- Email: [email protected]
Abnormal processing of procollagen VII → collagen VII underlies dystrophic epidermolysis bullosa in some families
Fifth International Conference on the Molecular Biology and Pathology of Matrix for JK-132 could be observed along the sinusoidal wall sporadically. Over 10 months of CCI 4 intoxication, the sinusoidal wall was stained intensely by JK-132. Electron microscopically, continuous endothelial cells with lamina densa were lined along the sinusoidal wall. Collagen fibrils were also increased in number in the perisinusoidal space as well as fibrotic septa to form bundles. Flocculent material filled the perisinusoidal space. Immunoelectron microscopically, dense reaction products for JK-199 were observed on the collagen fibrils. The concentration of BCC in serum increased again 2 to 3 times as high as that in normal condition. Taking account of these findings, a deposit of BCC in the perisinusoidal space in liver appears to be the first step in the development of hepatic fibrosis.
U t i l i z a t i o n o f a P C R - B a s e d Strategy for t h e
Isolation o f c D N A Clones Encoding t h e Human 0~3(IX) Chain
357
nucleotide primers were designed and used to amplify a 213-bp PCR product from human chondrocyte first strand cDNA. The identity of the 213bp PCR product was confirmed by DNA sequencing and the PCR product was subsequently used to screen a human chondrocyte cDNA library constructed in ~.UniZAP XR. Two overlapping cDNAs of 847 and 913 bp that extend from COL2 to the poly A tail were isolated and sequenced. A 1061-bp PCR product was generated by designing a sense primer based on bovine 0t3(IX) amino acid sequences within COL2 and pairing this primer with the antisense primer used to generate the PCR product 213. A 5' PCR product of 545 bp was generated by pairing a primer based on the signal peptide and NC4 domain of chicken 0t3(IX) with a primer based on bovine 0t3(IX) amino acid sequences from the 5' end of COL2. A third PCR product of 625 bp was then generated that overlaps the PCR products 1061 and 545 and closes the gap between these sequences. PCR products 545 and 625 were then used to rescreen the human chondrocyte cDNA library and a 1934-bp insert was identified and characterized that hybridized to both probes.
R. Brewton, B. Wood, Z-X. Ren, B. Lee, W. Horton, J. Baker and R. Mayne Department of Cell Biology and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294; Department of Pediatrics, Baylor College of Medicine, Houston, TX; and Shriner's Hospital for Crippled Children, Portland, OR, USA
Abnormal Processing of Procollagen VII --) Collagen VII underlies Dystrophic Epidermolysis Bullosa in some Families
Leena Bruckner-Tuderman, Dieter R. Zimmermann, Maria T. Dours-Zimmermann, Type IX collagen is located on the surface of Jan-Olof Winberg, Ulrike Kalinke and Tobias collagen fibrils in hyaline cartilage and vitreous Gedde-Dahl, Jr. humor and is stabilized by covalent crosslinks to type II collagen. In order to complete the primary Departments of Dermatology and Pathology, University of structure of human type IX collagen, we have uti- Ziirich, Ziirich, Switzerland; Department of Dermatology lized the polymerase chain reaction (PCR) to obtain University of Miinster, MOnster, Germany; and overlapping PCR products and cDNA clones that Department of Biochemistry, University of Troms6, cover the coding region of the 0t3(IX) chain. Norway Pepsin-resistant fragments of type IX collagen (called HMW and LMW) were isolated from The major constituent of the anchoring fibrils in human newborn sterna and costal cartilage the skin, collagen VII, is synthesized as a procollaobtained at autopsy. Unreduced HMW and LMW gen which has been presumed to be processed to were separated by molecular sieve chromatog- collagen by proteolytic cleavage of the C-terminal raphy. Reduced and alkylated LMW peptides were globular NC-2 domain, but details of this process then separated by C18 reverse-phase HPLC. The have remained unknown. We have investigated 0t3(IX) L M W peptide was digested with the processing of procollagen VII in cultured kerTPCK-trypsin, the tryptic peptides were separated atinocytes and in the skin. For this purpose, bacteby reverse-phase HPLC and select fractions were rial fusion proteins containing unique sequences of subjected to N-terminal amino acid sequencing. A the NC-2 domain of collagen VII were prepared. contiguous stretch of 123 amino acids extending Polyclonal antibodies were raised against the fusion from COL1 into NC1 was obtained from six over- proteins and affinity purified. Immunoblotting lapping peptide sequences. Degenerate oligo- showed that the antibodies reacted with procolla-
358
Fifth International Conference on the Molecular Biology and Pathology of Matrix
gen VII isolated from cultured keratinocytes but not with collagen VII extracted from the skin. Processing of procollagen -+ collagen VII occurred very slowly in vitro, addition of dextran sulfate and divalent cations to the cultures for 48 h results in only minimal conversion. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes but the basement membrane zone of normal skin was negative, indicating that the NC-2 domain is not present in collagen VII deposited at the dermalepidermal junction. Chemical or enzymatic unmasking did not reveal hidden epitopes. In contrast, a positive staining was observed in the skin of three patients of two unrelated families with dystrophic epider-molysis bullosa (EBD). In a further eight patients, a very faint positive reaction was observed, and the skin of 20 patients and 20 controls remained negative. This suggests that in some patients abnormal processing of procollagen -+ collagen VII underlies the EBD phenotype. The retention of the C-propeptide presumably disturbs the fibrillogenesis of collagen VII and leads to structurally and functionally abnormal anchoring fibrils.
recognition sites implicated are located in both the triple helix and globular domains. In vivo size heterogeneity at the protein level for tile 0t3(VI) chain that comprises most at the N-terminal globular precludes the possibility of obtaining type VI collagen molecules with defined 0~3(VI) variants for functional studies. In an effort to understand the biology of type VI collagen assembly and cell-matrix interactions, a model system was initially developed in which murine NIH/3T3 cells were stably transfected with cDNAs encoding chicken type VI collagen chains and no self-association of the different chains was observed. NIH/3T3 cells were then successfully cotransfected with the cDNAs (3.15, 3.2, and 8.1 kb) coding for the three chicken type VI collagen chains and for a selectable marker. Stable co-expression of all three chains was obtained and tile three transfected chains asssembled into pepsinresistant type VI collagen molecules that were secreted as monomers and higher order multimers of tile proper size and became deposited into the extracellular matrix. This approach demonstrates for the first time the production, albeit with low efficiency of expression, of a recombinant nonfibrillar collagen composed of three genetically different chains.
Secretion and Matrix Assembly of Recombinant Type VI Collagen A. Colombatti*t, M.T. Mucignat* and P. Bonaldol:
Amplification and Characterization of Type XII Collagen cDNAs from Human Fetal Chondrocytes and Fibroblasts
*Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico, Aviano; tDipartimento di Scienze e Tecnologie Biomediche, Universit& di Udine, Udine; and Rita M. Dharmarvaram and Sergio A. Jimenez ~tIstituto di Istologia, Universita di Padova, Padova, Italy Department of Medicine, Division of Rheumatology, Type VI collagen is a component of a microfila- Thomas Jefferson University, Philadelphia, PA mentous meshwork that is believed to play a signif- 19107-5541, USA icant role in cell-matrix interactions. Unique among the collagens discovered to date, type VI The extracellular matrix is a complex structure collagen constituent chains have significantly consisting of several distinct sub-families of tissue different sizes. The 0tl(VI) and 0t2(VI) chains are specific collagens. The sub-family of fibrilabout 130-140 kDa, the 0t3(VI) chain is much associated collagens with interrupted triple helices larger, ranging from 250 to 350 kDa. Cloning and (FACIT) is composed of collagen types IX, XII and sequencing of the cDNAs for the chicken and XIV. Type XII collagen has been characterized in human chains has shown that ~I(VI) and 0t2(VI) type I collagen containing tissues of chick embryos chains consist of a short collagenous sequence but was shown to be absent from bone and flanked at the N- and C-terminus by one and two cartilage based on immunohistological studies. type A modules, respectively. A mayor portion of However, it has recently been demonstrated that in the additional sequences of the 0t3(VI) chain bovine fetal epiphyseal cartilage a larger form of consists of six-to-nine type A modules. Tissue type XII collagen, XIIA, is predominant, whereas, e x t r a c t i o n and b i o s y n t h e t i c studies have in human epithelial ceils a smaller form, XIIB, is demonstrated that the three chains occur in predominant. When primers corresponding to the stoichiometric proportions in a 1:1:1 ratio. NC3 domain of the chicken type XII collagen were Type VI collagen promotes cell adhesion and cell utilized for RT-PCR of RNA from human fetal
U t i l i z a t i o n o f a P C R - B a s e d Strategy for t h e
Isolation o f c D N A Clones Encoding t h e Human 0~3(IX) Chain
357
nucleotide primers were designed and used to amplify a 213-bp PCR product from human chondrocyte first strand cDNA. The identity of the 213bp PCR product was confirmed by DNA sequencing and the PCR product was subsequently used to screen a human chondrocyte cDNA library constructed in ~.UniZAP XR. Two overlapping cDNAs of 847 and 913 bp that extend from COL2 to the poly A tail were isolated and sequenced. A 1061-bp PCR product was generated by designing a sense primer based on bovine 0t3(IX) amino acid sequences within COL2 and pairing this primer with the antisense primer used to generate the PCR product 213. A 5' PCR product of 545 bp was generated by pairing a primer based on the signal peptide and NC4 domain of chicken 0t3(IX) with a primer based on bovine 0t3(IX) amino acid sequences from the 5' end of COL2. A third PCR product of 625 bp was then generated that overlaps the PCR products 1061 and 545 and closes the gap between these sequences. PCR products 545 and 625 were then used to rescreen the human chondrocyte cDNA library and a 1934-bp insert was identified and characterized that hybridized to both probes.
R. Brewton, B. Wood, Z-X. Ren, B. Lee, W. Horton, J. Baker and R. Mayne Department of Cell Biology and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294; Department of Pediatrics, Baylor College of Medicine, Houston, TX; and Shriner's Hospital for Crippled Children, Portland, OR, USA
Abnormal Processing of Procollagen VII --) Collagen VII underlies Dystrophic Epidermolysis Bullosa in some Families
Leena Bruckner-Tuderman, Dieter R. Zimmermann, Maria T. Dours-Zimmermann, Type IX collagen is located on the surface of Jan-Olof Winberg, Ulrike Kalinke and Tobias collagen fibrils in hyaline cartilage and vitreous Gedde-Dahl, Jr. humor and is stabilized by covalent crosslinks to type II collagen. In order to complete the primary Departments of Dermatology and Pathology, University of structure of human type IX collagen, we have uti- Ziirich, Ziirich, Switzerland; Department of Dermatology lized the polymerase chain reaction (PCR) to obtain University of Miinster, MOnster, Germany; and overlapping PCR products and cDNA clones that Department of Biochemistry, University of Troms6, cover the coding region of the 0t3(IX) chain. Norway Pepsin-resistant fragments of type IX collagen (called HMW and LMW) were isolated from The major constituent of the anchoring fibrils in human newborn sterna and costal cartilage the skin, collagen VII, is synthesized as a procollaobtained at autopsy. Unreduced HMW and LMW gen which has been presumed to be processed to were separated by molecular sieve chromatog- collagen by proteolytic cleavage of the C-terminal raphy. Reduced and alkylated LMW peptides were globular NC-2 domain, but details of this process then separated by C18 reverse-phase HPLC. The have remained unknown. We have investigated 0t3(IX) L M W peptide was digested with the processing of procollagen VII in cultured kerTPCK-trypsin, the tryptic peptides were separated atinocytes and in the skin. For this purpose, bacteby reverse-phase HPLC and select fractions were rial fusion proteins containing unique sequences of subjected to N-terminal amino acid sequencing. A the NC-2 domain of collagen VII were prepared. contiguous stretch of 123 amino acids extending Polyclonal antibodies were raised against the fusion from COL1 into NC1 was obtained from six over- proteins and affinity purified. Immunoblotting lapping peptide sequences. Degenerate oligo- showed that the antibodies reacted with procolla-
358
Fifth International Conference on the Molecular Biology and Pathology of Matrix
gen VII isolated from cultured keratinocytes but not with collagen VII extracted from the skin. Processing of procollagen -+ collagen VII occurred very slowly in vitro, addition of dextran sulfate and divalent cations to the cultures for 48 h results in only minimal conversion. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes but the basement membrane zone of normal skin was negative, indicating that the NC-2 domain is not present in collagen VII deposited at the dermalepidermal junction. Chemical or enzymatic unmasking did not reveal hidden epitopes. In contrast, a positive staining was observed in the skin of three patients of two unrelated families with dystrophic epider-molysis bullosa (EBD). In a further eight patients, a very faint positive reaction was observed, and the skin of 20 patients and 20 controls remained negative. This suggests that in some patients abnormal processing of procollagen -+ collagen VII underlies the EBD phenotype. The retention of the C-propeptide presumably disturbs the fibrillogenesis of collagen VII and leads to structurally and functionally abnormal anchoring fibrils.
recognition sites implicated are located in both the triple helix and globular domains. In vivo size heterogeneity at the protein level for tile 0t3(VI) chain that comprises most at the N-terminal globular precludes the possibility of obtaining type VI collagen molecules with defined 0~3(VI) variants for functional studies. In an effort to understand the biology of type VI collagen assembly and cell-matrix interactions, a model system was initially developed in which murine NIH/3T3 cells were stably transfected with cDNAs encoding chicken type VI collagen chains and no self-association of the different chains was observed. NIH/3T3 cells were then successfully cotransfected with the cDNAs (3.15, 3.2, and 8.1 kb) coding for the three chicken type VI collagen chains and for a selectable marker. Stable co-expression of all three chains was obtained and tile three transfected chains asssembled into pepsinresistant type VI collagen molecules that were secreted as monomers and higher order multimers of tile proper size and became deposited into the extracellular matrix. This approach demonstrates for the first time the production, albeit with low efficiency of expression, of a recombinant nonfibrillar collagen composed of three genetically different chains.
Secretion and Matrix Assembly of Recombinant Type VI Collagen A. Colombatti*t, M.T. Mucignat* and P. Bonaldol:
Amplification and Characterization of Type XII Collagen cDNAs from Human Fetal Chondrocytes and Fibroblasts
*Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico, Aviano; tDipartimento di Scienze e Tecnologie Biomediche, Universit& di Udine, Udine; and Rita M. Dharmarvaram and Sergio A. Jimenez ~tIstituto di Istologia, Universita di Padova, Padova, Italy Department of Medicine, Division of Rheumatology, Type VI collagen is a component of a microfila- Thomas Jefferson University, Philadelphia, PA mentous meshwork that is believed to play a signif- 19107-5541, USA icant role in cell-matrix interactions. Unique among the collagens discovered to date, type VI The extracellular matrix is a complex structure collagen constituent chains have significantly consisting of several distinct sub-families of tissue different sizes. The 0tl(VI) and 0t2(VI) chains are specific collagens. The sub-family of fibrilabout 130-140 kDa, the 0t3(VI) chain is much associated collagens with interrupted triple helices larger, ranging from 250 to 350 kDa. Cloning and (FACIT) is composed of collagen types IX, XII and sequencing of the cDNAs for the chicken and XIV. Type XII collagen has been characterized in human chains has shown that ~I(VI) and 0t2(VI) type I collagen containing tissues of chick embryos chains consist of a short collagenous sequence but was shown to be absent from bone and flanked at the N- and C-terminus by one and two cartilage based on immunohistological studies. type A modules, respectively. A mayor portion of However, it has recently been demonstrated that in the additional sequences of the 0t3(VI) chain bovine fetal epiphyseal cartilage a larger form of consists of six-to-nine type A modules. Tissue type XII collagen, XIIA, is predominant, whereas, e x t r a c t i o n and b i o s y n t h e t i c studies have in human epithelial ceils a smaller form, XIIB, is demonstrated that the three chains occur in predominant. When primers corresponding to the stoichiometric proportions in a 1:1:1 ratio. NC3 domain of the chicken type XII collagen were Type VI collagen promotes cell adhesion and cell utilized for RT-PCR of RNA from human fetal