Spatial position is a key determinant of N-glycan functionality of the scavenger receptor cysteine-rich domain of human hepsin.

The scavenger receptor cysteine-rich (SRCR) domain is a key constituent in diverse proteins. N-glycosylation is important in protein expression and function. In the SRCR domain of different proteins, N-glycosylation sites and functionality vary substantially. In this study, we examined the importance of N-glycosylation site positions in the SRCR domain of hepsin, a type II transmembrane serine protease involved in many pathophysiological processes. We analysed hepsin mutants with alternative N-glycosylation sites in the SRCR and protease domains using three-dimensional modelling, site-directed mutagenesis, HepG2 cell expression, immunostaining, and western blotting. We found that the N-glycan function in the SRCR domain in promoting hepsin expression and activation on the cell surface cannot be replaced by alternatively created N-glycans in the protease domain. Within the SRCR domain, the presence of an N-glycan in a confined surface area was essential for calnexin-assisted protein folding, endoplasmic reticulum (ER) exiting, and zymogen activation of hepsin on the cell surface. Hepsin mutants with alternative N-glycosylation sites on the opposite side of the SRCR domain were trapped by ER chaperones, resulting in the activation of the unfolded protein response in HepG2 cells. These results indicate that the spatial N-glycan positioning in the SRCR domain is a key determinant in the interaction with calnexin and subsequent cell surface expression of hepsin. These findings may help to understand the conservation and functionality of N-glycosylation sites in the SRCR domains of different proteins.

Scavenging of bacteria or bacterial products by magnetic particles functionalized with a broad-spectrum pathogen recognition receptor motif offers diagnostic and therapeutic applications.

Sepsis is a dysregulated host response of severe bloodstream infections, and given its frequency of occurrence and high mortality rate, therapeutic improvements are imperative. A reliable biomimetic strategy for the targeting and separation of bacterial pathogens in bloodstream infections involves the use of the broad-spectrum binding motif of human GP-340, a pattern-recognition receptor of the scavenger receptor cysteine rich (SRCR) superfamily that is expressed on epithelial surfaces but not found in blood. Here we show that these peptides, when conjugated to superparamagnetic iron oxide nanoparticles (SPIONs), can separate various bacterial endotoxins and intact microbes (E. coli, S. aureus, P. aeruginosa and S. marcescens) with high efficiency, especially at low and thus clinically relevant concentrations. This is accompanied by a subsequent strong depletion in cytokine release (TNF, IL-6, IL-1ß, Il-10 and IFN-γ), which could have a direct therapeutic impact since escalating immune responses complicates severe bloodstream infections and sepsis courses. SPIONs are coated with aminoalkylsilane and capture peptides are orthogonally ligated to this surface. The particles behave fully cyto- and hemocompatible and do not interfere with host structures. Thus, this approach additionally aims to dramatically reduce diagnostic times for patients with suspected bloodstream infections and accelerate targeted antibiotic therapy. STATEMENT OF SIGNIFICANCE: Sepsis is often associated with excessive release of cytokines. This aspect and slow diagnostic procedures are the major therapeutic obstacles. The use of magnetic particles conjugated with small peptides derived from the binding motif of a broad-spectrum mucosal pathogen recognition protein GP-340 provides a highly efficient scavenging platform. These peptides are not found in blood and therefore are not subject to inhibitory mechanisms like in other concepts (mannose binding lectine, aptamers, antibodies). In this work, data are shown on the broad bacterial binding spectrum, highly efficient toxin depletion, which directly reduces the release of cytokines. Host cells are not affected and antibiotics not adsorbed. The particle bound microbes can be recultured without restriction and thus be used directly for diagnostics.

Assessment of Scavenger Receptor Cysteine-Rich Domain Binding to Bacteria.

The scavenger receptor cysteine-rich SRCR domain is an ancient protein domain found in SR-A and SR-I scavenger receptors, which is characterized by a conserved arrangement of cysteines (Martinez et al., Pharmacol Rev 63(4):967-1000, 2011; Sarrias et al., Crit Rev Immunol 24(1):1-37, 2004; Telfer and Baldwin, Cell Immunol 296(1):76-86, 2015; PrabhuDas et al., J Immunol, 2017. 198(10):3775-3789). SRCR domains are divided into group A and group B SRCR domains by virtue of how many cysteines they contain and the resulting disulfide bonding pattern. Group B SRCR domains, found in WC1, CD163, CD5, CD6, Spα and DMBT1, are approximately 100-110 amino acids long and contain 6-8 cysteines predicted to form 3-4 disulfide bonds. The crystal structure of a CD5 group B SRCR domain predicts a fold of two beta-sheets and an alpha helix (Rodamilans et al., J Biol Chem 282(17):12669-12677, 2007; Wang et al., Mol Immunol 48:801-809, 2011). SRCR domains bind to many different types of chemical compounds found on cells, viruses, and microbes and are usually found in multiples in the extracellular domains of transmembrane proteins or in secreted proteins. Small amino acid differences between these SRCR domains lead to significant differences in binding affinity. In addition, SRCR domain genes contain allelic polymorphisms and can be extensively duplicated. Thus, single and duplicated SRCR domain protein gene loci encode a large tunable binding potential. Binding to pathogen-associated molecular patterns (PAMPs) combined with signaling potential predicts an important role for these molecules in the immune response. WC1 SRCR domains bind to the spirochetes Leptospira and Borrelia (Hsu et al., J Immunol 194(5):2280-2288, 2015). CD6 (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007), Spα (Sarrias et al., J Biol Chem 280(42):35391-35398, 2005), CD163A (Fabriek et al., Blood 113(4):887-892, 2009) and DMBT1 (Madsen et al., Eur J Immunol 33(8):2327-2336, 2003) bind to Gram-positive and Gram-negative bacteria; CD5 binds to yeast (Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009). Identified ligands include lipoteichoic acid, lipopolysaccharide, poly-phosphorylated, and -sulfated compounds such as dextran sulfate sodium, leucine-rich repeat proteins, and fungal mannose (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007; Sarrias et al., J Biol Chem 280(42):35391-35398, 2005; Fabriek et al., Blood 113(4):887-892, 2009; Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009; End et al., Eur J Immunol 39(3):833-842, 2009; Loimaranta et al., J Biol Chem 284(28):18614-18623, 2009). A conserved linear binding motif (VEVLXXXXW) in an external loop in the SRCR domain has been identified in CD163A and DMBT1 and can be used as a peptide that aggregates bacteria (Fabriek et al., Blood 113(4):887-892, 2009; Bikker et al., J Biol Chem 279(46):47699-47703, 2004; Leito et al., Biol Chem 389(9):1193-1200, 2008). In contrast, WC1 binding to bacteria is mediated by a noncontinuous motif in the native protein, and mutation of the VEVLXXXXW motif has no effect upon bacterial binding (Hsu et al., J Immunol 194(5):2280-2288, 2015). Thus, bacterial binding studies with WC1 SRCR domains must be done with native, correctly disulfide bonded, protein, ideally posttranslationally modified in mammalian cells.WC1 is found in the genomes of most mammals, reptiles, and birds and is expressed exclusively on γδ T cells in ruminants. The 13 bovine WC1 genes encode up to 11 extracellular SRCR domains, organized in the SRCR domain pattern of a1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d'11], where the alphabet designations indicate homology between genes and across species (Chen et al., BMC Genet 13:86, 2012; Herzig et al., BMC Evol Biol 10:181, 2010; Herzig and Baldwin, BMC Genomics 10:191, 2009). Some of the signaling co-receptor WC1 molecules are required for the γδ T cell response to Leptospira (Wang et al., Mol Immunol 48:801-809, 2011; Rogers et al., J Immunol 174(6):3386-3393, 2005; Wang et al., Eur J Immunol 39(1):254-266, 2009). The WC1 expressed on responsive γδ T cells is correlated with its direct binding to Leptospira via some of its SRCR domains (Hsu et al., J Immunol 194(5):2280-2288, 2015). Because WC1+ γδ T cells share a restriction in their γδ TCRs and WC1 has TCR co-receptor activity, we hypothesize that WC1 co-ligation with the TCR plays the determining role in the activation of WC1+ γδ T cells by pathogens. Classification of the binding of WC1 SRCR domains, their ligands, and their role in the interaction of 𝛾δ T cells with pathogens relevant to the host will allow these cells to be recruited in next-generation vaccines to pathogens that have significant negative economic and health impact.

TLR2 Potentiates SR-Marco-Mediated Neuroinflammation by Interacting with the SRCR Domain.

Microglial activation-induced neuroinflammation is critical in the pathogenesis of neurodegenerative diseases. Activated microglia are regulated mainly by innate pattern recognition receptors (PRRs) on their surface, of which macrophage receptor with collagenous structure (Marco) is a well-characterized scavenger receptor constitutively expressed on specific subsets of macrophages, including microglia. Increasing evidence has shown that Marco is involved in the pathogenesis of a range of inflammatory processes. However, research on the role of Marco in regulating neuroinflammation has reported conflicting results. In the present study, we examined the role Marco played in triggering neuroinflammation and its underlying mechanisms. The results demonstrated that silencing the Marco gene resulted in a significantly reduced neuroinflammatory response and vice versa. α-Syn stimulation in Marco overexpressing cells induced a pronounced inflammatory response, suggesting that Marco alone could trigger an inflammatory response. We also found that TLR2 significantly promoted Marco-mediated neuroinflammation, indicating TLR2 was an important co-receptor of Marco. Knocking down the TLR2 gene in microglia and mouse substantia nigra resulted in decreased expression of Marco. Subsequent mechanistic studies showed that deleting the SRCR domain of Marco resulted in disruption of the inflammatory response and the interaction between TLR2 and Marco. This suggested that TLR2 binds directly to the SRCR domain of Marco and regulates Marco-mediated neuroinflammation. In summary, this investigation revealed that TLR2 could potentiate Marco-mediated neuroinflammation by interacting with the SRCR domain of Marco, providing a new target for inhibiting neuroinflammation in neurodegenerative diseases.

Recognition of lipoproteins by scavenger receptor class A members.

Scavenger receptor class A (SR-A) proteins are type II transmembrane glycoproteins that form homotrimers on the cell surface. This family has five known members (SCARA1 to 5, or SR-A1 to A5) that recognize a variety of ligands and are involved in multiple biological pathways. Previous reports have shown that some SR-A family members can bind modified low-density lipoproteins (LDLs); however, the mechanisms of the interactions between the SR-A members and these lipoproteins are not fully understood. Here, we systematically characterize the recognition of SR-A receptors with lipoproteins and report that SCARA1 (SR-A1, CD204), MARCO (SCARA2), and SCARA5 recognize acetylated or oxidized LDL and very-low-density lipoprotein in a Ca2+-dependent manner through their C-terminal scavenger receptor cysteine-rich (SRCR) domains. These interactions occur specifically between the SRCR domains and the modified apolipoprotein B component of the lipoproteins, suggesting that they might share a similar mechanism for lipoprotein recognition. Meanwhile, SCARA4, a SR-A member with a carbohydrate recognition domain instead of the SRCR domain at the C terminus, shows low affinity for modified LDL and very-low-density lipoprotein but binds in a Ca2+-independent manner. SCARA3, which does not have a globular domain at the C terminus, was found to have no detectable binding with these lipoproteins. Taken together, these results provide mechanistic insights into the interactions between SR-A family members and lipoproteins that may help us understand the roles of SR-A receptors in lipid transport and related diseases such as atherosclerosis.

Interactions of ferritin with scavenger receptor class A members.

Scavenger receptors are a superfamily of membrane-bound receptors that recognize both self and nonself targets. Scavenger receptor class A (SR-A) has five known members (SCARA1 to -5 or SR-A1 to -A5), which are type II transmembrane proteins that form homotrimers on the cell surface. SR-A members recognize various ligands and are involved in multiple biological pathways. Among them, SCARA5 can function as a ferritin receptor; however, the interaction between SCARA5 and ferritin has not been fully characterized. Here, we determine the crystal structures of the C-terminal scavenger receptor cysteine-rich (SRCR) domain of both human and mouse SCARA5 at 1.7 and 2.5 Å resolution, respectively, revealing three Ca2+-binding sites on the surface. Using biochemical assays, we show that the SRCR domain of SCARA5 recognizes ferritin in a Ca2+-dependent manner, and both L- and H-ferritin can be recognized by SCARA5 through the SRCR domain. Furthermore, the potential binding region of SCARA5 on the surface of ferritin is explored by mutagenesis studies. We also examine the interactions of ferritin with other SR-A members and find that SCARA1 (SR-A1, CD204) and MARCO (SR-A2, SCARA2), which are highly expressed on macrophages, also interact with ferritin. By contrast, SCARA3 and SCARA4, the two SR-A members without the SRCR domain, have no detectable binding with ferritin. Overall, these results provide a mechanistic view regarding the interactions between the SR-A members and ferritin that may help to understand the regulation of ferritin homeostasis by scavenger receptors.

The scavenging capacity of DMBT1 is impaired by germline deletions.

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

AIM/CD5L: a key protein in the control of immune homeostasis and inflammatory disease.

CD5L, a soluble protein belonging to the SRCR superfamily, is expressed mostly by macrophages in lymphoid and inflamed tissues. The expression of this protein is transcriptionally controlled by LXRs, members of the nuclear receptor family that play major roles in lipid homeostasis. Research undertaken over the last decade has uncovered critical roles of CD5L as a PRR of bacterial and fungal components and in the control of key mechanisms in inflammatory responses, with involvement in processes, such as infection, atherosclerosis, and cancer. In this review, we summarize the current knowledge of CD5L, its roles at the intersection between lipid homeostasis and immune response, and its potential use as a diagnostic biomarker in a variety of diseases, such as TB and liver cirrhosis.