Soluble Collectin-12 (CL-12) Is a Pattern Recognition Molecule Initiating Complement Activation via the Alternative Pathway

Abstract

Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to ∼120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca2+-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.

A journey through the lectin pathway of complement—MBL and beyond

“…The cleavage site was suggested to be located at its N‐terminus in close vicinity of the plasma membrane. Soluble CL‐12 was found in human umbilical cord plasma with a mean plasma concentration of around 0.1 μg/ml (range 0.05–0.12 μg/ml) . Moreover, we observed that soluble CL‐12 mediated complement activation.…”

“…It has been suggested that the similarities between CL‐12 and the above‐described collectins, MBL, CL‐10, and CL‐11 appear to result from random genetic rearrangements of the different structural domains more than a common evolutionary origin . We hypothesized that membrane‐anchored CL‐12 might be cleaved into a soluble version of CL‐12 . The cleavage site was suggested to be located at its N‐terminus in close vicinity of the plasma membrane.…”

“…However, recent findings suggest that properdin by itself may provide a platform for the assembly of C3bBb complex on target surfaces, providing new insights into properdin function . We hypothesized that a soluble analog of the transmembrane receptor CL‐12 might activate the lectin pathway via interaction with MASPs as it activated the complement system on A. fumigatus . However, this turned out to be incorrect as CL‐12 did not interact with the MASPs but rather recruited and interacted with properdin, thereby allowing alternative pathway activation .…”

“…The MASPs possess somewhat promiscuous substrate‐binding specificities, thereby involving the lectin pathway in a multitude of biological processes that reach beyond the conventional text book view on the complement system. Recently, another collectin named collectin‐12 (CL‐12 or CL‐P1) has, in its soluble form, been shown to mediate complement activation . This appears not to involve interaction with the MASPs, but rather with molecules from the alternative pathway.…”

“…Recent studies link the initiation of the alternative pathway to the lectin pathway as factor D, the key activation enzyme of the alternative pathway, can be cleaved by MASP‐3 . The alternative pathway also serves as an amplification loop for the classical and lectin pathways and the presence of ligand bound pentraxins seems to mediate interaction between all three pathways . Overall, the notion of three separate pathways is fairly oversimplified .…”