Horse complement protein C9: primary structure and cytotoxic activity.
Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. 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 two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative 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.
Esser
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AF
Department of Comparative and Experimental Pathology, University of Florida Health Science Center, Gainesville, USA.
Tarnuzzer
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RW
Tomlinson
S
S
Tatar
L D
LD
Stanley
K K
KK
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers / Q000235:genetics / Q000302:isolation & purification; D001770:Blood Bactericidal Activity; D002417:Cattle; D003001:Cloning, Molecular; D003186:Complement C9 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003602:Cytotoxicity, Immunologic; D000939:Epitopes / Q000235:genetics / Q000302:isolation & purification; D006736:Horses / Q000097:blood / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data
D001704:Biopolymers; D003186:Complement C9; D000939:Epitopes