Floris J. Bikker

Immunohistochemical Detection of Salivary Agglutinin/gp-340 in Human Parotid, Submandibular, and Labial Salivary Glands

Salivary agglutinin is a Streptococcus mutans binding protein and a member of the scavenger receptor cysteine-rich superfamily. It is identical to lung gp-340 and brain DMBT1, which possibly play a role in innate immunity and tumor suppression, respectively. The goal of this study was to localize salivary agglutinin in human salivary glands. Two monoclonal antibodies, directed against gp-340, were characterized. mAb 213-1 reacted with sialic acid epitopes and cross-reacted with MUC7. The reaction with mAb 213-6 disappeared after reduction, suggesting that a protein epitope was recognized. In the parotid gland, immunohistochemical labeling with mAb 213-6 was found in the duct cells. In the submandibular gland and labial gland, both serous acini and demilune cells were labeled. In the labial gland, labeling was found at the luminal side of the duct cells. Salivary agglutinin was distinctly localized in salivary glands, but in distinct glandular secretions, no differences in electrophoretic behavior were observed.

DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial‐binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS‐induced TLR4‐mediated NF‐κB activation and to the pathogenesis of Crohns disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen‐binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1‐mediated bacterial aggregation via interaction with its bacterial‐recognition motif. Competition and ELISA studies identify poly‐sulfated and poly‐phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose–response studies in Dmbt1−/− and Dmbt1+/+ mice utilizing the DSS‐induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern‐recognition molecule for poly‐sulfated and poly‐phosphorylated ligands providing a molecular basis for its broad bacterial‐binding specificity and its inhibitory effects on LPS‐induced TLR4‐mediated NF‐κB activation.

Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer

DMBT1 and galectin‐3 are potential interacting proteins with presumably complex roles in tumorigenesis. While at present a variety of mechanisms are discussed for DMBT1 and its participation in cancer, galectin‐3 is commonly known to exert tumor‐promoting effects. However, in vitro studies in a rodent system have suggested that DMBT1/galectin‐3 interaction in the ECM triggers epithelial differentiation, which would point to tumor‐suppressive properties. To improve the understanding of DMBT1/galectin‐3 action in cancer, we carried out studies in skin cancer of different origins. Mutational analyses of DMBT1 identified a missense mutation in 1 of 13 melanoma cell lines. It led to an exchange of an evolutionary conserved proline residue for serine and located within the second CUB domain of DMBT1. Immunohistochemical analyses demonstrated absence of DMBT1/galectin‐3 expression from melanocytes but induction of DMBT1 expression in 1 of 8 nevi and 1 of 11 melanomas and of galectin‐3 expression in 3 of 8 nevi and 4 of 8 melanomas. These data suggest that DMBT1 and galectin‐3 are unlikely to act as classical tumor suppressors in melanomas. DMBT1 and galectin‐3 appear to be secreted to the ECM by epithelial cells within the epidermis and the hair follicle. Compared to the flanking normal epidermis, skin tumors of epithelial origin frequently displayed downregulation of DMBT1 (18 of 19 cases) and galectin‐3 (12 of 12 cases). Thus, loss of DMBT1/galectin‐3 expression may play a role in the genesis of epithelial skin cancer. This would support the view that galectin‐3 can exert tumor‐suppressive effects in certain scenarios, and DMBT1/galectin‐3‐mediated differentiation represents a candidate mechanism for this effect.

The SRCR/SID region of DMBT1 defines a complex multi-allele system representing the major basis for its variability in cancer.

Deleted in malignant brain tumors 1 (DMBT1) at 10q25.3–q26.1 has been proposed as a candidate tumor‐suppressor gene for brain and epithelial cancer. DMBT1 encodes a multifunctional mucin‐like protein presumably involved in epithelial differentiation and protection. The gene consists of highly homologous and repeating exon and intron sequences. This specifically applies to the region coding for the repetitive scavenger receptor cysteine‐rich (SRCR) domains and SRCR‐interspersed domains (SIDs) that constitutes the major part of the gene. This particular structure may previously have interfered with the delineation of DMBT1 alterations in cancer. Uncovering these, however, is of mechanistic importance. By a combined approach, we conducted a detailed mutational analysis, starting from a panel of 51 tumors, including 46 tumor cell lines and five primary tumors. Alterations in the repetitive region were present in 22/31 (71%) tumors that were investigated in detail. Six tumors showed presumably de novo mutations, among these three with point mutations in combination with a loss of heterozygosity. However, none of the alterations unambiguously would be predicted to lead to an inactivation of DMBT1. We define seven distinct DMBT1 alleles based on variable numbers of tandem repeats (VNTRs). At least 11 tumors exclusively harbored these VNTRs. The data suggest that the SRCR/SID region defines a complex multi‐allele system that has escaped previous analyses and that represents the major basis for the variability of DMBT1 in cancer. DMBT1 thus compares to mucins rather than to conventional tumor suppressors.

Identification of chicken cathelicidin-2 core elements involved in antibacterial and immunomodulatory activities

Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomodulatory activities and consists of two alpha-helices connected by a hinge region. Here we report the biological properties of the separate alpha-helical segments and the importance of the proline residue in the hinge region. Substitution of proline-14 in the CATH-2 hinge region by leucine, but not by glycine, strongly reduced antibacterial and hemolytic activity. Furthermore, substitution by leucine strongly reduced the neutralization of LPS-induced cytokine production and peptide-induced monocyte chemotactic protein-1 (MCP-1) production by human peripheral blood mononuclear cells (PBMCs). This indicates that the hinge region is important for rapid penetration of the bacterial membrane as well as indirect and direct immunomodulatory activities. The highly cationic and amphipathic N-terminal segment (C1-15) exhibited very potent antibacterial activity and fast killing kinetics, while displaying low cytotoxicity towards chicken erythrocytes and PBMCs. The N-terminal and, to a lesser extent, the C-terminal helical regions potently neutralized LPS-induced release of TNFalpha, IL-6 and IL-10 by PBMCs, while IL-8 production was only moderately affected. These results indicate that core elements within mature CATH-2 can be identified that are linked to antibacterial and/or immunomodulatory activities. Further studies may lead to the development of peptide antibiotics with specific properties that can be used for prophylactic and/or therapeutic applications.

Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species

We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis but also for several other Bacillus spp. and for both vegetative cells and spores. With the use of mass spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, with the use of these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids.

Carcinogen inducibility in vivo and down-regulation of DMBT1 during breast carcinogenesis

Deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain and epithelial cancer. Initial studies suggested loss of expression rather than mutation as the predominant mode of DMBT1 inactivation. However, in situ studies in lung cancer demonstrated highly sophisticated changes of DMBT1 expression and localization, pointing to a chronological order of events. Here we report on the investigation of DMBT1 in breast cancer in order to test whether these principles might also be attributable to other tumor types. Comprehensive mutational analyses did not uncover unambiguous inactivating DMBT1 mutations in breast cancer. Expression analyses in the human and mouse mammary glands pointed to the necessity of DMBT1 induction. While age‐dependent and hormonal effects could be ruled out, 9 of 10 mice showed induction of Dmbt1 expression after administration of the carcinogen 7,12‐dimethybenz(α)anthracene prior to the onset of tumorigenesis or other histopathological changes. DMBT1 displayed significant up‐regulation in human tumor–flanking tissues compared to in normal breast tissues (P < 0.05). However, the breast tumor cells displayed a switch from lumenal secretion to secretion to the extracellular matrix and a significant down‐regulation compared to that in matched normal flanking tissues (P < 0.01). We concluded that loss of expression also is the predominant mode of DMBT1 inactivation in breast cancer. The dynamic behavior of DMBT1 in lung carcinoma is fully reflected in breast cancer, which suggests that this behavior might be common to tumor types arising from monolayered epithelia.

Binding of salivary agglutinin to IgA

SAG (salivary agglutinin), which is identical to gp-340 (glycoprotein-340) from the lung, is encoded by DMBT1 (deleted in malignant brain tumours 1). It is a member of the SRCR (scavenger receptor cysteine-rich) superfamily and contains 14 SRCR domains, 13 of which are highly similar. SAG in saliva is partially complexed with IgA, which may be necessary for bacterial binding. The goal of the present study was to characterize the binding of purified SAG to IgA. SAG binds to a variety of proteins, including serum and secretory IgA, alkaline phosphatase-conjugated IgGs originating from rabbit, goat, swine and mouse, and lactoferrin and albumin. Binding of IgA to SAG is calcium dependent and is inhibited by 0.5 M KCl, suggesting that electrostatic interactions are involved. Binding of IgA was destroyed after reduction of SAG, suggesting that the protein moiety is involved in binding. To pinpoint further the binding domain for IgA on SAG, a number of consensus-based peptides of the SRCR domains and SRCR interspersed domains were designed and synthesized. ELISA binding studies with IgA indicated that only one of the peptides tested, comprising amino acids 18-33 (QGRVEVLYRGSWGTVC) of the 109-amino-acid SRCR domain, exhibited binding to IgA. This domain is identical to the domain of SAG that is involved in binding to bacteria. Despite this similar binding site, IgA did not inhibit binding of Streptococcus mutans to SAG or peptide. These results show that the binding of IgA to SAG is specifically mediated by a peptide sequence on the SRCR domains.

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.

ABSTRACT Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 105 CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO2 showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.