HORSE COMPLEMENT PROTEIN C9 - PRIMARY STRUCTURE AND CYTOTOXIC ACTIVITY

Lack of hemolytic activity of horse serum is an inherent property of h orse C9. To understand the molecular reasons for this deficiency we ha ve cloned C9 cDNA from a horse liver cDNA library and have sequenced t he cDNA yielding the complete coding sequence for horse C9. Purificati on of C9 from horse plasma and microsequencing established the N-termi nus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds t o a mature protein of 526 amino acids that is 77% identical to human C 9. It has the same domain structure as human C9 and contains 22 cystei nes and four invariant tryptophanes. The few differences include the N -terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to tw o in human C9. The N-terminal difference is unimportant since microseq uencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obv ious structural differences apparent that could resolve the difference s in hemolytic potency between the two molecules. Aside from a few con servative replacements, both C9 sequences are identical between positi ons 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to cons titute the wall of a putative transmembrane pore and, therefore, shoul d be required for cytotoxicity. In agreement with this prediction we h ave observed that, in contrast to the marked decrease in hemolytic act ivity, horse C9 is very efficient in killing a variety of Gram-negativ e bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes. Copyright (C) 1996 Elsevier Science Ltd.