Complement component C5: engineering of a mutant that is specifically cleaved by the C4-specific C1s protease.
Previous studies showed that simply inserting or substituting a few amino acid residues immediately downstream of the proteolytic activation site in C component C3 renders that site susceptible to the C4-specific C1s protease. This report describes the results of extending those studies to the closely related component C5. We found that small changes, similar to those that made C3 susceptible to C1s, were insufficient to render C5 C1s-sensitive; and neither more extensive substitution downstream of the cleavage site with a 14 residue long segment from C4, nor upstream substitution with an 8 residue long C4 segment gave C1s cleavage. However, substitution of both the upstream and downstream segments gave a hybrid C5 protein, designated ASC4, which was cleaved by C1s. The protease sensitivity of ASC4 was curious, however, in that C1s was more active against the secreted extracellular biosynthetic precursor, pro-ASC4(E) than mature ASC4, whereas a C5-specific convertase cleaved the mature protein but not the precursor. In contrast, both mature and precursor forms of wild-type C5 were cleaved by the C5 convertase, but neither of course is recognized by C1s. These results demonstrate that a mutant C5 molecule can be constructed that is cleaved at the activation site by both C1s and C5 convertase. This suggests that the structures necessary for specific recognition by the two proteases have little or no overlap and that recognition by C5 convertase involves residues that are distant from the activation site itself.
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Medical Biology Institute, La Jolla, CA 92037, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D003174:Complement C1 Inactivator Proteins; D003173:Complement C1s / Q000502:physiology*; D003176:Complement C3 / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism; D003182:Complement C5 / Q000235:genetics* / Q000378:metabolism*; D011415:Complement Factor B / Q000502:physiology; D005818:Genetic Engineering; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011817:Rabbits
D003174:Complement C1 Inactivator Proteins; D003176:Complement C3; D003181:Complement C4; D003182:Complement C5; D003173:Complement C1s; D011415:Complement Factor B