Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold.

The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain-ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.

An inhibitor of complement C5 provides structural insights into activation.

The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5-CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

Complement in Human Pre-implantation Embryos: Attack and Defense.

It is essential for early human life that mucosal immunological responses to developing embryos are tightly regulated. An imbalance of the complement system is a common feature of pregnancy complications. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus, far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation against developing human embryos takes place already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products on healthy developing embryos, which highlights the need for strict complement regulation. We show that embryos express complement membrane inhibitors and bind soluble regulators. These findings show that mucosal complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing revealed cellular expression of complement activators. This shows that the embryonic cells themselves have the capacity to express and activate C3 and C5. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and furthermore may impact cellular homeostasis and differentiation at the earliest stages of life.

The salivary scavenger and agglutinin binds MBL and regulates the lectin pathway of complement in solution and on surfaces.

The salivary scavenger and agglutinin (SALSA), also known as gp340, salivary agglutinin and deleted in malignant brain tumor 1, is a 340-kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A, and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan-binding lectin (MBL) as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit Candida albicans-induced complement activation. Thus, SALSA has a dual complement activation modifying function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid phase. These activities are mediated via a direct interaction with MBL. This suggests that SALSA could target the innate immune responses to certain microorganisms and simultaneously limit complement activation in the fluid phase.

CD163-L1 is an endocytic macrophage protein strongly regulated by mediators in the inflammatory response.

CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.