Sven Ulrik Gorr

Antimicrobial peptides of the oral cavity

Antimicrobial proteins and peptides constitute a diverse class of host-defense molecules that act early to combat invasion and infection with bacteria and other microorganisms. These peptides have engendered considerable interest in the past decade as a biological paradigm in innate immunity and as a potential source of novel antibiotics. Many recent reviews have explored the biological diversity and function of this class of peptides [e.g. (36, 50, 73, 80, 103, 126, 127, 191, 209, 221, 226, 255)]. This review focuses especially on antimicrobial proteins and peptides of the oral cavity and on their role in periodontal disease. The potential use of antimicrobial peptides as templates for novel antibiotics is also discussed.

Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.

Antimicrobial peptides and periodontal disease

AIMS The goal of this review is to identify the antimicrobial proteins in the oral fluids, saliva and gingival crevicular fluid and identify functional families and candidates for antibacterial treatment. RESULTS Periodontal biofilms initiate a cascade of inflammatory and immune processes that lead to the destruction of gingival tissues and ultimately alveolar bone loss and tooth loss. Treatment of periodontal disease with conventional antibiotics does not appear to be effective in the absence of mechanical debridement. An alternative treatment may be found in antimicrobial peptides and proteins, which can be bactericidal and anti-inflammatory and block the inflammatory effects of bacterial toxins. The peptides have co-evolved with oral bacteria, which have not developed significant peptide resistance. Over 45 antibacterial proteins are found in human saliva and gingival crevicular fluid. The proteins and peptides belong to several different functional families and offer broad protection from invading microbes. Several antimicrobial peptides and proteins (AMPs) serve as templates for the development of therapeutic peptides and peptide mimetics, although to date none have demonstrated efficacy in human trials. CONCLUSIONS Existing and newly identified AMPs may be developed for therapeutic use in periodontal disease or can serve as templates for peptide and peptide mimetics with improved therapeutic indices.

Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

Bio-inspired stable antimicrobial peptide coatings for dental applications.

We developed a novel titanium coating that has applications for preventing infection-related implant failures in dentistry and orthopedics. The coating incorporates an antimicrobial peptide, GL13K, derived from parotid secretory protein, which has been previously shown to be bactericidal and bacteriostatic in solution. We characterized the resulting physicochemical properties, resistance to degradation, activity against Porphyromonas gingivalis and in vitro cytocompatibility. Porphyromonas gingivalis is a pathogen associated with dental peri-implantitis, an inflammatory response to bacteria resulting in bone loss and implant failure. Our surface modifications obtained a homogeneous, highly hydrophobic and strongly anchored GL13K coating that was resistant to mechanical, thermochemical and enzymatic degradation. The GL13K coatings had a bactericidal effect and thus significantly reduced the number of viable bacteria compared to control surfaces. Finally, adequate proliferation of osteoblasts and human gingival fibroblasts demonstrated the GL13K coatings cytocompatibility. The robustness, antimicrobial activity and cytocompatibility of GL13K-biofunctionalized titanium make it a promising candidate for sustained inhibition of bacterial biofilm growth. This surface chemistry provides a basis for development of multifunctional bioactive surfaces to reduce patient morbidities and improve long-term clinical efficacy of metallic dental and orthopedic implants.

Secretory granule biogenesis and chromogranin A: master gene, on/off switch or assembly factor?

Secretory granules are found in specialized cell types, including endocrine cells, suggesting that a coordinated programme of gene expression is involved in their biogenesis. Indeed, it has been proposed that chromogranin A (CgA) acts as an on/off switch for secretory granule biogenesis. However, this proposed function is difficult to reconcile with the large body of evidence suggesting that secretory granules exist in the absence of CgA and that cells can synthesize CgA in the absence of secretory granules. Indeed, recent evidence suggests that, rather than a master gene or universal on/off switch, a series of on/off switches combines to induce expression of subsets of secretory granule-associated genes. The assembly of newly synthesized proteins and the inclusion of existing granule proteins would produce functional secretory granules. CgA and related proteins might act as assembly factors in this process.

Comparative sorting of neuroendocrine secretory proteins: a search for common ground in a mosaic of sorting models and mechanisms.

Endocrine, neuroendocrine and exocrine cells store regulated secretory proteins in secretory granules, while constitutive and constitutive-like secretory proteins are secreted directly without storage. Sorting of secretory proteins takes place in the trans-Golgi network (sorting for entry) or immature secretory granules (sorting by retention). The relative contribution of these sorting steps and the sorting signals and mechanisms involved in each step has been the subject of intense studies and debate in recent years. New evidence now suggests that: (1) two proteins with structurally similar sorting signals can use different sorting mechanisms; (2) one protein with multiple sorting signals can be sorted differently in different cell types; and (3) one cell type can recognize different sorting signals and use different sorting mechanisms. The latter finding suggests that sorting must be a regulated event. While the current image of sorting is complex, recent findings are pointing to common features that form a mosaic of related sorting mechanisms.

Expression and anti-bacterial activity of human parotid secretory protein (PSP)

Parotid secretory protein (PSP) is an abundant protein in mouse and rat parotid glands. A related sequence (C20orf70) was identified on human chromosome 20. The goal of this study was to determine if PSP is expressed in the human parotid gland. The cDNA for human PSP was amplified from a human parotid cDNA sample. A peptide antibody, raised to the C-terminal peptide of PSP, identified the protein in human parotid tissue by immunofluorescence microscopy. Immunoaffinity chromatography suggested that PSP was expressed in human saliva. PSP is related to bactericidal/permeability-increasing protein (BPI). To test if PSP exhibits anti-bacterial activity, epitope-tagged PSP was expressed in rat GH4C1 cells. The secretion medium exhibited bacteristatic or bactericidal effects on Pseudomonas aeruginosa in a colony-forming assay when compared with secretion medium from GH4C1 cells that did not express PSP. These results suggest that PSP is expressed in the human parotid gland and saliva, where it functions as a BPI-like anti-bacterial protein.

Antimicrobial peptides in periodontal innate defense.

The development of oral biofilms and the host response to biofilm bacteria and their toxins are important factors in the development of periodontal disease. An early component of the host response is the secretion of antimicrobial proteins and peptides (AMPs) by salivary glands, oral epithelial cells and neutrophils. Over 45 AMPs have been identified in the oral cavity. All are found in saliva and several are also present in gingival crevicular fluid. Of these, 13 are up regulated in periodontal disease while 11 are downregulated. However, the concentrations of most AMPs found in oral fluids are below the effective in vitro concentrations, suggesting that local concentrations must be higher for effect or that additional biological functions are important in the oral cavity. Thus, in addition to direct antibacterial activity (e.g. bactericidal activity, bacterial agglutination), AMPs may affect the course of periodontal disease by inactivating bacterial or host proteases (e.g. secretory leukoprotease inhibitor) or bind bacterial toxins, including lipopolysaccharides (e.g. LL-37). Several AMPs (e.g. defensins) also act as immune system alarmins, i.e. endogenous mediators that recruit and activate antigen-presenting cells to enhance innate and adaptive immune responses. The differential regulation of AMP expression in periodontal disease suggests that AMP panels, including up- and downregulated proteins, can be used in oral fluid diagnosis of periodontal disease and to monitor treatment outcome.

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH(4)C(1) cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, in GH(4)C(1) cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH(4)C(1) cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH4C1cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J.12: 2159-2168, 1993). However, in GH4C1cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH4C1cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.