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

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Abstract
A fragment of human C1q [q fragment of complement component 1] was prepared by limited proteolysis with pepsin at 37.degree. C for 20 h, and at pH 4.4, followed by gel filtration on Sephadex G-200. This fragment contains 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. The 230 and 223 nm bands had a substantially lower magnitude than that obtained for the unaggregated single fibers of totally triple-helical collagen. Thermal-transition temperatures obtained for C1q, the pepsin fragment and the reduced and alkylated pepsin fragment were 48.degree., 48.degree. and 39.degree. C, respectively, compared with a value of 38.degree. 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. EM studies of C1q and the collagen-like pepsin-resistant fragment of C1q showed that the 6 peripheral globular regions of the molecule were fragmented by pepsin leaving the 6 collagen-like connecting strands and fibril-like central portion intact.