Circular dichroism and electron microscopy studies of human subcomponent C1q before and after limited proteolysis by pepsin

Abstract

A fragment of human subcomponent C1q was prepared by limited proteolysis with pepsin at 37°C for 20h, and at pH 4.4, followed by gel filtration on Sephadex G 200. This fragment was shown to contain all the collagen like features known to be present in the intact molecule. Circular dichroism studies showed the presence of positive bands at 230 and 223 nm in the intact subcomponent C1q and pepsin fragment respectively, compared with a positive band at 220 nm obtained for lathyritic rat skin collagen. These bands were abolished by collagenase treatment, which suggested that there may be some collagen like triple helical structure in subcomponent C1q and that this structure resides in the pepsin resistant portion of the molecule. However, the 230 and 223 nm bands had a substantially lower magnitude than that obtained for the unaggregated single fibres of totally triple helical collagen. Thermal transition temperatures obtained for subcomponent C1q, the pepsin fragment and the reduced and alkylated pepsin fragment were 48°, 48° and 39°C respectively, compared with a value of 38°C obtained for lathyritic rat skin collagen. Only the unreduced pepsin fragment regained significant amounts (up to 60%) of collagen like structure, after heat denaturation and cooling, as estimated by circular dichroism measurements. Electron microscopy studies of subcomponent C1q and the collagen like pepsin resistant fragment of subcomponent C1q showed that the six peripheral globular regions of the molecule were fragmented by pepsin leaving the six collagen like connecting strands and fibril like central portion intact.