Figure 3
SDS-PAGE analysis of secreted 14C-labeled (pro)collagen molecules from dermal fibroblasts. Secreted procollagen molecules were extracted from the medium fraction of the cell cultures. The globular N- and C-propeptide of the procollagen molecules were removed by pepsin-digestion, resulting in mature triple helical collagen proteins. Lane numbers correspond to the patient’s numbers in the text; “C” corresponds to control samples. panel A: medium fraction. P2: note the extra band of higher molecular weight, suggestive for presence of mutant α1(I)-collagen dimers ( indicated by *). EDS VIIA: patient with EDS arthrochalasis type, due to a COL1A1 exon 6 skip: note the “doublet” for the α1(I) collagen chain that corresponds to mature α-chains and the incompletely cleaved pNα1(I)-chains (indicated by **) respectively. This doublet is absent in the OI/EDS patients. panel B: medium procollagen fraction. Compared to the control sample, the intensity of the bands representing the pNα1(I) chain is increased, whereas the intensity of the bands representing the pCα1(I) and the mature α1(I)-chain is decreased. This is caused by a disturbed balance in the normal kinetics of the delayed N-processing and the unhampered C-processing of type I procollagen. This pattern is similar to what is observed in the patient with EDS arthrochalasis type (EDS VIIA). In the right panel, the medium procollagen fraction is shown from a patient with classic OI, without EDS-signs, with p.(Gly257Arg) substitution in exon 11 of COL1A1. Here, intensity of the bands representing the pNα1-, pCα1- and mature α1 chains of type I collagens is comparable to that of the control.