Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain.

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.

Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal/permeability increasing-like protein family.

Parotid secretory protein (PSP) (SPLUNC2), a potential host-defense protein related to bactericidal/permeability-increasing protein (BPI), was used as a template to design antibacterial peptides. Based on the structure of BPI, new PSP peptides were designed and tested for antibacterial activity. The peptides did not exhibit significant bactericidal activity or inhibit growth but the peptide GL-13 induced bacterial matting, suggesting passive agglutination of bacteria. GL-13 was shown to agglutinate the Gram negative bacteria Pseudomonas aeruginosa and Aggregatibacter (Actinobacillus) actinomycetemcomitans, Gram positive Streptococcus gordonii and uncoated sheep erythrocytes. Bacterial agglutination was time and dose-dependent and involved hydrophobic interactions. Variant forms of GL-13 revealed that agglutination also depended on the number of amine groups on the peptide. GL-13 inhibited the adhesion of bacteria to plastic surfaces and the peptide prevented the spread of P. aeruginosa infection in a lettuce leaf model, suggesting that GL-13 is active in vivo. Moreover, GL-13-induced agglutination enhanced the phagocytosis of P. aeruginosa by RAW 264.7 macrophage cells. These results suggest that GL-13 represents a class of antimicrobial peptides, which do not directly kill bacteria but instead reduce bacterial adhesion and promote agglutination, leading to increased clearance by host phagocytic cells. Such peptides may cause less bacterial resistance than traditional antibiotic peptides.