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Horse complement protein C9: primary structure and cytotoxic activity
A F Esser1, R W Tarnuzzer, S Tomlinson
1Department of Comparative and Experimental Pathology, University of Florida Health Science Center, Gainesville, USA.
Insights
Horse C9 protein lacks hemolytic activity, but efficiently kills bacteria. Structural analysis reveals no obvious differences from hemolytic bovine C9, suggesting other factors influence red blood cell lysis.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Horse serum C9 (Complement 9) protein is known for its lack of hemolytic activity.
- Understanding the molecular basis for this deficiency is crucial for comprehending complement system function.
Purpose of the Study:
- To elucidate the molecular reasons behind the deficiency in hemolytic activity of horse C9.
- To compare the structure and function of horse C9 with other species, particularly human and bovine C9.
Main Methods:
- Cloning and sequencing of horse C9 cDNA from a horse liver cDNA library.
- Purification of C9 protein from horse plasma and subsequent microsequencing.
- Amino acid sequence analysis and comparison with human and bovine C9.
Main Results:
- Horse C9 cDNA encodes a mature protein of 526 amino acids, 77% identical to human C9.
- Identical domain structure and conserved regions, including the membrane interaction domain, were observed.
- Horse C9 exhibits efficient cytotoxic activity against Gram-negative bacteria, despite lacking hemolytic activity.
Conclusions:
- Horse C9 is a structurally sound molecule with potent antibacterial cytotoxic activity.
- The inability of horse C9 to lyse erythrocytes may be due to interactions with regulatory proteins or target cell membranes, rather than intrinsic structural defects.
Abstract:
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.