Recognition protein C1q of innate immunity agglutinates nanodiamonds without activating complement.

Nanodiamonds are promising nanomedicines for diagnostic and therapeutic applications. As nanodiamonds are mainly administered intravenously, it is critical to understand the humoral immune response upon exposure to nanodiamonds. Here, we report the interactions of pristine, oxidized, and PEG-functionalized nanodiamonds with human complement, an important part of our humoral innate immunity. In particular, we report the nanodiamond binding properties of the recognition protein of the classical complement pathway: C1q, which also takes part in many other physiological and pathological processes. Our results show similar trends in the effects of C1q on the three types of nanodiamonds. Complement activation assays using human serum show that the nanodiamonds trigger slight activities via the alternative pathway and no response via the classical pathway. Nevertheless, surface plasmon resonance shows that C1q binds the nanodiamonds and transmission electron microscopy reveals their agglutination. Studies with macrophages further show that C1q attachment affects their phagocytosis and cytokine response.

Proteins of the innate immune system crystallize on carbon nanotubes but are not activated.

The classical pathway of complement is an essential component of the human innate immune system involved in the defense against pathogens as well as in the clearance of altered self-components. Activation of this pathway is triggered by C1, a multimolecular complex comprising a recognition protein C1q associated with a catalytic subunit C1s-C1r-C1r-C1s. We report here the direct observation of organized binding of C1 components C1q and C1s-C1r-C1r-C1s on carbon nanotubes, an ubiquitous component in nanotechnology research. Electron microscopy imaging showed individual multiwalled carbon nanotubes with protein molecules organized along the length of the sidewalls, often over 1 µm long. Less well-organized protein attachment was also observed on double-walled carbon nanotubes. Protein-solubilized nanotubes continued to attract protein molecules after their surface was fully covered. Despite the C1q binding properties, none of the nanotubes activated the C1 complex. We discuss these results on the adsorption mechanisms of macromolecules on carbon nanotubes and the possibility of using carbon nanotubes for structural studies of macromolecules. Importantly, the observations suggest that carbon nanotubes may interfere with the human immune system when entering the bloodstream. Our results raise caution in the applications of carbon nanotubes in biomedicine but may also open possibilities of novel applications concerning the many biochemical processes involving the versatile C1 macromolecule.

CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site.

CD91 plays an important role in the scavenging of apoptotic material, possibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found to bind CD91. The MBL-CD91 interaction was time- and concentration-dependent and could be inhibited by known ligands of CD91. MBL-associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP-MBL interaction site. This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic material by CD91.

Direct interaction between CD91 and C1q.

C1q-mediated removal of immune complexes and apoptotic cells plays an important role in tissue homeostasis and the prevention of autoimmune conditions. It has been suggested that C1q mediates phagocytosis of apoptotic cells through a receptor complex assembled from CD91 (alpha-2- macroglobulin receptor, or low-density lipoprotein receptor-related protein) and calreticulin, with CD91 being the transmembrane part and calreticulin acting as the C1q-binding molecule. In the present study, we observe that C1q binds cells from a CD91 expressing monocytic cell line as well as monocytes from human blood. C1q binding to monocytes was shown to be correlated with CD91 expression and could be inhibited by the CD91 chaperone, receptor-associated protein. We also report data showing a direct interaction between CD91 and C1q. The interaction was investigated using various protein interaction assays. A direct interaction between purified C1q and CD91 was observed both by ELISA and a surface plasmon resonance assay, with either C1q or CD91 immobilized. The interaction showed characteristics of specificity because it was time-dependent, saturable and could be inhibited by known ligands of both CD91 and C1q. The results obtained show for the first time that CD91 recognizes C1q directly. On the basis of these findings, we propose that CD91 is a receptor for C1q and that this multifunctional scavenger receptor uses a subset of its ligand-binding sites for clearance of C1q and C1q bound material.

Trimeric reassembly of the globular domain of human C1q.

C1q is a versatile recognition protein which binds to a variety of targets and consequently triggers the classical pathway of complement. C1q is a hetero-trimer composed of three chains (A, B and C) arranged in three domains, a short N-terminal region, followed by a collagenous repeat domain that gives rise to the formation of (ABC) triple helices, each ending in a C-terminal hetero-trimeric globular domain, called gC1q, which is responsible for the recognition properties of C1q. The mechanism of the trimeric assembly of C1q and in particular the role of each domain in the process is unknown. Here, we have investigated if the gC1q domain was able to assemble into functional trimers, in vitro, in the absence of the collagenous domain, a motif known to promote obligatory trimers in other proteins. Acid-mediated gC1q protomers reassembled into functional trimers, once neutralized, indicating that it is the gC1q domain which possesses the information for trimerization. However, reassembly occurred after neutralization, only if the gC1q protomers had preserved a residual tertiary structure at the end of the acidic treatment. Thus, the collagenous domain of C1q might initialize the folding of the gC1q domain so that subsequent assembly of the entire molecule can occur.