Production in mammalian cells of chimeric human/sea urchin procollagen molecules displaying distinct versions of the minor triple helix

One of the mechanisms involved in the regulation of the fibril diameter is the retention of the N-propeptide. In sea urchin embryo, thin collagen fibr ils harbor numerous extensions at their surface, which we have suggested co rrespond to the large N-propeptide of the 2 alpha collagen chain. To invest igate the function of the N-propeptide during fibrillogenesis, we engineere d constructs coding for the globular region of the 2 alpha N-propeptide, To obtain homotrimeric molecules, the N-telopeptide, the central triple helix and the C-propeptide of the 2 alpha chain were replaced by human domains o f the pro alpha1(I) chain. A single restriction site allowed insertion of d istinct versions of the minor triple helix of the N-propeptide, Several hum an cell lines were transfected, and with one of them we were able to produc e intact homotrimeric procollagen molecules. Rotary shadowing of these puri fied molecules indicates the presence of three large 2 alpha N-propeptides that are similar to the extensions present at the surface of the sea urchin thin fibrils, This cassette-vector will be useful in determining the respe ctive contributions of the globular and minor triple helical domains of the N-propeptide in the regulation of fibril diameter.