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Reconstitution of banding fibrils from the subtypes of type V collagen with the chain compositions of [α1(V)I2α2(V) and α1(V)α2(V)α3(V)
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The Third Pan Pacific Connective Tissue Societies Symposium
lar to, but genetically distinct from Marfan syndrome. Genetic linkage analysis implicated the fibrillin-2 gene (FBN2) as the CCA locus. Mutation analysis of single CCA patients indicate that defects in FBN2 may be responsible for that disorder. However, co-segregation of a mutant allele with the disease phenotype has not been established. We have investigated the primary cause of CCA in a large, well characterized kindred with four documented generations of 18 affected individuals. Previous studies showed linkage of the CCA phenotype in this family to FBN2. Mutation analysis of the proband's eDNA using Non-Isotopic Rnase Cleavage Assay identified the presence of a skipped exon that was subsequently identified as exon 31. The occurrence of exon skipping was confirmed in the cDNA of an affected sibling. Additional analyses indicate that mis-splicing is partial and that the mutant allele makes up only about 25% of the fibrillin-2 transcript. DNA sequence analysis of genomic DNA identified a mutation at the -26 position of intron 30. The mutation affects the branch point region upstream from the 3' splice site, resulting in a lowered efficiency of splicing of exon 31. Genomic DNA from 29 additional available family members, both affected and unaffected, were also analyzed for the mutation. The resuits clearly demonstrate co-segregation of the abberant splice site with the CCA phenotype. This unequivocally establishes that mutations in FBN2 are responsible for the CCA phenotype. It appears that the low level of mutant transcript seen here is sufficient to result in a CCA phenotype seen in this family. We propose that the significant intrafamilial variability is due to the degree of missplicing in a given individual.
collagen is directly involved in connecting fine collagen fibrils with basal lamina composed of the type IV collagen polygonal meshwork. Hence, we compared characteristic structures formed from the isolated two subtypes obtained by pepsin treatment. The electron micrographs showed several intriguing characteristics. It was reconfirmed that either subtype of the type V collagen could form D-periodic banding fibrils with a thinner diameter than the length of D-periodicity. However, the two subtypes appeared to require different temperatures for the stable formation of banding pattern. The subtype of J~l(V)]2c~2(V) essentially reconstituted the fibrils with Dperiodic banding pattern at 37 °C, while the fibrils formed from the subtype of cd(V)c~2(V)c~3(V) did not show the banding pattern at this temperature. Instead, the fibrillar aggregates from (x3(V) chain-containing type V collagen subtype showed a distinct banding pattern at a temperature lower by than 37 °C. Since the two subtypes were obtained from pepsin-treated human placenta, and that the proteins are essentially composed of the helical domains alone, the characteristic aggregate structures with banding fibrils in a smaller diameter must be due to the self-assembly through the triple-helical domains of the type V collagen subtypes. Thus the essential limiting factor of the fibril diameter may reside m the specific helical structure of type V collagen. The fibrils of otl(V)ot2(V)0t3(V) formed at 37 °C appeared to fray into finer fibrils at the end, suggesting that this subtype would be a candidate molecules to interact with the type IV collagen meshwork through frayed fine fibrils at the end.
Membrane-Type Metalloproteinase Digests Extracellular Matrix Macromolecules including Interstitial Collagens Reconstitution of Banding Fibrils from the Subtypes of Type V Collagen with the Chain Compositions of [~I(V)]2c~2(V) and al(V)c~2(V)cG(V) Kazunori Mizuno, Eijiro Adachi, and Toshihiko Hayashi Dept. of Life Sciences, Graduate School of Arts & Sciences, The University of Tokyo, Meguro, Tokyo. The biochemical characteristics of the fine fibrils reconstituted from isolated type V collagen molecules suggested that the type V collagen has not only a fibril-forming character and thus interacting property with type I collagen, but may also have a potential of interacting with type IV collagen. The implication is consistent with the histochemical distribution of the protein. The third a chain of type V collagen, c~3(V), was discovered from the tissues rich in vascular systems such as placenta. Thus, a question is raised, if the 0t3(V) chain-containing type V
Ohuchi, E., Imai K., Fujii Y., Sato H.+, Seiki, M.+ And Okada Y.* Departments of Molecular Immunology and Pathology, and +Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, ::Fuji Chemical Industries, Ltd., and Department of Chemistry, Fukui Medical School, Japan. Membrane-type I Matrix Metalloproteinase (MT1) is expressed on cancer cell membranes and activates proMMP-2 (progelatinase A). In this study, we purified a deletion mutant of MT1 (DMT1) lacking the transmembrane domain (DAIaS36-VaP 82) and native MT1 secreted from a human breast carcinoma cell line (MDA-MB-231 cells), and examined their substrate specificities. Both proteinases were active without any treatment for activation, and their activities were identical. DMT1 digested
The Third Pan Pacific Connective Tissue Societies Symposium
lar to, but genetically distinct from Marfan syndrome. Genetic linkage analysis implicated the fibrillin-2 gene (FBN2) as the CCA locus. Mutation analysis of single CCA patients indicate that defects in FBN2 may be responsible for that disorder. However, co-segregation of a mutant allele with the disease phenotype has not been established. We have investigated the primary cause of CCA in a large, well characterized kindred with four documented generations of 18 affected individuals. Previous studies showed linkage of the CCA phenotype in this family to FBN2. Mutation analysis of the proband's eDNA using Non-Isotopic Rnase Cleavage Assay identified the presence of a skipped exon that was subsequently identified as exon 31. The occurrence of exon skipping was confirmed in the cDNA of an affected sibling. Additional analyses indicate that mis-splicing is partial and that the mutant allele makes up only about 25% of the fibrillin-2 transcript. DNA sequence analysis of genomic DNA identified a mutation at the -26 position of intron 30. The mutation affects the branch point region upstream from the 3' splice site, resulting in a lowered efficiency of splicing of exon 31. Genomic DNA from 29 additional available family members, both affected and unaffected, were also analyzed for the mutation. The resuits clearly demonstrate co-segregation of the abberant splice site with the CCA phenotype. This unequivocally establishes that mutations in FBN2 are responsible for the CCA phenotype. It appears that the low level of mutant transcript seen here is sufficient to result in a CCA phenotype seen in this family. We propose that the significant intrafamilial variability is due to the degree of missplicing in a given individual.
collagen is directly involved in connecting fine collagen fibrils with basal lamina composed of the type IV collagen polygonal meshwork. Hence, we compared characteristic structures formed from the isolated two subtypes obtained by pepsin treatment. The electron micrographs showed several intriguing characteristics. It was reconfirmed that either subtype of the type V collagen could form D-periodic banding fibrils with a thinner diameter than the length of D-periodicity. However, the two subtypes appeared to require different temperatures for the stable formation of banding pattern. The subtype of J~l(V)]2c~2(V) essentially reconstituted the fibrils with Dperiodic banding pattern at 37 °C, while the fibrils formed from the subtype of cd(V)c~2(V)c~3(V) did not show the banding pattern at this temperature. Instead, the fibrillar aggregates from (x3(V) chain-containing type V collagen subtype showed a distinct banding pattern at a temperature lower by than 37 °C. Since the two subtypes were obtained from pepsin-treated human placenta, and that the proteins are essentially composed of the helical domains alone, the characteristic aggregate structures with banding fibrils in a smaller diameter must be due to the self-assembly through the triple-helical domains of the type V collagen subtypes. Thus the essential limiting factor of the fibril diameter may reside m the specific helical structure of type V collagen. The fibrils of otl(V)ot2(V)0t3(V) formed at 37 °C appeared to fray into finer fibrils at the end, suggesting that this subtype would be a candidate molecules to interact with the type IV collagen meshwork through frayed fine fibrils at the end.
Membrane-Type Metalloproteinase Digests Extracellular Matrix Macromolecules including Interstitial Collagens Reconstitution of Banding Fibrils from the Subtypes of Type V Collagen with the Chain Compositions of [~I(V)]2c~2(V) and al(V)c~2(V)cG(V) Kazunori Mizuno, Eijiro Adachi, and Toshihiko Hayashi Dept. of Life Sciences, Graduate School of Arts & Sciences, The University of Tokyo, Meguro, Tokyo. The biochemical characteristics of the fine fibrils reconstituted from isolated type V collagen molecules suggested that the type V collagen has not only a fibril-forming character and thus interacting property with type I collagen, but may also have a potential of interacting with type IV collagen. The implication is consistent with the histochemical distribution of the protein. The third a chain of type V collagen, c~3(V), was discovered from the tissues rich in vascular systems such as placenta. Thus, a question is raised, if the 0t3(V) chain-containing type V
Ohuchi, E., Imai K., Fujii Y., Sato H.+, Seiki, M.+ And Okada Y.* Departments of Molecular Immunology and Pathology, and +Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, ::Fuji Chemical Industries, Ltd., and Department of Chemistry, Fukui Medical School, Japan. Membrane-type I Matrix Metalloproteinase (MT1) is expressed on cancer cell membranes and activates proMMP-2 (progelatinase A). In this study, we purified a deletion mutant of MT1 (DMT1) lacking the transmembrane domain (DAIaS36-VaP 82) and native MT1 secreted from a human breast carcinoma cell line (MDA-MB-231 cells), and examined their substrate specificities. Both proteinases were active without any treatment for activation, and their activities were identical. DMT1 digested