Comparative Immunology Reports · Published 2026-05-29 · DOI 10.1016/j.cirep.2026.200292
Akhil Kizhakkumpat, Michiyo Kimura, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao
The complement system of jawed vertebrates comprises three activation routes—classical (CP), lectin (LP), and alternative (AP) pathways—yet the functional architecture of these cascades in early vertebrates remain insufficiently defined. Although the AP is often considered dominant in early vertebrates, direct functional evidence supporting this view has been limited, leaving key questions regarding the ancestral roles of complement pathways unresolved. Here, we investigated the hemolytic reaction of common carp (Cyprinus carpio) complement using serum selectively depleted of factor D (Df), an essential AP serine protease. Df-depleted serum completely lost hemolytic activity not only via AP but also via CP, and this defect was fully restored by re-addition of purified carp Df. Quantitative ELISA and flow cytometry demonstrated that CP activation in the absence of Df deposited only trace amounts of C3b on target surfaces, insufficient to support C5-convertase formation. Consistently, Df-depleted serum failed to deposit C5, indicating a complete loss of C5-convertase activity. Nevertheless, these minimally opsonized intermediates efficiently triggered AP activation and terminal pathway–mediated lysis when exposed to Mg–EGTA-buffered normal serum, revealing that even small amounts of CP-derived C3b can initiate AP amplification. Collectively, our findings demonstrate that teleost CP is intrinsically incapable of forming a functional C5-convertase and relies absolutely on AP to complete the complement cascade. We propose that the ancestral role of CP was not direct C5 activation, but the initiation of AP-mediated amplification—a functional configuration that persists in modern bony fish.
Abstract from DOAJ. Public domain (CC0 1.0).
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Kizhakkumpat, A., Kimura, M., Nagasawa, T., et al. (2026). Ancestral roles of the classical complement pathway uncovered through functional analysis of factor D-depleted carp serum. Comparative Immunology Reports. https://doi.org/10.1016/j.cirep.2026.200292