Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta |
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Authors: | Galicka Anna Wo?czyński S?awomir Gindzieński Andrzej |
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Institution: | Department of Medical Chemistry, Medical Academy of Bia?ystok, 15-230 Bia?ystok 8, Poland. angajko@amb.ac.bialystok.pl |
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Abstract: | Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells. |
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