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Antimicrobial peptides may be alternatives to traditional antibiotics with reduced bacterial resistance. The antimicrobial peptide GL13K was derived from the salivary protein BPIFA2. This study determined the relative activity of the L-and D-enantiomers of GL13K to wild-type and drug-resistant strains of three gram-negative species and against Pseudomonas aeruginosa biofilms. DGL13K displayed in vitro activity against ESBL and KPC-producing Klebsiella pneumoniae (MICs 16-32 μg/ml), MDR and XDR P. aeruginosa, and XDR Acinetobacter baumannii carrying metallo-beta-lactamases (MICs 8-32 μg/ml). P. aeruginosa showed low inherent resistance to DGL13K and the increased metabolic activity and growth caused by sub-MIC concentrations of GL13K peptides did not result in acquired bacterial resistance. Daily dosing for approximately two weeks did not increase the MIC of DGL13K or cause cross-resistance between LGL13K and DGL13K. These data suggest that DGL13K is a promising candidate antimicrobial peptide for further development.

BPIFA2 (PSP, SPLUNC2, C20orf70) is a major salivary protein of uncertain physiological function. BPIFA2 is downregulated in salivary glands of spontaneously hypertensive rats, pointing to a role in blood pressure regulation. This study used a novel Bpifa2 knockout mouse model to test the role of BPIFA2 in sodium preference and blood pressure. Blood pressure did not differ between wild-type male and female mice but was significantly lower in male knockout mice compared to male wild-type mice. In contrast, blood pressure was increased in female knockout mice compared to female wild-type mice. Female wild-type mice showed a significant preference for 0.9% saline compared to male mice. This difference was reduced in the knockout mice. BPIFA2 is an LPS-binding protein but it remains to be determined if the reported effects are mediated by the LPS-binding activity of BPIFA2.

Antimicrobial peptides have been evaluated as possible alternatives to traditional antibiotics. The translational potential of the antimicrobial peptide DGL13K was tested with focus on peptide toxicity and in vivo activity in two animal models. DGL13K was effective against Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus with minimal bactericidal concentrations similar to the minimal inhibitory concentration. The peptide showed low toxicity to human red blood cells and HEK cells with median lethal dose around 1 mg/ml. The median lethal dose in greater wax moth larvae (Galleria mellonella) was about 125mg/kg while the peptide caused no skin toxicity in a mouse model. A novel high-throughput luminescence assay was used to test peptide activity in infected G. mellonella, thus reducing vertebrate animal use. DGL13K killed P. aeruginosa in both the G. mellonella model and a mouse burn wound infection model, with bacterial viability 3-10-fold lower than in untreated controls. Future experiments will focus on optimizing peptide delivery, dose and frequency to further improve the antibacterial effect.

Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH 2 (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa , 8 μg/ml [5.6 μM]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 μg/ml (44.8 μM) GL13K reduced biofilm bacteria by 10 2 , and 100 μg/ml (70 μM) GL13K reduced bacteria by 10 3 . Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 μg/ml [22.4 μM]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a d amino acid version of the peptide.

Gorr S-U, Abdolhosseini M. Antimicrobial peptides and periodontal disease. J Clin Periodontol 2011; 38 (Suppl. 11): 126–141. doi: 10.1111/j.1600-051X.2010.01664.x. Abstract 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.

Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a "sorting escort" (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as "sorting escorts" to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells.

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins.

Background: Parotid secretory protein (PSP) is a major salivary protein that is thought to possess both antibacterial and anti-inflammatory activity. A major question is whether PSP expression can be regulated by humoral factors and bacteria. Periodontitis is an inflammatory lesion initiated by interaction between gingival keratinocytes and periodontopathogenic microorganisms such as the Gram-negative anaerobe Porphyromonas gingivalis. Cytokines and sex hormones have been implicated in the progression of various forms of periodontal diseases. Materials and methods: We investigated the expression of PSP and its regulation in primary cultures of human gingival keratinocytes (HGK). HGK at the third or fourth passage were exposed to heat-killed P. gingivalis, tumor necrosis factor-α (TNF-α) and 17β-estradiol. The PSP mRNA levels were examined by real-time polymerase chain reaction (PCR). The protein expression of PSP was confirmed by immunofluorescence. Results: Heat-killed P. gingivalis, TNF-α and 17β-estradiol all resulted in increased HGK levels of mRNA for PSP as determined by real-time PCR analysis. Immunofluorescence demonstrated increased PSP localized within the cytoplasm of HGK following exposure to killed P. gingivalis. Conclusion: The present study has demonstrated for the first time that PSP is expressed in keratinocytes and that it can be up-regulated by bacteria and humoral factors. Thus PSP may have a role in the innate defense system at the gingival epithelial surface.

Parotid secretory protein (PSP) and palate-lung-nasal epithelium clone (PLUNC) are novel secretory proteins that are expressed in the oral cavity and upper airways. Both proteins are related to bactericidal/permeability increasing protein (BPI). Cationic peptides derived from BPI exhibit anti-inflammatory activity. To test if PSP (C20orf70 gene product) also contains anti-inflammatory peptides, we designed 3 cationic peptides based on the predicted structure of PSP and known active regions of BPI. Each peptide inhibited the lipopolysaccharide (LPS)-stimulated secretion of TNFα from RAW 264.7 macrophage cells. At 200 μg/mL, the peptide GK-7 exhibited inhibition similar to that achieved with 10 μg/mL of polymyxin B. PSP peptides directly inhibited the binding of LPS to LPS-binding protein. The cationic peptide Substance P had no inhibitory effect in these assays, confirming the specificity of the PSP peptides. These findings suggest that PSP peptides can serve as templates for the design of novel anti-inflammatory peptides.

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although

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.

Regulated secretory proteins are stored in secretory granules. While the sorting and storage process appears similar in endocrine and exocrine cells, the extent of overlap of sorting between endocrine and exocrine cell types is not clear. It is predicted that exocrine regulated secretory proteins that are stored with high efficiency in exocrine granules would also be stored efficiently in endocrine granules. To test this hypothesis, parotid secretory protein (PSP), which is stored efficiently in parotid acinar cells, was expressed in the endocrine cell lines GH4C1 and PC12. PSP undergoes stimulated secretion in both cell types. Secretion is similar to that of the endocrine regulated secretory protein chromogranin A but distinct from secreted alkaline phosphatase, a marker for the constitutive secretory pathway in endocrine cells. Subcellular fractionation of GH4C1 cells revealed that PSP co-fractionates with chromogranin A but not with secreted alkaline phosphatase.

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

Chromogranin A (CgA) is a glycoprotein stored in secretory granules of many endocrine and neuroendocrine cells. CgA undergoes tissue specific processing to release regulatory peptides. In the parathyroid, although processing is limited and variable, several CgA-derived peptides have been characterized including parastatin and betagranin. An early stage of CgA processing is the generation of a 64-kDa fragment (CgA64). In this study, we have purified CgA64 from porcine parathyroid glands by chromatographic separations. Edman degradation of this CgA64 yielded the N-terminal sequence NDQAELKEGTEEASSKEAAEKRGDXAVEKND corresponding to pCgA(94-125). Amino acid composition suggests that CgA64 corresponds to CgA(94-430) (i.e. the entire CgA molecule, less the N-terminal residues 1-93). To determine the origin of CgA64, we fractionated parathyroid membrane vesicles by sucrose gradient centrifugation. Intact CgA is predominantly located in dense sucrose fractions (secretory granules), whereas CgA64 is located near the top of the gradient (soluble protein fraction). In vitro incubation of these fractions revealed that the conversion of CgA did not occur in intact granules. These results indicate that CgA64 is not present in intact granules suggesting that it is not a naturally occurring secretory product in parathyroid cells.

Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75–80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N-glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information.

Exocrine cells secrete granule proteins by regulated or constitutive-like secretory pathways. It is thought that all secretory proteins can enter immature secretory granules in exocrine cells. To test this hypothesis, we expressed the constitutive secretory protein secreted alkaline phosphatase (SEAP) in the exocrine cell line AR42J and compared its secretion to that of amylase, an endogenous regulated secretory protein. Secretion of SEAP and amylase were stimulated about 1.5-fold by substance P and 2-fold by barium chloride. In dexamethasone-treated cells, SEAP and amylase secretion were stimulated about 1.8-fold by substance P, 5-fold by barium chloride, and 4-fold by cholecystokinin-8. Cycloheximide reduced basal secretion of SEAP and amylase by 50%, increasing cholecystokinin-stimulated secretion to about 10-fold. Sodium butyrate induced expression of SEAP 2-fold but had no effect on stimulated secretion. These results suggest that SEAP is stored in secretory granules in AR42J cells.

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 coating's 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.

S.-U. Gorr and S. S. Pence . Pancreastatin Stimulates Secretion from Neonatal Rat Atrial Cells. Journal of Molecular and Cellular Cardiology (1995) 27, 767–771. Atrial myocardial cells store atrial natriuretic factor and secrete the peptide in response to extracellular stimulation, Ventricular myocardial cells also exhibit stimulated secretion of atrial natriuretic factor but with little or no intracellular storage. The stimulated secretion of other proteins from these cells is poorly characterized. In the present study the secretion of sulfated macromolecules from Na235SO4 labeled atrial and ventricular myocardial cells was quantitated. Both cell types secreted a chondroitin sulfate proteoglycan, as evidenced by glycanase digestion. Endothelin-1, a known stimulator of ANF secretion, stimulated secretion from atrial cells about 70% but had no effect on ventricular cell secretion. The differential stimulation of atrial and ventricular cell secretion was not due to the absence of a regulated secretory pathway in the latter cell type since phorbol ester stimulated secretion from both cell types. This result indicates that differences exist in the early steps of signal transduction between atrial and ventricular cells. Pancreastatin, a regulatory peptide derived from chromogranin A, stimulated atrial cell secretion 90%. This result suggests that chromogranin A, which has been identified in atrial secretory granules, may play an autoregulatory role in atrial secretion.

GL13NH2 is a bacteria-agglutinating peptide derived from the sequence of the salivary protein parotid secretory protein (PSP, BPIFA2, SPLUNC2, C20orf70). The peptide agglutinates both Gram negative and Gram positive bacteria, and shows anti-lipopolysaccharide activity in vitro and in vivo. However, GL13NH2 does not exhibit bactericidal activity. To generate a more cationic peptide with potential bactericidal activity, three amino acid residues were replaced with lysine residues to generate the peptide GL13K. In this report, the antibacterial and anti-inflammatory activities of GL13K were characterized. GL13K had lost the ability to agglutinate bacteria but gained bactericidal activity. Substitution of individual amino acids in GL13K with alanine did not restore bacterial agglutination. GL13K was bactericidal against Pseudomonas aeruginosa, Streptococcus gordonii and Escherichia coli but not Porphyromonas gingivalis. Unlike the agglutinating activity of GL13NH2, the bactericidal activity of GL13K against Pseudomonas aeruginosa was retained in the presence of saliva. Both GL13NH2 and GL13K exhibited anti-lipopolysaccharide activity. In GL13K, this activity appeared to depend on a serine hydroxyl group. GL13K protected mice from lipopolysaccharide- induced sepsis and the peptide exhibited a low level of hemolysis, suggesting that it may be suitable for in vivo application.

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.

PSP (parotid secretory protein)/SPLUNC2 (short palate, lung and nasal epithelium clone 2) is expressed in human salivary glands and saliva. The protein exists as an N-glycosylated and non-glycosylated form and both appear to induce agglutination of bacteria, a major antibacterial function for salivary proteins. Both forms of PSP/SPLUNC2 bind LPS (lipopolysaccharide), suggesting that the protein may also play an anti-inflammatory role. Based on the predicted structure of PSP/SPLUNC2 and the location of known antibacterial and anti-inflammatory peptides in BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), we designed GL13NH2 and GL13K, synthetic peptides that capture these proposed functions of PSP/SPLUNC2. GL13NH3 agglutinates bacteria, leading to increased clearance by macrophages and reduced spread of infection in a plant model. GL13K kills bacteria with a minimal inhibitory concentration of 5–10 μg/ml, kills bacteria in biofilm and retains activity in 150 mM NaCl and 50% saliva. Both peptides block endotoxin action, but only GL13K appears to bind endotoxin. The peptides do not cause haemolysis, haemagglutination in serum, inhibit mammalian cell proliferation or induce an inflammatory response in macrophages. These results suggest that the GL13NH2 and the modified peptide GL13K capture the biological activity of PSP/SPLUNC2 and can serve as lead compounds for the development of novel antimicrobial and anti-inflammatory peptides.

Parotid secretory protein (PSP) (C20orf70) is a salivary protein of unknown function. The protein belongs to the palate, lung, and nasal epithelium clone (PLUNC) family of mucosal secretory proteins that are predicted to be structurally similar to lipid-binding and host-defense proteins including bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein. However, the PLUNC proteins exhibit significant sequence variation and different biological functions have been proposed for different family members. This study tested the functional implications of the proposed similarity of PSP to the acute phase protein lipopolysaccharide-binding protein (LBP). PSP was identified in human saliva and was soluble in 70% ethanol, as shown for other PLUNC proteins. PSP binds lipopolysaccharide and can be eluted by non-ionic detergent, but not by urea or high salt. A synthetic PSP peptide, GL13NH2, which corresponds to a lipopolysaccharide-inhibiting peptide from LBP, inhibited the binding of lipopolysaccharide to both PSP and lipopolysaccharide-binding protein. Peptides from other regions of PSP and the control peptide polymyxin B showed no effect on the binding of PSP to lipopolysaccharide. GL13NH2 also inhibited lipopolysaccharide-stimulated secretion of tumor necrosis factor from macrophages. The other PSP peptides had no effect in this assay. PSP peptides had no or only minor effect on macrophage cell viability. These results indicate that PSP is a lipopolysaccharide-binding protein that is functionally related to LBP, as suggested by their predicted structural similarities.

Summary Chromogranin A (CgA, Secretory Protein-I) is a protein of about 450 amino acids representing a major soluble component of the secretory granules of parathyroid and other endocrine and neuroendocrine cells. In the parathyroid, CgA is costored and cosecreted with parathormone (PTH). We earlier found that CgA and the derived peptide, pancreastatin, inhibited secretion of PTH and CgA by parathyroid cells in culture and that CgA antiserum stimulated secretion above the maximum achieved at low (0.5 mM) Ca2+. In the present study, porcine parathyroid cells were incubated at different cell concentrations at low Ca2+. The amount of secreted CgA increased over the 6-h incubation period at 1 × 106 to 4 × 106 cells/ml, but plateaued after 3 h at 6 × 106 cells/ml. Secretion did not plateau when antiserum to CgA and pancreastatin were present at the start of incubation or resumed when the antisera were added at 3 h. Conditioned medium contained a factor or factors that blocked secretion by fresh parathyroid cells at 0.5 mM Ca2+. Pulse-chase studies revealed that 40% of the secreted CgA was processed after 6 h of chase, α-2-macroglobulin, an inhibitor of proteolytic processing, increased the amount of CgA in the medium by 30% at 1 h of chase and decreased the amount processed to 20% by 6 h. Other protease inhibitors similarly enhanced the amount of CgA in the medium. These data indicate that proteolytic processing of intact CgA is requisite for its autocrine inhibitory activity.

Chromogranin A (secretory protein-I) is an acidic sulfated glycoprotein found in secretory granules of most endocrine and neuroendocrine cells. In the parathyroid it is co-stored and secreted with parathormone in response to hypocalcemia. Differences in post-translational modifications have been reported between chromogranin A from the bovine adrenal and porcine parathyroid glands. The former has been reported to be sulfated mainly on oligosaccharide residues and apparently includes a proteoglycan form, whereas the latter was previously reported to be tyrosine sulfated with little of the proteoglycan form present. Here we have directly compared 35SO4-labeled parathyroid chromogranin A from the pig and the cow to determine if these reported differences were tissue or species specific. We find that the chromogranin A secreted by the bovine gland contains a proteoglycan form, whereas that from the porcine gland does not. Moreover, chromogranin A of both species is primarily sulfated on oligosaccharide residues with little if any tyrosine sulfate detected. Differences were detected in the structure of sulfated O-linked oligosaccharides in bovine and porcine parathyroid chromogranin A.

Chromogranin-A, also referred to as secretory protein-I, is a 50-kDa protein present in and secreted by most endocrine cells together with the native hormone. Porcine chro- mogranin-A contains a sequence identical to pancreastatin, sug- gesting that it is the precursor of pancreastatin. Pancreastatin is a potent inhibitor of parathyroid gland secretion, and it and chromogranin-A inhibit glucose-stimulated insulin release by the pancreas. It is possible that chromogranin-A, pancreastatin, or a related peptide is a physiological inhibitor of secretion by the parathyroid as well as other endocrine glands. As a test of this hypothesis, parathyroid cells in culture were incubated with purified porcine chromogranin-A or antisera to chromogranin- A and pancreastatin. In the absence of exogenous chromogranin- A or antisera, secretion of chromogranin-A and PTH at 0.5 mM Ca 2+ was about twice that at 3.0 mM Ca 2+ . When intact chro- mogranin-A was added to the incubation medium at 0.5 mM Ca 2+ , secretion was reduced to the basal level obtained at 3.0 mM Ca 2+ . Chromogranin-A did not affect the secretion of cells incubated at 3.0 mM Ca 2+ . At 1 h of incubation, 100 nM chro- mogranin-A was equivalent in potency to 1 nM pancreastatin, but after 3 h the two agents were equipotent. This suggests that chromogranin-A was processed into biologically active peptide(s) during incubation. Antisera directed against chromogranin-A or pancreastatin potentiated the secretion of both chromogranin- A and PTH at 0.5 mM, but not 3.0 mM, Ca 2+ . This stimulatory action of the antisera was dose dependent from 1:3200 to 1:400 final dilution, was effective within 2 h, and did not shift the Ca 2+ set-point for glandular secretion. These results are consonant with chromogranin-A-derived peptides serving as an autocrine inhibitor of parathyroid gland secretion. (Endocrinology 127: 1329-1335, 1990)

Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and [3H]Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.

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.

Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation.

Infection is one of the most prevalent causes for dental implant failure. We have developed a novel antimicrobial peptide coating on titanium by immobilizing the antimicrobial peptide GL13K. GL13K was developed from the human salivary protein BPIFA2. The peptide exhibited MIC of 8 µg/ml against planktonic Pseudonomas aeruginosa and their biofilms were reduced by three orders of magnitude with 100 µg/ml GL13K. This peptide concentration also killed 100% of Streptococcus gordonii. At 1 mg/ml, GL13K caused less than 10% lysis of human red blood cells, suggesting low toxicity to mammalian cells. Our GL13K coating has also previously showed bactericidal effect and inhibition of biofilm growth against peri-implantitis related pathogens, such as Porphyromonas gingivalis. The GL13K coating was cytocompatible with human fibroblasts and osteoblasts. However, the bioactivity of antimicrobial coatings has been commonly tested under (quasi)static culture conditions that are far from simulating conditions for biofilm formation and growth in the oral cavity. Oral salivary flow over a coating is persistent, applies continuous shear forces, and supplies sustained nutrition to bacteria. This accelerates bacteria metabolism and biofilm growth. In this work, the antimicrobial effect of the coating was tested against Streptococcus gordonii, a primary colonizer that provides attachment for the biofilm accretion by P. gingivalis, using a drip-flow biofilm bioreactor with media flow rates simulating salivary flow. The GL13K peptide coatings killed bacteria and prevented formation and growth of S. gordonii biofilms in the drip-flow bioreactor and under regular mild-agitation conditions. Surprisingly the interaction of the bacteria with the GL13K peptide coatings ruptured the cell wall at their septum or polar areas leaving empty shell-like structures or exposed protoplasts. The cell wall rupture was not detected under regular culture conditions, suggesting that cell wall rupture induced by GL13K peptides also requires media flow and possible attendant biological sequelae of the conditions in the bioreactor. info:eu-repo/semantics/publishedVersion

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.

Aims/hypothesis. Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. Methods. SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. Results. Secreted alkaline phosphatase showed high basal secretion (6.6 % total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8 % total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. Conclusion/interpretation. These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both. [Diabetologia (2000) 43: 1157–1164]

Chromogranins are a family of regulated secretory proteins that are stored in secretory granules in endocrine and neuroendocrine cells and released in response to extracellular stimulation (regulated secretion). A conserved N-terminal disulfide bond is necessary for sorting of chromogranins in neuroendocrine PC12 cells. Surprisingly, this disulfide bond is not necessary for sorting of chromogranins in endocrine GH4C1 cells. To investigate the sorting mechanism in GH4C1 cells, we made several mutant forms removing highly conserved N- and C-terminal regions of bovine chromogranin A. Removing the conserved N-terminal disulfide bond and the conserved C-terminal dimerization and tetramerization domain did not affect the sorting of chromogranin A to the regulated secretory pathway. In contrast, removing the C-terminal 90 amino acids of chromogranin A caused rerouting to the constitutive secretory pathway and impaired aggregation properties as compared with wild-type chromogranin A. Since this mutant was sorted to the regulated secretory pathway in PC12 cells, these results demonstrate that chromogranins contain independent N- and C-terminal sorting domains that function in a cell type-specific manner. Moreover, this is the first evidence that low pH/calcium-induced aggregation is necessary for sorting of a chromogranin to the regulated secretory pathway of endocrine cells.

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.

The rat pituitary cell line GH4C1 secretes granins (chromogranin B and secretogranin II) and prolactin by the regulated secretory pathway. The intracellular storage of prolactin is preferentially induced by hormone treatment with estradiol, insulin, and epidermal growth factor. The goal of this study was to determine the effect of hormone treatment on storage of granins and constitutive secretory markers. The granins were efficiently stored in both hormone-treated and -untreated cells (17% of total secreted in 4 h). Secreted alkaline phosphatase (SEAP), a truncated membrane protein that would not be expected to enter secretory granules, and glycosaminoglycan, a marker for the constitutive secretory pathway, exhibited 70 80% secretion under both conditions. In comparison, the relative prolactin secretion was 31 and 68% from hormone-treated and -untreated cells, respectively. Phorbol ester and KCl stimulated prolactin secretion 2.3-fold from untreated cells and 5. 5-fold from hormone-treated cells. In contrast, SEAP secretion was stimulated 1.5-fold from both treated and untreated cells, consistent with secretion by the constitutive secretory pathway. Stimulated secretion of granins was detected from both hormone-treated and -untreated cells. These results suggest that granin and prolactin storage are differentially regulated and that the constitutive secretory pathway is not affected by hormone treatment.

Sorting between the regulated and the constitutive secretory pathway in exocrine cells is thought to involve aggregation of regulated secretory proteins. This study demonstrates that, unlike endocrine secretory proteins, exocrine secretory proteins, including amylase, do not undergo homotypic aggregation under the conditions found in the sorting organelles. Also, unlike other exocrine proteins, amylase does not aggregate with chondroitin sulfate. Since amylase exhibits heterotypic aggregation, the role of protein concentration in amylase sorting was tested in AR42J cells. Secretion was stimulated with substance P and cholecystokinin from both untreated and dexamethasone-treated cells, with more efficient stimulation from dexamethasone-treated cells. These results indicate that amylase sorting is enhanced when its expression is stimulated by dexamethasone treatment.

Dental plaque, a microbial biofilm that accumulates on teeth and initiates periodontal disease, is composed of hundreds of different bacterial species within an organized structure. The biofilm bacteria and their byproducts irritate the gingival epithelium and induce an "inflammatory response". The perturbation of epithelial cells by bacteria is the first stage in the initiation of inflammatory and immune processes which eventually cause destruction of the tissues surrounding and supporting the teeth, and ultimately result in tooth loss. This review addresses the early bacterial-epithelial cell interactions and the subsequent responses of the epithelial cell. It includes discussion of how epithelial Toll-like receptors (TLRs) respond to different bacterial challenges, the variable antimicrobial peptides released and the host signaling responses which trigger release of these molecules and the overall fate of these cells in terms of survival, apoptosis, or cell lysis.

Chromogranin-A, also referred to as secretory protein-I, is a 50K protein found in and secreted by endocrine cells, in which it is costored with the native hormone. Porcine chromogranin-A contains a sequence identical to pancreastatin, a 49-amino acid, C-terminally amidated peptide that has been isolated from porcine pancreas, suggesting that chromogranin-A is the precursor of pancreastatin. Pancreastatin has been found to be a potent inhibitor of glucose-stimulated insulin release. As it is possible that pancreastatin inhibits secretion from other chromogranin-A-containing tissues in which it may be formed, we tested its action on dispersed porcine parathyroid cells in culture. Secretion of chromogranin-A and PTH was up to 6-fold greater at 0.5 mM Ca2+ than at 3.0 mM Ca2+. Pancreastatin (1 nM) reduced the secretion of both chromogranin-A and PTH at 0.5 mM Ca2+ to approximately the levels found at 3.0 mM Ca2+, but did not affect secretion at 3.0 mM Ca2+. Pancreastatin (0.01-1.0 nM) inhibited secretion of chromogranin-A in a dose-dependent fashion. Preincubation of the cells with pancreastatin was not required for inhibition. Transfer of inhibited cells to medium without pancreastatin led to restoration of secretion within 90 min. Phorbol myristate acetate (1.6 microM) stimulated secretion of PTH and chromogranin-A at 3.0 mM Ca2+, but not at 0.5 mM Ca2+. Pancreastatin reversed this stimulation, demonstrating that its inhibition was independent of Ca2+ concentration. These results are consonant with pancreastatin playing a physiological role in modulation of secretion by the parathyroid and, by extension, other endocrine tissues.

Endocrine and exocrine cells each contain a regulated and constitutive secretory pathway. The presence of two distinct secretory pathways in the same cell type requires a sorting step to direct secretory proteins to the correct pathway. It is thought that regulated secretory proteins contain a specific sorting signal. However, this signal has not been identified. Amino acid sequence comparisons have not revealed any significant similarity between different regulated secretory proteins, suggesting that the sorting signal does not consist of a conserved primary sequence. In the present report, we have analyzed the predicted secondary structures of regulated secretory proteins and identified an N-terminal hydrophobic peak (NHP) which is located approximately from amino acids 9–26, overlaps with a predicted α-helix and contains charged amino acid residues. This signal is present in regulated secretory proteins that exhibit an N-terminal sorting sequence, but it is absent from constitutively secreted proteins and proteins where the sorting sequence is not located near the N-terminus. It appears that the NHP is both necessary and sufficient for sorting of many secretory proteins to the regulated secretory pathway.