A common binding motif for various bacteria of the bacteria-binding peptide SRCRP2 of DMBT1/gp-340/salivary agglutinin

Abstract Salivary agglutinin (DMBT1SAG) is identical to lung glycoprotein-340 and encoded by deleted in malignant brain tumors-1. It is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, proteins that have one or more SRCR domains. Salivary agglutinin plays a role in oral innate immunity by the binding and agglutination of oral streptococci. S. mutans has been shown to bind to a 16-mer peptide (QGRVEVLYRGSWGTVC) located within the SRCR domains. Within this peptide, designated SRCR Peptide 2, residues VEVL and W were critical for binding. The aim of this study was to investigate binding of DMBT1SAG to other bacteria. Therefore, interaction between a series of bacteria and DMBT1(SAG), SRCR peptide 2 and its alanine substitution variants was studied in adhesion and agglutination assays. For different bacteria there was a highly significant correlation between adhesion to DMBT1SAG and adhesion to SRCR peptide 2 suggesting that SRCR peptide 2 is the major bacteria binding site. An alanine substitution scan showed that 8 amino acids were involved in binding (xRVEVLYxxSWxxxx). The binding motifs varied for different species were found, but the residues VxVxY and W were always present. In conclusion, a common binding motif (RVEVLYxxxSW) within the SRCR domains is responsible for the broad bacteria-binding spectrum of DMBT1SAG.     

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.     

Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains

The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.     

DMBT1, a regulator of mucosal homeostasis through the linking of mucosal defense and regeneration?

DMBT1 (deleted in malignant brain tumor 1), which encodes a large scavenger receptor cysteine rich (SRCR) B protein, has been proposed to be a tumor suppressor gene, due to the high frequency of its homozygous deletion and the lack of expression in a variety of cancers. However, studies on its physiological functions and its relationship with tumorigenesis are still at an initial stage. Two mucosal defense-related molecules, gp-340 and salivary agglutinin, have been identified to be alternatively spliced products of DMBT1, which suggests that DMBT1 is a pattern recognition receptor in innate immunity. Meanwhile, results from immunohistochemical staining and studies at the cellular level, began to associate DMBT1 with a proliferation to differentiation switching process in gastrointestinal epithelial cells. Together with its up-regulation in inflammation, these findings suggest that DMBT1 might be a local regulator of homeostasis, possibly through linking mucosal inflammation to the modulation of epithelial regeneration, and whose abnormality is a frequent cause of malignancy.     

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.     

Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation

Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n = 7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements. Western blots of saliva samples, and of gp-340 purified, from the seven donors using a gp-340 specific antibody distinguished four gp-340 size variants, designated I to IV (n = 2,2,2 and 1). While saliva gp-340 variants I to III had single bands of increasing sizes, variant IV and lung gp-340 had double bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galβ1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4) and smaller size grouping I/IV correlated with a secretor, Se(+), and a non secretor, Se(−), dependent glycoform of gp-340, respectively (p = 0.03). The Se(+) glycoforms contained ABH, Le^b, Le^y and polylactosamine structures, while the Se(−) glycoforms lacked ABH antigens but expressed Le^a, Le^x and lactosamine structures. By contrast, lung gp-340 completely lacked ABH, Le^a/b, Le^x/y or sLe^x structures. Gp-340 and secretor typing of saliva from additional donors (n = 29) showed gp-340 glycoforms I to IV for 6, 16, 4 and 0 donors, respectively, and 3 non-typeable donors, and verified that gp-340 glycoforms I and II/III correlate with Se(−) and Se(+) phenotypes, respectively (p < 0.0001). The glycoforms of saliva and lung gp-340 mediated differential aggregation of Le^b- (Helicobacter pylori), sialylpolylactosamine- (Streptococcus suis) or sialic acid- (Streptococcus mutans) binding bacteria. In conclusion, variant size- and glycoforms of gp-340 are expressed by different individuals and may modulate the biological properties of gp-340 pertinent to health and disease.     

Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses

The lung scavenger receptor-rich protein glycoprotein-340 (gp-340) is present in bronchoalveolar lavage (BAL) fluids and saliva and mediates specific adhesion to and aggregation of bacteria. It also binds to surfactant proteins A and D (SP-A and -D). Prior studies demonstrated that SP-A and SP-D contribute to innate defense against influenza A virus (IAV). We now show that lung and salivary gp-340 inhibit the hemagglutination activity and infectivity of IAV and agglutinate the virions through a mechanism distinct from that of SP-D. As in the case of SP-A, the antiviral effects of gp-340 are mediated by noncalcium-dependent interactions between the virus and sialic acid-bearing carbohydrates on gp-340. Gp-340 inhibits IAV strains that are resistant to SP-D. Concentrations of gp-340 present in saliva and BAL fluid of healthy donors are sufficient to bind to IAV and inhibit viral infectivity. On the basis of competition experiments using competing saccharide ligands, it appears that SP-D does not entirely mediate that anti-IAV activity of BAL fluid and contributes little to that of saliva. Furthermore, removal of gp-340 from BAL fluid and saliva significantly reduced anti-IAV activity. Hence, gp-340 contributes to defense against IAV and may be particularly relevant to defense against SP-D-resistant viral strains.     

Cloning of S4D-SRCRB,a new soluble member of the group B scavenger receptor cysteine-rich family (SRCR-SF) mapping to human chromosome 7q11.23

The scavenger receptor cysteine-rich superfamily (SRCR-SF) is a highly conserved group of membrane and/or secreted proteins related to the innate and adaptive immune system. Here, we report the cloning of the gene encoding human S4D-SRCRB, a novel soluble member of the SRCR-SF, which is composed of four group B SRCR domains separated by Pro-, Ser- and Thr-rich polypeptides. The longest cDNA sequence found is 2,806 bp in length and encodes a mature protein of 528 aa, with a predicted molecular mass of M(r) 55,600. The S4D-SRCRB gene is located at Chromosome 7q11.23, telomeric to the Williams-Beuren syndrome deletion. It extends over 20 kb and consists of 11 exons, with each SRCR domain being encoded by a single exon. Northern blot analysis indicated that S4D-SRCRB has a broad tissue distribution and is expressed as two major mRNA species: one of 2.8 kb, with a restricted tissue expression pattern (mainly kidney and placenta), and another of 1.5 kb, with a broader distribution. A similar mRNA expression pattern was observed during the analysis of several tumor cell lines. The highest degree of similarity found between S4D-SRCRB and other group B SRCR-SF members was with human DMBT1 (a mosaic protein composed of fourteen SRCR domains, which is involved in innate defense and epithelia polarization) and chicken 18-B (a turpentine-induced soluble acute-phase protein composed of four SRCR domains). Our data indicate that S4D-SRCRB constitutes a novel SRCR-SF member, which could be involved in basic homeostatic functions such as innate host defense.     

Crystal structure of the third extracellular domain of CD5 reveals the fold of a group B scavenger cysteine-rich receptor domain

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 A of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel beta-sheets and one alpha-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.     

Interaction between DMBT1 and galectin 3 is modulated by the structure of the oligosaccharides carried by DMBT1

DMBT1 (deleted in malignant brain tumor 1), a human mucin-like glycoprotein, belonging to the scavenger receptor cystein-rich (SRCR) superfamily, is mainly secreted from mucosal epithelia. It has been shown previously that interaction of hensin, the rabbit ortholog of DMBT1, with galectin 3, a β-galactoside-binding lectin, induces a terminal differentiation of epithelial cells. In this paper, we have used surface plasmon resonance (SPR), to analyse the binding of galectin 3 to two purified samples of human DMBT1:recombinant DMBT1 produced in CHO cells and DMBT1 isolated from intestinal tissues. Characterization of their glycosylation profile by nano-ESI-Q-TOF tandem mass spectrometry showed significant differences in O-glycans between the two DMBT1 samples. Results obtained by SPR demonstrated that the oligosaccharide side chains of DMBT1 are recognized by the carbohydrate-recognition domain (CRD) of galectin 3 and modification in the pattern of oligosaccharides modulates the binding parameters of DMBT1 with galectin 3. Moreover, using immunohistochemistry on paraffin-embedded colonic tissue sections, we could show a co-localisation of DMBT1 and galectin 3 in human intestine, suggesting a potential physiological interaction.     

A role for human Sp alpha as a pattern recognition receptor

Human Sp alpha is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, which suggests that it may play an important role in the regulation of the innate and adaptive immune systems. In the present study we show that recombinant human Sp alpha (rSp alpha) binds to the surface of several gram-positive and gram-negative bacterial strains. Competition studies indicated that such binding is mediated by the recognition of lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, through nonoverlapping sites on the Sp alpha molecule. The most conserved part of LPS (2-keto-3-deoxyoctulosonic acid and lipid A) was shown to be involved in the recognition by Sp alpha. Bacterial binding studies using the SRCR domain 1 of Sp alpha showed that this domain retains both the LPS and LTA binding activities, indicating that both bacterial interacting sites are retained in a single SRCR domain. Furthermore, rSp alpha induced aggregation of gram-positive and gram-negative bacteria strains. On the other hand, rSp alpha inhibited tumor necrosis factor-alpha secretion by human monocytes stimulated with LPS or LTA. Binding of Sp alpha to conserved components of bacterial surfaces and modulation of the monocyte response indicate that this molecule is an active constituent of the innate immune response of the host.     

Identification of human immunodeficiency virus type 1 (HIV-1) gp120-binding sites on scavenger receptor cysteine rich 1 (SRCR1) domain of gp340

Background

gp340, a member of scavenger receptor cysteine rich family encoded by Deleted in Malignant Brain Tumors 1 (DMBT1), is an important component in innate immune defense. The first scavenger receptor cysteine rich domain (SRCR1) of gp340 has been shown to inhibit HIV-1 infection through binding to the N-terminal flank of the V3 loop of HIV-1 gp120.

Results

Through homology modeling and docking analysis of SRCR1 to a gp120-CD4-X5 antibody complex, we identified three loop regions containing polar or acidic residues that directly interacted with gp120. To confirm the docking prediction, a series of over-lapping peptides covering the SRCR1 sequence were synthesized and analyzed by gp120-peptide binding assay. Five peptides coincide with three loop regions showed the relative high binding index. An alanine substitution scan revealed that Asp34, Asp35, Asn96 and Glu101 in two peptides with the highest binding index are the critical residues in SRCR1 interaction with gp120.

Conclusion

We pinpointed the vital gp120-binding regions in SRCR1 and narrowed down the amino acids which play critical roles in contacting with gp120.     

HIV envelope binding by macrophage-expressed gp340 promotes HIV-1 infection

The scavenger receptor cysteine-rich protein gp340 functions as part of the host innate immune defense system at mucosal surfaces. In the genital tract, its expression by cervical and vaginal epithelial cells promotes HIV trans-infection and may play a role in sexual transmission. Gp340 is an alternatively spliced product of the deleted in malignant brain tumors 1 (DMBT1) gene. In addition to its innate immune system activity, DMBT1 demonstrates instability in multiple types of cancer and plays a role in epithelial cell differentiation. We demonstrate that monocyte-derived macrophages express gp340 and that HIV-1 infection is decreased when envelope cannot bind it. Inhibition of infection occurred at the level of fusion of M-, T-, and dual-tropic envelopes. Additional HIV-1 envelope binding molecules, such as dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose-binding lectin, and heparan sulfate, enhance the efficiency of infection of the cells that express them by increasing the local concentration of infectious virus. Our data suggest that gp340, which is expressed by macrophages in vivo, may function to enhance infection in much the same manner. Its expression on tissue macrophages and epithelial cells suggests important new opportunities for HIV-1 pathogenesis investigation and therapy.     

The 5' flanking region of the Rhesus monkey H3 (DMBT1) gene contains putative progesterone response elements

DMBT1 (deleted in malignant brain tumors) encodes a large scavenger receptor cysteine rich (SRCR) protein with proposed tumor suppressor properties due to its frequent deletion or lack of expression in a variety of different tumors including endometrial cancers. The gene is alternatively spliced to produce a number of related proteins with suspected functions in mucosal inflammation and epithelial regeneration. Expression of DMBT1 has been demonstrated in a wide variety of cell types, mostly of epithelial origin, including tissues of the respiratory system, the alimentary system, brain, and reproductive system. We have previously identified a Rhesus monkey cDNA clone H3 (homologous to human DMBT1) as a progesterone-induced gene in Rhesus monkey endometrium during the secretory phase of the menstrual cycle. As an initial step in understanding the molecular mechanisms of H3 (DMBT1) regulation we have cloned and sequenced 1.5 kb of the 5'-flanking region expected to contain promoter sequences of the Rhesus monkey gene and identified six putative progesterone receptor binding sites in the 5'-upstream region.     

The Calcium-induced Conformation and Glycosylation of Scavenger-rich Cysteine Repeat (SRCR) Domains of Glycoprotein 340 Influence the High Affinity Interaction with Antigen I/II Homologs

Oral streptococci adhere to tooth-immobilized glycoprotein 340 (GP340) via the surface protein antigen I/II (AgI/II) and its homologs as the first step in pathogenesis. Studying this interaction using recombinant proteins, we observed that calcium increases the conformational stability of the scavenger-rich cysteine repeat (SRCRs) domains of GP340. Our results also show that AgI/II adheres specifically with nanomolar affinity to the calcium-induced SRCR conformation in an immobilized state and not in solution. This interaction is significantly dependent on the O-linked carbohydrates present on the SRCRs. This study also establishes that a single SRCR domain of GP340 contains the two surfaces to which the apical and C-terminal regions of AgI/II noncompetitively adhere. Compared with the single SRCR domain, the three tandem SRCR domains displayed a collective/cooperative increase in their bacterial adherence and aggregation. The previously described SRCRP2 peptide that was shown to aggregate several oral streptococci displayed limited aggregation and also nonspecific adherence compared to SRCR domains. Finally, we show distinct species-specific adherence/aggregation between Streptococcus mutans AgI/II and Streptococcus gordonii SspB in their interaction with the SRCRs. This study concludes that identification of the metal ion and carbohydrate adherence motifs on both SRCRs and AgI/II homologs could lead to the development of anti-adhesive inhibitors that could deter the adherence of pathogenic oral streptococci and thereby prevent the onset of infections.     

First evidence of the interaction between deleted in malignant brain tumor 1 and galectin-3 in the mammalian oviduct

The oviduct supports the transport and final maturation of gametes, and harbors fertilization and early embryo development. The oviductal epithelium is responsible for providing the correct environment for these processes. Deleted in malignant brain tumor 1 (DMBT1) is expressed by multiple organisms and several cell types, and the interaction of the rabbit ortholog of DMBT1 with galectin-3 (gal-3) modulates the polarity of epithelial cells. This interaction has not yet been shown in locations other than rabbit kidney and human-cultured endothelial cells. DMBT1 and gal-3 also protect epithelial layers from pathogens and trauma, and are innate immunity components. DMBT1 has been detected in the porcine oviduct, and gal-3 has been reported in the Fallopian tube and in the cow oviduct. Interaction between both proteins would show a probable physiological function in the female reproductive tract. This work describes the presence and co-localization of DMBT1 and gal-3 mainly in the apical region of the epithelial cells of the Fallopian tube and the porcine oviduct, and co-immunoprecipitation in membrane-enriched epithelial cell extracts from the porcine oviduct. The findings strongly support a functional interaction in the mammalian oviduct, suggestive of a role on epithelial protection and homeostasis, which might be related to epithelium–gamete interaction.     

Molecular identification of the sperm selection involved porcine sperm binding glycoprotein (SBG) as deleted in malignant brain tumors 1 (DMBT1)

Porcine sperm binding glycoprotein (SBG) is involved in sperm-oviduct interaction. Here we use mass spectrometry to identify SBG, finding peptides corresponding to deleted in malignant brain tumors 1 (DMBT1), at scavenger receptor cysteine-rich (SRCR) and CUB domains. RT-PCR allowed the cloning of unique sequences, belonging to porcine DMBT1. Western blot and immunofluorescence of oviductal tissues using anti-SBG and anti-hDMBT1 antibodies showed identical results. The biochemical characteristics of both proteins are coincident. We conclude that porcine SBG is an oviductal form of DMBT1, and thus assign this protein a novel location and function.