Origin of the classical complement pathway: Lamprey orthologue of mammalian C1q acts as a lectin

Abstract

The lectin complement pathway in innate immunity is closely related to the classical complement pathway in adaptive immunity, with respect to the structures and functions of their components. Both pathways are initiated by complexes consisting of collagenous proteins and serine proteases of the mannose-binding lectin (MBL)-associated serine protease (MASP)/C1r/C1s family. It has been speculated that the classical pathway emerged after the lectin pathway, and that the activation mechanism of the latter was partially conserved. The classical and lectin pathways can be traced back to at least cartilaginous fish and ascidian (urochordata), respectively. To elucidate the evolution of the complement system, we isolated and characterized a GlcNAc-binding lectin from sera of lamprey (agnathans), the most primitive vertebrate that lacks the classical pathway. Lamprey GlcNAc-binding lectin was an oligomer consisting of 24-kDa subunits. cDNA and phylogenetic analyses revealed that the lamprey GlcNAc-binding lectin is an orthologue of mammalian C1q, a collagenous subcomponent of the first component involved in binding to immunoglobulins in the classical pathway. Lamprey C1q copurified with MASP-A, a serine protease of the MASP/C1r/C1s family, which exhibited proteolytic activity against lamprey C3. Surface plasmon resonance analysis showed that lamprey C1q specifically bound to GlcNAc, but not various other carbohydrates tested. These results suggest that C1q may have emerged as a lectin and may have functioned as an initial recognition molecule of the complement system in innate immunity before the establishment of adaptive immunity such as immunoglobulins in the cartilaginous fish.

Fig. 1.

Gel filtration and SDS/PAGE analysis of lamprey GlcNAc-binding lectin. Lamprey serum was subjected to Mono Q column chromatography, followed by gel filtration on Superose 6 in the presence of Ca²⁺, as described in Materials and Methods. (A) SDS/PAGE profile under reducing conditions. Arrows indicate fractions in which standard substances were eluted. (B) Protease activity of each fraction.

Fig. 2.

Characterization of lamprey GlcNAc-binding lectin and its associated protease. (A) SDS/PAGE of purified GlcNAc-binding lectin (lane 1) and its associated protease (lane 2, arrows). Samples were run under reducing (Left) or nonreducing (Right) conditions and stained for proteins with Coomassie brilliant blue R-250. (B) Incorporation of tritium-labeled diisopropylfluorophosphate into GlcNAc-binding lectin-associated protease. The purified protease was treated with tritium-labeled diisopropylfluorophosphate and subjected to SDS/PAGE under reducing (lane 1) or nonreducing (lane 2) conditions, followed by autoradiography. (C) Collagenase digestion of GlcNAc-binding lectin. Lamprey GlcNAc-binding lectin was incubated in the absence (lane 1) or presence (lane 2) of collagenase and subjected to SDS/PAGE, followed by protein staining.

Fig. 3.

Alignment of amino acid sequences of LC1q and mammalian C1q. Sequences were aligned by using clustalw software. Residues that were identical in all proteins are indicated by asterisks. LC1q contains a collagen-like domain (residues 29–110) and a gC1q domain (residues 111–240).

Fig. 4.

Phylogenetic tree of gC1q domains of proteins that have both collagen-like and gC1q domains. The tree was constructed by the neighbor-joining method, based on the alignment of amino acid sequences from 50 proteins by using clustalw software. Numbers on branches are bootstrap percentages supporting a given partitioning. C1q, adiponectin, type VIII collagen, type X collagen, and chipmunk hibernation-associated plasma protein are grouped as indicated, but other proteins are not defined.

Fig. 5.

Cleavage of lamprey C3 by MASP-A. Lamprey C3 (lane 1) was incubated with LC1q–MASP-A complex (lane 2) or MASP-A (lane 3) and subjected to SDS/PAGE under reducing conditions, followed by protein staining.