Horse complement protein C9: primary structure and cytotoxic activity.

The research article involves a study on the construction and functionality of horse complement protein C9. The scientists cloned, sequenced, and studied the components of the protein finding it to be surprisingly efficient at killing bacteria even though it lacks some of the hemolytic activity found in humans’ C9 version.

Objective and Methodology

• The researchers were curious about why horse C9 lacked hemolytic activity, a mechanism by which certain organisms destroy red blood cells.
• They attempted to understand the molecular cause of this by cloning C9 cDNA from a horse liver cDNA library and sequencing it to get the full coding sequence for the horse C9 protein.
• Horse C9 was purified from horse plasma and microsequencing helped establish the N-terminus of the mature protein. This process confirmed that the correct horse C9 cDNA clone was isolated.

Findings

• The amino acid sequence deduced corresponds to a mature protein that has 526 amino acids and is 77% similar to human C9.
• Similar to the human C9, the horse’s version also has the same domain structure, carries 22 cysteine residues and four tryptophans that don’t change.
• Key differences lie in the N-terminus where horse C9 possesses an unblocked glycine, compared to the pyroglutamine in human C9. It also has three potential N-glycosylation sites as compared to human’s which only has two.
• Despite these differences, the N-terminus variation seems non-significant when considering the hemolytic activity, as validated by a comparison to bovine C9 with similar N-terminus but possessing strong hemolytic activity.

Cytotoxic Activity

• There were no detectable structural differences that explain the difference in hemolytic activity between horse and human C9.
• The two proteins have nearly identical sequences in specific regions thought to influence cytotoxicity, indicating that horse C9 should be capable of this activity.
• Importantly, horse C9 demonstrated an excellent ability to kill off Gram-negative bacteria, despite poor hemolytic activity. This suggested that the hemolytic activity in horse’s C9 might be less influential overall or tempered by the action of control proteins on target cell membranes.