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Purification and Characterization of a Single Chain Precursor C3-Protein (PRO-C3) from Normal Human Plasma¹

From the Department of Pathology and the Institute of Immunology, University of Toronto, Medical Sciences Building, Toronto, Ontario, Canada M5S 1A8 and the Rheumatic Diseases Unit, the Wellesley Hospital, Toronto, Ontario, Canada M4Y 1J3

Abstract

Fresh human plasma was treated with proteinase inhibitors and passed through an immunoadsorbent column of Sepharose anti-C3 globulin. The insolubilized C3 was eluted with 5 M guanidine and, after extensive dialysis, was reduced with 0.2 M 2-mercaptoethanol and electrophoresed on SDS-polyacrylamide gels. The bulk of the eluted C3 dissociated into two major protein bands, m.w. 125,000 and 75,000 corresponding to the -and -chains of C3. In addition, a nonreducible single polypeptide chain (SPC) with a m.w. value of 197,000 ± 2,000 similar to the apparent m.w. of unreduced C3 was consistently present. SPC has been purified by elution from SDS (SPC*) and found to remain a single polypeptide chain upon re-electrophoresis on SDS gels in the presence of 0.2 M 2-Mercaptoethanol. The purified SPC reacted with antisera to denatured C3, C3, and C3 chains. Additionally, antisera to SPC*, also reacted with denatured C3, C3-, and C3-chains, revealed a reaction of identity between SPC and C3, and detected partial identity between SPC and C3- as well as C3-chains. This suggested that SPC and C3 are antigenically related. The amino acid compositions and tryptic peptide maps of SPC and C3 were similar. Based on these findings, it is suggested that SPC must represent a single-chain precursor C3 (pro-C3) in plasma that escaped post-synthetic proteolytic cleavage into a two-subunit chain C3 molecule.

Footnotes

¹ This work was supported by the Canadian Medical Research Council Grant MT-5063 and a grant from the Ontario Heart Foundation. Part of this work was presented at the Meetings of the Federation of American Societies for Experimental Biology held in Atlanta, Georgia, June 4–8, 1978.

² Research Associate, Canadian Arthritis and Rheumatism Society.