Scavenger Receptor Cysteine-rich Research Articles

DON/DRP-104 as potent serine protease inhibitors implicated in SARS-CoV-2 infection: Comparative binding modes with human TMPRSS2 and novel therapeutic approach.

Human transmembrane serine protease 2 (TMPRSS2) is an important member of the type 2 transmembrane serine protease (TTSP) family with significant therapeutic markings. The search for potent TMPRSS2 inhibitors against severe acute respiratory syndrome coronavirus 2 infection with favorable tissue specificity and off-site toxicity profiles remains limited. Therefore, probing the anti-TMPRSS2 potential of enhanced drug delivery systems, such as nanotechnology and prodrug systems, has become compelling. We report the first in silico study of TMPRSS2 against a prodrug, [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] also known as DRP-104 synthesized from 6-Diazo-5-oxo-l-norleucine (DON). We performed comparative studies on DON and DRP-104 against a clinically potent TMPRSS2 inhibitor, nafamostat, and a standard serine protease inhibitor, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) against TMPRSS2 and found improved TMPRSS2 inhibition through synergistic binding of the S1/S1' subdomains. Both DON and DRP-104 had better thermodynamic profiles than AEBSF and nafamostat. DON was found to confer structural stability with strong positive correlated inter-residue motions, whereas DRP-104 was found to confer kinetic stability with restricted residue displacements and reduced loop flexibility. Interestingly, the Scavenger Receptor Cysteine-Rich (SRCR) domain of TMPRSS2 may be involved in its inhibition mechanics. Two previously unidentified loops, designated X (270-275) and Y (293-296) underwent minimal and major structural transitions, respectively. In addition, residues 273-277 consistently transitioned to a turn conformation in all ligated systems, whereas unique transitions were identified for other transitioning residue groups in each TMPRSS2-inhibitor complex. Intriguingly, while both DON and DRP-104 showed similar loop transition patterns, DRP-104 preserved loop structural integrity. As evident from our systematic comparative study using experimentally/clinically validated inhibitors, DRP-104 may serve as a potent and novel TMPRSS2 inhibitor and warrants further clinical investigation.

Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection

CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16 amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main physiological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection.

The Transmembrane Protease Serine 2 (TMPRSS2) Non-Protease Domains Regulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike-Mediated Virus Entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2's cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

Surface-expressed phosphoglycerate mutase of Candida albicans binds to salivary DMBT1.

Candida albicans colonizes oral tissues and causes infectious diseases. Colonization of C. albicans on the oral mucosa and tooth enamel surfaces is established via the interaction between C. albicans adhesins and salivary proteins, forming a film on the oral tissues. Deleted in malignant brain tumors 1 (DMBT1), also known as salivary agglutinin or gp-340, belongs to the scavenger receptor cysteine-rich (SRCR) superfamily. In the oral cavity, immobilized DMBT1 on oral tissues causes microbial adherence. Recently, we demonstrated that C. albicans binds to DMBT1 and isolated a 25-kDa C. albicans adhesin involved in the interaction with the binding domain of DMBT1, namely, SRCRP2. In the present study, we searched for additional DMBT1-binding adhesins in C. albicans. The component isolated here had a molecular mass of 29kDa and was found to be phosphoglycerate mutase (Gpm1). Isolated Gpm1 inhibited C. albicans binding to SRCRP2 and directly bound to SRCRP2 in a dose-dependent manner. Gpm1 localization on the C. albicans cell wall surface was confirmed by immunostaining. These results suggest that surface-expressed Gpm1 functions as an adhesin for the establishment of C. albicans cells on the oral mucosa and tooth enamel by binding to DMBT1.

Species-specific MARCO-alphavirus interactions dictate chikungunya virus viremia

Arboviruses are public health threats that cause explosive outbreaks. Major determinants of arbovirus transmission, geographic spread, and pathogenesis are the magnitude and duration of viremia in vertebrate hosts. Previously, we determined that multiple alphaviruses are cleared efficiently from murine circulation by the scavenger receptor MARCO (Macrophage receptor with collagenous structure). Here, we define biochemical features on chikungunya (CHIKV), o'nyong 'nyong (ONNV), and Ross River (RRV) viruses required for MARCO-dependent clearance invivo. Invitro, MARCO expression promotes binding and internalization of CHIKV, ONNV, and RRV via the scavenger receptor cysteine-rich (SRCR) domain. Furthermore, we observe species-specific effects of the MARCO SRCR domain on CHIKV internalization, where those from known amplification hosts fail to promote CHIKV internalization. Consistent with this observation, CHIKV is inefficiently cleared from the circulation of rhesus macaques in contrast with mice. These findings suggest a role for MARCO in determining whether a vertebrate serves as an amplification or dead-end host following CHIKV infection.

Effect of maleylation and denaturation of human serum albumin on its interaction with scavenger receptors.

The specific recognition of serum proteins by scavenger receptors is critical and fundamental in many biological processes. However, the underlying mechanism of scavenger receptor-serum protein interaction remains elusive. In this work, taking scavenger receptors class A1 (SR-A1) as an example, we systematically investigate its interaction with human serum albumin (HSA) at different states through a combination of molecular docking and all-atom molecular dynamics simulations. It is found that native HSA can moderately bind to collagen-like (CL) region or scavenger receptor cysteine-rich (SRCR) region, with both electrostatic (ELE) and van der Waals (VDW) interactions, playing important roles. After maleylation, the binding energy, particularly the ELE energy, between HSA and CL region is significantly enhanced, while the binding energy between HSA and SRCR region remains nearly unchanged. Additionally, we also observe that unfolding of the secondary structures in HSA leads to a larger contact surface area between denatured HSA and CL region, but has little impact on the HSA-SRCR region interaction. Therefore, similar to maleylated HSA, denatured HSA is also more likely to bind to the CL region of SR-A1.

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.

Polymorphism of tmprss2 (rs12329760) but not ace2 (rs4240157), tmprss11a (rs353163) and cd147 (rs8259) is associated with the severity of COVID-19 in the Ukrainian population.

Angiotensin-converting enzyme 2 (ACE2), transmembrane serine 2 and serine 11A proteases (TMPRSS2, TMPRSS11A), and a cell surface cluster of differentiation 147 (CD147) might be a gene candidate that exerts the susceptibility to and mortality from coronavirus disease 19 (COVID-19). The aim of this study was to investigate the associations between ace2, tmprss2, tmprss11a, and cd147 polymorphic variants and the severity of COVID-19 in the Ukrainian population. The study population consisted of the Ukrainian population with COVID-19: patients without oxygen therapy (n=62), with non-invasive (n=92) and invasive (n=35) oxygen therapy, as well as control subjects (n=92). Allelic polymorphisms of ace2 rs4240157, tmprss2 rs12329760, and tmprss11a rs353163 were determined by real-time PCR, and cd147 rs8259 polymorphism was detected by PCR with subsequent restrictase analysis. We compared investigated polymorphisms distribution with other populations by meta-analysis. Our study is the first to obtain data about the distribution of investigated gene polymorphisms in the Ukrainian population: tmprss2 rs12329760 - CC 60.9%, CT 35.9%, TT 3.2%; tmprss11a rs353163 - CC 46.7%, CT 40.2%, TT 13.1%; ace2 rs4240157 - CC 7.6%, C 18.5%, CT 22.8%, TT 19.6%, T 31.5%; cd147 rs8259 - TT 60.9%, AT 32.6%, AA 6.5%. This distribution was similar to the Northern, Western and Southern European populations. There was a statistically significant difference in the frequency of tmprss2 polymorphic genotypes CC 57.1%, CT 28.6%, and TT 14.3% (P<0.05) in COVID-19 patients with invasive oxygen therapy in comparison with non-invasive oxygen therapy. This tmprss2 mutation occurs in the scavenger receptor cysteine-rich (SRCR) domain and might be important for protein-protein interaction in a calcium-dependent manner. Our study indicated the presence of an association between the tmprss2 rs12329760 polymorphism and the severity of COVID-19 in the Ukrainian population.

Loss of LOXL2 Promotes Uterine Hypertrophy and Tumor Progression by Enhancing H3K36ac-Dependent Gene Expression.

LOXL2 loss reprograms the epigenetic landscape to promote uterine cancer initiation and progression and repress the efficacy of anti-PD-1 immunotherapy, indicating that LOXL2 is a tumor suppressor.

Lysyl Oxidase-like Protein Recognizes Viral Envelope Proteins and Bacterial Polysaccharides against Pathogen Infection via Induction of Expression of Antimicrobial Peptides

Lysyl oxidases (LOXs) are copper-dependent monoamine oxidases, and they play critical roles in extracellular matrix (ECM) remodeling. The LOX and LOX-like (LOXL) proteins also have a variety of biological functions, such as development and growth regulation, tumor suppression, and cellular senescence. However, the functions of LOXLs containing repeated scavenger receptor cysteine-rich (SRCR) domains in immunity are rarely reported. In this study, we characterized the antiviral and antibacterial functions of a lysyl oxidase-like (LOXL) protein containing tandem SRCR domains in Marsupenaeus japonicus. The mRNA level of LoxL was significantly upregulated in the hemocytes and intestines of shrimp challenged using white spot syndrome virus (WSSV) or bacteria. After the knockdown of LoxL via RNA interference, WSSV replication and bacterial loads were apparently increased, and the survival rate of the shrimp decreased significantly, suggesting that LOXL functions against pathogen infection in shrimp. Mechanistically, LOXL interacted with the envelope proteins of WSSV or with lipopolysaccharide and peptidoglycan from bacteria in shrimp challenged using WSSV or bacteria, and it promoted the expression of a battery of antimicrobial peptides (AMPs) via the induction of Dorsal nuclear translocation against viral and bacterial infection. Moreover, LOXL expression was also positively regulated by Dorsal in the shrimp challenged by pathogens. These results indicate that, by acting as a pattern recognition receptor, LOXL plays vital roles in antiviral and antibacterial innate immunity by enhancing the expression of AMPs in shrimp.

Activating Fc Gamma Receptors and Viral Receptors Are Required for Antibody-Dependent Enhancement of Porcine Reproductive and Respiratory Syndrome Virus Infection.

Simple SummaryPorcine reproductive and respiratory syndrome virus (PRRSV)-specific sub- or non-neutralizing antibodies promote the adhesion and internalization of the virion into host cells. This phenomenon is known as antibody-dependent enhancement (ADE) of PRRSV infection. It has long been accepted that Fc gamma receptors (FcγRs) are responsible for mediating ADE of virus infection. However, few researchers pay attention to the role of the virus receptors in the ADE of virus infection. In this study, we showed that activating FcγRs (FcγRI and FcγRIII) were responsible for mediating PRRSV-ADE infection. Simultaneously, we showed that the viral receptors (sialoadhesin and CD163) were involved in FcγR-mediated PRRSV-ADE infection. The extracellular domains 1-6 of sialoadhesin and the scavenger receptor cysteine-rich 5 domain of CD163 might play central roles in PRRSV-ADE infection. In conclusion, our studies indicated that activating FcγRs and virus receptors were required for PRRSV-ADE infection. Our findings should allow a more precise understanding of the structural basis for the mechanism of PRRSV-ADE infection, which would provide references for screening targets of novel PRRS vaccines or antiviral drugs against the PRRSV.Antibody-dependent enhancement (ADE) is an event in preexisting sub-, or non-neutralizing antibodies increasing the viral replication in its target cells. ADE is one crucial factor that intensifies porcine reproductive and respiratory syndrome virus (PRRSV) infection and results in PRRSV-persistent infection. Nevertheless, the exact mechanisms of PRRSV-ADE infection are poorly understood. In the current research, the results of the ADE assay showed that porcine immunoglobulin G (IgG) specific for the PRRSV significantly enhanced PRRSV proliferation in porcine alveolar macrophages (PAMs), suggesting that the ADE activity of PRRSV infection existed in pig anti-PRRSV IgG. The results of the RNA interference assay showed that knockdown of the Fc gamma receptor I (FcγRI) or FcγRIII gene significantly suppressed the ADE activity of PRRSV infection in PAMs, suggesting that FcγRI and FcγRIII were responsible for mediating PRRSV-ADE infection. In addition, the results of the antibody blocking assay showed that specific blocking of the Sn1, 2, 3, 4, 5, or 6 extracellular domain of the sialoadhesin (Sn) protein or selective blockade of the scavenger receptor cysteine-rich (SRCR) 5 domain of the CD163 molecule significantly repressed the ADE activity of PRRSV infection in PAMs, suggesting that Sn and CD163 were involved in FcγR-mediated PRRSV-ADE infection. The Sn1–6 domains of porcine Sn protein and the SRCR 5 domain of porcine CD163 molecule might play central roles in the ADE of PRRSV infection. In summary, our studies indicated that activating FcγRs (FcγRI and FcγRIII) and viral receptors (Sn and CD163) were required for ADE of PRRSV infection. Our findings provided a new insight into PRRSV infection that could be enhanced by FcγRs and PRRSV receptors-mediated PRRSV-antibody immune complexes (ICs), which would deepen our understanding of the mechanisms of PRRSV-persistent infection via the ADE pathway.

Identification of CD163 regions that are required for porcine reproductive and respiratory syndrome virus (PRRSV) infection but not for binding to viral envelope glycoproteins

CD163, a receptor for porcine reproductive and respiratory syndrome virus (PRRSV), possesses nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. To identify CD163 regions involved in PRRSV infection, CD163 mutants were generated. Infection experiments showed resistance to infection following deletion of the SRCR4/5 interdomain or the Exon 13 that encodes a portion of PSTII. The mutation of a pentapeptide domain in SRCR5 and SRCR7 also conferred resistance. Mutant CD163 proteins that resisted infection retained the ability to interact with GP2, GP3, GP4 and GP5 viral glycoproteins. The contribution of multiple domains to infection but not to the binding of viral glycoproteins suggests that the envelope proteins may form multiple interactions with CD163, or that receptor regions important for infection have other cellular binding partners required for PRRSV infection. Finally, we mapped the localization the anti-CD163 2A10 antibody epitope.

LGALS3BP in Microglia Promotes Retinal Angiogenesis Through PI3K/AKT Pathway During Hypoxia

PurposeRetinal microglia promote angiogenesis and vasculopathy in oxygen-induced retinopathy (OIR); however, its specific molecular mechanism in the formation of retinal angiogenesis remains unclear. The lectin galactoside-binding soluble 3 binding protein (LGALS3BP), a member of the scavenger receptor cysteine-rich (SRCR) domain protein family, is involved in tumor neovascularization, and we therefore hypothesized that LGALS3BP plays an active role in microglia-induced angiogenesis.MethodsThe expression of LGALS3BP in microglia was detected by immunofluorescence, RT-qPCR, and western blotting. Functional assays of human umbilical vein endothelial cells (HUVECs) such as migration, proliferation, and tube formation were measured by Transwell, EdU, and Matrigel assays. Angiogenesis-related factors and PI3K/AKT levels were detected by western blotting. The relationship between LGALS3BP and PI3K or HIF-1α was investigated by immunoprecipitation.ResultsOur results showed that the expression of LGALS3BP was significantly increased in microglia surrounding neovascularization of the OIR mice and was also upregulated in human microglial clone 3 (HMC3) cells after hypoxia. Moreover, HUVECs co-cultured with hypoxic HMC3 cells showed increased migration, proliferation, and tube formation, as well as levels of angiogenesis-related factor. However, the proangiogenic ability and angiogenesis-related factor expression of HMC3 cells was suppressed after silencing LGALS3BP. LGALS3BP induces the upregulation of angiogenesis-related factors through the PI3K/AKT pathway and then promotes angiogenesis in microglia.ConclusionsCollectively, our findings suggest that LGALS3BP in microglia plays an important role in angiogenesis, suggesting a potential therapeutic target of LGALS3BP for angiogenesis.

TMPRSS13 zymogen activation, surface localization, and shedding is regulated by proteolytic cleavage within the non-catalytic stem region.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Here we characterize a novel post-translational mechanism important for TMPRSS13 function: proteolytic cleavage within the extracellular TMPRSS13 stem region located between the transmembrane domain and the first site of N-linked glycosylation at asparagine (N)-250 in the scavenger receptor cysteine rich (SRCR) domain. Importantly, the catalytic competence of TMPRSS13 is essential for stem region cleavage, suggesting an autonomous mechanism of action. Site-directed mutagenesis of the 10 basic amino acids (four arginine and six lysine residues) in this region abrogated zymogen activation and catalytic activity of TMPRSS13, as well as phosphorylation, cell surface expression, and shedding. Mutation analysis of individual arginine residues identified R223, a residue located between the low-density lipoprotein receptor class A domain and the SRCR domain, as important for stem region cleavage. Mutation of R223 causes a reduction in the aforementioned functional processing steps of TMPRSS13. These data provide further insight into the roles of different post-translational modifications as regulators of the function and localization of TMPRSS13. Additionally, the data suggest the presence of complex interconnected regulatory mechanisms that may serve to ensure the proper levels of cell-surface and pericellular TMPRSS13-mediated proteolysis under homeostatic conditions.

A novel mannose-containing sialoprotein adhesin involved in the binding of Candida albicans cells to DMBT1.

Candida albicans colonizes the oral cavity and causes oral candidiasis and early childhood caries synergistically with cariogenic Streptococcus mutans. Colonization of oral tissues with C. albicans is an essential step in the initiation of these infectious diseases. Deleted in malignant brain tumors 1 (DMBT1), also known as salivary agglutinin or gp-340, belongs to the scavenger receptor cysteine-rich (SRCR) superfamily and has important functions in innate immunity. In the oral cavity, DMBT1 causes microbial adherence to tooth enamel and oral mucosa surfaces, but the adherence of C. albicans to DMBT1 has not been examined. In this study, we investigated the binding of C. albicans to DMBT1 and isolated the fungal components responsible for the binding. Candida albicans specifically bound to DMBT1 and strongly bound to the peptide domain SRCRP2. Binding to SRCRP2 was inhibited by N-acetylneuraminic acid and mannose and by lectins recognizing these sugars. The isolated component had a molecular mass of 25kDa, contained sialic acid and mannose residues, and inhibited C. albicans binding to SRCRP2. The localization of the 25-kDa protein on the surface of C. albicans cell walls was confirmed by immunostaining and a cell ELISA using an antiserum to the protein, and Western blotting revealed the presence of the 25-kDa protein in the cell wall fraction of C. albicans. These results suggest that the isolated adhesin is localized on the surface of C. albicans cell walls and that sialic acid and mannose residues in the adhesin play a significant role in the binding reaction.

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS).

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.

Balancing selection at the human salivary agglutinin gene (DMBT1) driven by host-microbe interactions

SummaryDiscovering loci under balancing selection in humans can identify loci with alleles that affect response to the environment and disease. Genome variation data have identified the 5′ region of the DMBT1 gene as undergoing balancing selection in humans. DMBT1 encodes the pattern-recognition glycoprotein DMBT1, also known as SALSA, gp340, or salivary agglutinin. DMBT1 binds to a variety of pathogens through a tandemly arranged scavenger receptor cysteine-rich (SRCR) domain, with the number of domains polymorphic in humans. We show that the signal of balancing selection is driven by one haplotype usually carrying a shorter SRCR repeat and another usually carrying a longer SRCR repeat. DMBT1 encoded by a shorter SRCR repeat allele does not bind a cariogenic and invasive Streptococcus mutans strain, in contrast to the long SRCR allele that shows binding. Our results suggest that balancing selection at DMBT1 is due to host-microbe interactions of encoded SRCR tandem repeat alleles.

Defining the caprine γδ T cell WC1 multigenic array and evaluation of its expressed sequences and gene structure conservation among goat breeds and relative to cattle.

Workshop cluster 1 (WC1) molecules are part of the scavenger receptor cysteine-rich (SRCR) superfamily and act as hybrid co-receptors for the γδ T cell receptor and as pattern recognition receptors for binding pathogens. These members of the CD163 gene family are expressed on γδ T cells in the blood of ruminants. While the presence of WC1+ γδ T cells in the blood of goats has been demonstrated using monoclonal antibodies, there was no information available about the goat WC1 gene family. The caprine WC1 multigenic array was characterized here for number, structure and expression of genes, and similarity to WC1 genes of cattle and among goat breeds. We found sequence for 17 complete WC1 genes and evidence for up to 30 SRCR a1 or d1 domains which represent distinct signature domains for individual genes. This suggests substantially more WC1 genes than in cattle. Moreover, goats had seven different WC1 gene structures of which 4 are unique to goats. Caprine WC1 genes also had multiple transcript splice variants of their intracytoplasmic domains that eliminated tyrosines shown previously to be important for signal transduction. The most distal WC1 SRCR a1 domains were highly conserved among goat breeds, but fewer were conserved between goats and cattle. Since goats have a greater number of WC1 genes and unique WC1 gene structures relative to cattle, goat WC1 molecules may have expanded functions. This finding may impact research on next-generation vaccines designed to stimulate γδ T cells.

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-1β, 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 significanceSepsis 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.

Physical Interactions With Bacteria and Protozoan Parasites Establish the Scavenger Receptor SSC4D as a Broad-Spectrum Pattern Recognition Receptor.

Since the pioneering discoveries, by the Nobel laureates Jules Hoffmann and Bruce Beutler, that Toll and Toll-like receptors can sense pathogenic microorganisms and initiate, in vertebrates and invertebrates, innate immune responses against microbial infections, many other families of pattern recognition receptors (PRRs) have been described. One of such receptor clusters is composed by, if not all, at least several members of the scavenger receptor cysteine-rich (SRCR) superfamily. Many SRCR proteins are plasma membrane receptors of immune cells; however, a small subset consists of secreted receptors that are therefore in circulation. We here describe the first characterization of biological and functional roles of the circulating human protein SSC4D, one of the least scrutinized members of the family. Within leukocyte populations, SSC4D was found to be expressed by monocytes/macrophages, neutrophils, and B cells, but its production was particularly evident in epithelial cells of several organs and tissues, namely, in the kidney, thyroid, lung, placenta, intestinal tract, and liver. Similar to other SRCR proteins, SSC4D shows the capacity of physically binding to different species of bacteria, and this opsonization can increase the phagocytic capacity of monocytes. Importantly, we have uncovered the capacity of SSC4D of binding to several protozoan parasites, a singular feature seldom described for PRRs in general and here demonstrated for the first time for an SRCR family member. Overall, our study is pioneer in assigning a PRR role to SSC4D.