Spatially conserved motifs in complement control protein domains determine functionality in regulators of complement activation-family proteins

Abstract

Regulation of complement activation in the host cells is mediated primarily by the regulators of complement activation (RCA) family proteins that are formed by tandemly repeating complement control protein (CCP) domains. Functional annotation of these proteins, however, is challenging as contiguous CCP domains are found in proteins with varied functions. Here, by employing an in silico approach, we identify five motifs which are conserved spatially in a specific order in the regulatory CCP domains of known RCA proteins. We report that the presence of these motifs in a specific pattern is sufficient to annotate regulatory domains in RCA proteins. We show that incorporation of the lost motif in the fourth long-homologous repeat (LHR-D) in complement receptor 1 regains its regulatory activity. Additionally, the motif pattern also helped annotate human polydom as a complement regulator. Thus, we propose that the motifs identified here are the determinants of functionality in RCA proteins.

The Sez6 family inhibits complement at the level of the C3 convertase

“…Ojha et al recently used AI-assisted computer learning along with significant functional annotation based off of decades of experimental research on RCA proteins to propose five short motifs that potentially confer complement inhibitory activity when conserved in a specific order across three tandem CCP domains[30]. Using their web-based CoreDo program, we found Sez6L2 has the correct motif pattern for predicted complement regulatory activity (SupplementalFigure 3;[30]). Thus, our results on Sez6L2 match their prediction.…”

“…However, the identity of complement regulators functioning on neurons throughout neuronal development or in disease with a potential role in modulating complement-mediated synaptic pruning has yet to be fully elucidated. A common feature of complement regulators, particularly those that work at the level of C3 convertases, is that they contain three or more tandem CCP domains [30,32].…”

“…CCP domains, also known as short consensus repeats or SUSHI repeats, contain ~ 60-70 amino acids with four invariant cysteines forming disulfide bonds. CCP domains are the primary components of several proteins of the complement system [30].…”

“…Complement activation on self cells is largely controlled by the regulators of complement activation (RCA) protein family that protect "self" surfaces by limiting the activity of the C3 convertases (C3bBb or C4b2a). RCAs bind C3b or C4b, then inactivate the C3 convertases by either removing the active protease or by recruiting the cleaving enzyme, complement Factor I, to destroy C3b or C4b [30][31][32]. Cleaved C3b fragments (iC3b, C3c, C3dg/d) can no longer participate in the complement cascade leading to cell lysis, but they still have opsonizing and immune signaling properties.…”

“…Although the amino acid sequences of CCP domains vary considerably, RCA proteins bind to C3b using a common binding platform on C3b with their 3-4 consecutive CCP domains in an extended orientation[32]. Ojha et al recently used AI-assisted computer learning along with significant functional annotation based off of decades of experimental research on RCA proteins to propose five short motifs that potentially confer complement inhibitory activity when conserved in a specific order across three tandem CCP domains[30]. Using their web-based CoreDo program, we found Sez6L2 has the correct motif pattern for predicted complement regulatory activity (SupplementalFigure 3;[30]).…”