated in the pathogenesis of acquired epidermolysis bullosa and congenital epidermolysis bullosa, two clinical diseases that affect subbasement membrane region and cause skin blistering (3,4). The noncollagenous domain of type VII collagen is primarily targeted by pathogenic autoantibodies that bind to anchoring fibril in acquired epidermolysis bullosa (3). Moreover, type VII collagen is not detected by indirect immunofluorescence in the basement membrane zone (BMZ) of patients with dystrophic type epidermolysis bullosa (4). Therefore, hereditary defect of type VII collagen or dysfunction of this molecule by circulating autoantibody causes similar blistering disorder in human skin. Because such biochemical and immunological studies have highlighted the importance of type VII collagen, as a step toward examining the genetic bases of the structure of type VII collagen, characterizing the acquired epidermolysis bullosa antigen, and further investigation of congenital epidermolysis bullosa, the present study was undertaken to clone the cDNA encoding human type VII collagen. 0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
958
Vol. 183, No. 3, 1992
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
EXPERIMENTAL PROCEDURES Reagents: All the reagents for library construction, RNA blot, polymerase chain reaction (PCR) and DNA sequence were purchased from Takara Shuzo Co. (Kyoto Japan). Reagents for immunoscreening were purchased from BRL (MD, U.S.A). Construction of oDNA library and immunoscreening Random primer library was constructed with poly(A) rich RNA from cultured human keratinocytes (5,6) and resultant cDNAs were inserted in Eco RI site of expression vector Xgt11. Library was immunoscreened with the monoclonal antibody (mAb) which specifically reacts with type VII collagen (Chemicon International Inc, CA, U.S.A.). This mAb recognizes ~300KDa type VII collagen by Western blot and is now used as a tool for the diagnosis and prenatal diagnosis of dystrophic type epidermolysis bullosa (EB) (7.8). Polvmerase Chain Reaction: The following oligonucleotides were synthesized according to the nucleotide sequence of our clone (primer A) and Parente's clone (primer B and C) (9): primer A: 5'-GGAGCCGGAAACTCCAC TTG-3', primer B; 5'-GAGTGGCCGCCAGGATAGGAT-3' and primer C; 5'-CCC AAGTGCCAACACCAGACG-3'. ~lpg of poly(A) rich RNA from keratinocytes was used as a template by using primer C as a reverse transcriptase primer. Primer C was removed from the reaction mixture by membrane filter (Takara Shuzo Co, Kyoto Japan) and aliquot of the purified cDNA was amplified by primer A and B under the PCR condition described by supplier. The resultant fragment was gel electrophoresed. This fragment was subcloned into pGEM3Z vector (Promega Wl U.S.A.) and nucleotide sequenced. RNA blot. nucleotide seauence and comouter analysis: RNA was extracted from primary cultured keratinocytes. Agarose gel electrophoresis of RNA, transfer to nitrocellulose filter and hybridization were performed as described (10). Double stranded cDNA inserted in pGEM3Z was sequenced by dideoxy chain termination method (5) and followed with computer analysis on EMBL and Genbank data base.
RESULTS AND DISCUSSION cDNA isolation and PCR A human primary cultured keratinocyte cDNA library was constructed in Xgtl 1 and was screened with a mAb against type VII collagen. ~10 6 plaques were screened and one clone, named R1 with an insert cDNA of ~lKb, was isolated from this library. We tentatively assign this clone corresponding to the non-collagenous domain because the mAb used in this screening reacts with the non-collagenous domain of type VII collagen. The positive signal of R1 clone was observed only when 13-thiogalactopyranoside (IPTG) was added to this screening system. This result indicates that synthesis of the fusion protein, which reacts to the mAb, is regulated by 13-galactosidase gene operon of ;Lgt11. Positive signals were observed only when R1 clone was incubated with this mAb, and normal mouse serum IgG does not bind to this clone. When reactivities of randomly picked up clone and R1 clone against this screening mAb were tested, there was marked difference between these two clones; R1 959
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clone showed clear positive signal and the clone randomly picked up showed clear negative. Therefore, R1 clone is specifically recognized by this mAb, and these observations thus indicate that R1 clone encodes type VII collagen. Very recently, Parente et aL reported the molecular cloning of type VII collagen cDNA ( named as K131) by screening an expression library with epidermolysis bullosa acquisita patient serum (9). They identified that their clone encodes the central portion of the type VII collagen molecule. The amino acid sequences of our clone and Parente's one are different from each other, reflecting the difference of recognition sites of these two screening antibodies. To confirm that the two cDNAs, R1 and K131, are derived from same mRNA species, analysis by PCR was performed. As shown in figure 1, primers A and B, corresponding to K131 and R1 respectively, gave rise to a 1.15Kb single band. Furthermore, the sequence analysis showed that this DNA fragment contains both R1 and K131 sequences ( data not shown ). These results clearly demonstrate that R1 clone encodes a part of type VII collagen. Northern blot of this clone The size of this mRNA was determined by Northern blot analysis with ~lKb fragment of R1 clone as a probe. ~9.5Kb single band was detected as shown in figure 2. This size is large enough to code type VII collagen of M.W. ~300KDa (11). A single polyadenylation signal may be utilized during transcription of this gene. This is at variance with type I collagen where several different sites are utilized leading to a length polymorphism of the mRNA products (12). Nucleotide sequence The nucleotide sequence and deduced amino acid sequence of this clone, representing approximately one-tenth of the ~9.5Kb mRNA, is presented in figure 3. There is a single open reading frame of 1020 bp encoding for 340
3
m
2
,----
9,5
- -
7.5
1.6 1 0.5
- -
m
4.4
'----"
2.4 m
Q
®
1.35
Fig. 1. PCR product by primer A and B. Left lane shows molecular weight in Kb. Fig. 2. RNA blot of total RNA from cultured keratinocytes. Left lane shows molecular weight in Kb. 960
Vol. 183, No. 3, 1992
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i
GAATTCCGAGCCCACAGCCTCCTGGTGGCCTGGCGGAGTGTGCCAGGTGCCACTGGCTAC GluPheArgAlaHisSerLeuLeuValAlaTrpArgSerValProGlyAlaThrGlyTyr
61
CGTGTGACATGGCGGGTCCTCAGTGGTGGGCCCACACAGCAGCAGGAGCTGGGCCCTGGG ArgValThrTrpArgValLeuSerGlyGlyProThrGlnGlnGlnGluLeuGlyProGly
121 CAGGGTTCAGTGTTGCTGCGTGACTTGGAGCCTGGCACGGACTATGAGGTGACCGTGAGC GlnGlySerValLeuLeuArgAspLeuGluProGlyThrAspTyrGluValTyrValSer 181 ACCCTATTTGGCCGCAGTGTGGGGCCCGCCACTTCCCTGATGGCTCGCACTGACGCTTCT TyrLeuPheGlyArgSerValGlyProAlaThrSerLeuMetAlaArgThrAspAlaSer 241 GTTGAGCAGACCCTGCGCCCGGTCATCCTGGGCCCCACATCCATCCTCCTTTCCTGGAAC ValGluGlnThrLeuArgProValIleLeuGlyProThrSerIleLeuLeuSerTrpAsn 301 TTGGTGCCTGAGGCCCGTGGCTACCGGTTGGAATGGCGGCGTGAGACTGGCTTGGAGCCA LeuValProGluAlaArgGlyTyrArgLeuGluTrpArgArgGluThrGlyLeuGluPro 361 CCGCAGAAGGTGGTACTGCCCTCTGATGTGACCCGCTACCAGTTGGATGGGCTGCAGCGG ProGlnLysValValLeuProSerAspValThrArgTyrGlnLeuAspGlyLeuGlnArg 421 GCACTGAGTACCGCCTCACACTCTACACTCTGCTGGAGGGCCACGAGGTGGCACCCCTGC AlaLeuSerThrAlaSerHisSerThrLeuCysTrpArgAlaThrArgTrpHisProCys 481 AACCGTGGTTCCCACTGGACCAGAGCTGCCTGTGAGCCCTGTAACAGACCTGCAAGCCAC AsnArgGlySerHisTrpThrArgAlaAlaCysGluProCysAsnArgProAlaSerHis 541 CGAGCTGCCCGGGCAGGGGTGCGAGTGTCCTGGTGCCCAGTCCCTGGTGCCACCCAGTAC ArgAlaAlaArgAlaGlyValArgValSerTrpCysProValProGlyAlaThrGlnTyr 601 CGCATCATTTGCGCAGACACCCAGGGGGTTGAGCGGACCCTGGTGCTTCCTGGGAGTCAG ArgIleIleCysAlaAspThrGlnGlyValGluArgThrLeuValLeuProGlySerGln 661 ACAGCATTCGACTTGGATGACGTTCAGGCTGGGCTTAGCTACACTGTGCGGGTGTCTGCT ThrAlaPheAspLeuAspAspValGlnAlaGlyLeuSerTyrThrValArgValSerAla 721 CGAGTGGGTCCCCGTGAGGGCAGTGCCAGTGTCCTCACTGTCCGCCGGGAGCCGGAAACT ArgValGlyProArgGluGlySerAlaSerValLeuThrValArgArgGluProGluThr 781 CCACTTGCTGTTCCAGGGCTGCGGGTTGTGGTGTCAGATGCAACGCGAGTGAGGGTGGCC ProLeuAlaValproGlyLeuArgValValValSerAspAlaThrArgValArgValAla 841 TGGGGACCCGTCCCTGGAGCCAGTGGATTTCGGATTAGCTGGAGCACAGGCAGTGGTCCG TrpGlyProValProGlyAlaSerGlyPheArgIleSerTrpSerThrGlySerGlyPro 901 GAGTCCAGCCAGACACTGCCCCCAGACTCTACTGCCACAGACATCACAGGGCTGCAGCCT GluSerSerGlnThrLeuProProAspSerThrAlaThrAspIleThrGlyLeuGlnPro 961 GGAACCACCTACCAGGTGGCTGTGTCGGTACTGCGAGGCAGAGAGGAGGGCCCGGAATTC GlyThrThrTyrGlnValAlaValSerValLeuArgGlyArgGluGluGlyProGluPhe
Fig. 3. Nucleotideand predi~ed amino acid sequences.
amino acids of expected MW of ~36.8 KDa, about one-fourth of the noncollagenous domain of type VII collagen. This sequence is also in frame to ~ galactosidase gene of kgt11.
The deduced structure does not contain a
repetitive sequence of Gly-X-Y unit that is a characteristic amino acid 961
Vol. 183, No. 3, 1992 Col
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HFNT Col
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EFRAHSLLVAWRSVPGA-TGYRVTWRVLS-GGPTQQQELGPGQGSVLLRDLE : : : : :::: : DITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLT
VII
P-EARGYRLEWRRETGLEPPQKVVLPSDVTRYQLDGLQRALS-TASHSTLCW
VII
:
:
:
::
:
:
:
:
:
:
•
::
RGDSPASSKPISINYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVT VII
CADTQ--GVERTLVLPGSQTAFDLDDVQAGLSYTVRVSARVGPREGSASVLT
TTPKNG PT KT GPD EMTZEG TV VOS YIQNPSG SQP Q
HFNT VII
HFNT
HFNT
:
RATRWHPCNRGSHWTRAACEPCNRPASHRAARAGVRVSWCPVPGA-TQYRII :
Col
::
AVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTG
HFNT
Col
:
PGTDYEVTVSTLFGRSVGPATSLMARTDASVEQTLRPVILGPTSILLSWNLV
•
Col
:
PGTEYVVSIVA{NGREESPLLIGQQSTVSDVPRD£EVVAATPTSL£ISWDAP
HFNT Col
:
VRREPETPLAVPGLRVVVSDATRVRVAWGPVPGASG-FRISWST--GSGPES : :: : :: : : :
:
AVTNIDRP---KGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHEL VII
SQTLPPDSTATDITGLQPGTTYQVAVSVLRGREEGPEF FPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPL
Fig. 4. Comparison of amino acid sequence of type VII collagen (Col VII) and fibronectin (HFNT) that corresponds to amino acid number 1334 to 1683 (13). Identical amino acids are indicated by :. RGD sequence of fibronectin is underlined.
sequence of a collagenous domain. Because the mAb used in this screening recognizes the peripheral end of a non-collagenous domain as demonstrated by the electron microscopic study (8), it is likely that the amino acid sequence encoded by R1 clone corresponds to the non-collagenous domain of type VII collagen. Computer analysis was performed to find a molecule which has a homology to our R1 clone encoded protein. As shown in figure 4, this part of type VII collagen has a weak homology (~23%) to the cell attachment domain of fibronectin, (13). Although the amino acid sequence of R1 clone does not contain RGD sequence, it is interesting that type VII collagen and fibronectin, both of which have similar function in aspect of cell binding to the underlying layer, have similar amino acid sequences.
The screening antibody reactive
with R1 clone is now used as a standard probe for the diagnosis as well as the prenatal diagnosis for the dystrophic type of epidermolysis bullosa because, in this disease, type VII collagen is not detected at BMZ with this mAb (4,7). Therefore, the isolation of this cDNA that contains coding region of the epitope for diagnostic mAb will provide a valuable tool for our understanding of normal and diseased skin. 962
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BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
ACKNOWLEDGMENTS This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan. We thank Dr. Ryoichiro Kageyama for the technical advices and criticisms.
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