Interactions between hyaluronic acid, lysozyme, and the glucose oxidase-mediated lactoperoxidase system in enzymatic and candidacidal activities
Introduction
Hyaluronic acid (HA) is present in human saliva and may contribute to the lubricating and healing properties of saliva, thereby assisting in protecting the oral mucosa.1, 2 HA also has anti-Candida activity.3 Due to its viscoelastic properties and non-immunogeneity,4 HA can be a candidate substance for effective saliva substitutes for patients with dry mouth as a solution containing HA has rheological properties which might mimic the viscosity of human saliva at shear rates for routine oral functions.5 A relationship between decreased levels of salivary HA and dry mouth symptoms has also been reported,6 suggesting that HA is important in protecting and lubricating the oral mucosa. In addition, the wound repair and potential anti-Candida activities of HA3, 7 can provide additional benefits to patients with dry mouth, who are susceptible to oral mucosal injuries and candidiasis.8
The most widely used antimicrobial host proteins in oral health care products are lysozyme and the peroxidase system.9 One lysozyme supplement in these products is hen egg white lysozyme (HEWL). The bactericidal activity of lysozyme is muramidase-dependent and uses cation-dependent or structure-related mechanisms.10, 11, 12 Its antifungal activity is also well known.13, 14, 15 The peroxidase system supplements are the glucose oxidase-mediated lactoperoxidase (GO-LPO) system, which comprises bovine lactoperoxidase (bLPO), glucose oxidase (GO), and SCN−. The GO-LPO system interacts with peroxidases (salivary peroxidase and myeloperoxidase), SCN−, H2O2, and glucose in whole saliva. GO degrades glucose and produces H2O2, which serves as a substrate for bLPO and peroxidase in saliva to form antimicrobial OSCN− from SCN−.16 Furthermore, by using glucose, GO can hamper the carbohydrate metabolism of glycolytic cariogenic bacteria.16, 17, 18 In addition, the GO-LPO system has antifungal activities.19
Many antimicrobial proteins in saliva interact with each other in vitro. These interactions can result in additive, synergistic, or inhibitory effects on mutans streptococci, lactobacilli, or fungi.20, 21, 22 For example, interactions have been reported between sIgA and peroxidase,23 lactoferrin and peroxidase,24, 25 lactoferrin and lysozyme,26 lysozyme and histatins,27 and lysozyme and the peroxidase system.15, 28
Although these observations are in vitro, such concerted effects likely exist in mixed saliva or oral health care products, where all the components are present simultaneously. Therefore, HA molecules may also interact with antimicrobial molecules in human saliva or saliva substitutes. HA and peroxidase have been suggested to form complex molecules,29 and HA affects the enzymatic and candidacidal activities of lysozyme and the peroxidase system.5, 30 However, there is no information as to how HA affects the enzymatic and anticandidal activities of the GO-LPO system or how lysozyme and the GO-LPO system affect each other. In this study, we investigated the influences of HA on the enzymatic and anticandidal activities of the GO-LPO system and the interactions between lysozyme and the GO-LPO system.
Section snippets
HA, lysozyme, and glucose oxidase-mediated lactoperoxidase system
HA (1630 kDa, Sigma–Aldrich Chemical Co., St Louis, MO, USA) was used at three concentrations: 0.5, 1.0, and 2.0 mg/mL. HEWL (final concentration of 30 μg/mL) (Sigma–Aldrich) served as the lysozyme source. The GO-LPO system included bLPO (final concentration of 25 μg/mL), potassium thiocyanate (KSCN, final concentration of 1 mM), GO (final concentration of 10 units/mL), and glucose (final concentration of 30 μg/mL) (Sigma–Aldrich). All components were solubilized with simulated salivary buffer (SSB,
Influence of HA and lysozyme on the enzymatic activity of the GO-LPO system
HA inhibited the enzymatic activity of the GO-LPO system in an HA or glucose concentration-dependent manner (Fig. 1). HEWL did not affect the enzymatic activity of the GO-LPO system. The mean (±SD) differences in OD between with HEWL and without HEWL was −0.86 ± 3.60% at 20 μg/mL glucose, 0.63 ± 2.60% at 40 μg/mL glucose, and −0.18 ± 2.17% at 60 μg/mL glucose.
Influence of the GO-LPO system on the enzymatic activity of lysozyme
GO, KSCN, and glucose did not affect the enzymatic activity of HEWL. bLPO alone and mixtures containing bLPO significantly enhanced the enzymatic
Discussion
Developing effective saliva substitutes requires understanding and mimicking both the rheological and biological properties of human saliva.32, 33 Therefore, a practical method to develop effective salivary substitutes for xerostomic patients is to identify or develop substances with viscoelastic patterns similar to human whole saliva and supplement them with antimicrobials that restore the diminished biochemical properties. The best salivary substitute in a viscoelastic aspect is one with
Funding
This work was supported by the National Research Foundation of Korea Grant through the Oromaxillofacial Dysfunction Research Center for the Elderly (No. 2012-000911) at Seoul National University in Korea.
Competing interests
None declared.
Ethical approval
Not required.
Acknowledgements
This work was supported by the National Research Foundation of Korea Grant through the Oromaxillofacial Dysfunction Research Center for the Elderly (No. 2012-000911) at Seoul National University in Korea.
References (42)
- et al.
Hyaluronan (hyaluronic acid) in human saliva
Archives of Oral Biology
(1996) - et al.
Rheological properties of hyaluronic acid and its effects on salivary enzymes and candida
Oral Diseases
(2010) - et al.
An update of the etiology and management of xerostomia
Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics
(2004) - et al.
Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function
FEBS Letters
(2001) - et al.
Origin, structure, and biological activities of peroxidases in human saliva
Archives of Biochemistry and Biophysics
(2006) - et al.
The effects of peroxidase on the enzymatic and candidacidal activities of lysozyme
Archives of Oral Biology
(2010) - et al.
Efficacy of enzymatic mouth rinses for immobilisation of protective enzymes in the in situ pellicle
Archives of Oral Biology
(2010) - et al.
Efficacy of enzymatic toothpastes for immobilisation of protective enzymes in the in situ pellicle
Archives of Oral Biology
(2010) - et al.
Candidacidal activities of the glucose oxidase-mediated lactoperoxidase system
Archives of Oral Biology
(2012) - et al.
Hyaluronan (hyaluronic acid) and its regulation in human saliva by hyaluronidase and its inhibitors
Journal of Oral Science
(2003)
Potential role of high molecular weight hyaluronan in the anti-Candida activity of human oral epithelial cells
Medical Mycology
Hyaluronan
Cellular and Molecular Life Sciences
Salivary levels of hyaluronic acid in female patients with dry mouth compared with age-matched controls: a pilot study
Biomedical Research
Functions of hyaluronan in wound repair
Wound Repair and Regeneration
Clinical applications of antimicrobial host proteins, lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety
Oral Diseases
Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides
Infection and Immunity
In vitro susceptibility of Candida species to lysozyme
Oral Microbiology and Immunology
Inhibition of growth and secreted aspartyl proteinase production in Candida albicans by lysozyme
Journal of Medical Microbiology
Enzymes in the acquired enamel pellicle
European Journal of Oral Sciences
Salivary lysozyme, lactoferrin and peroxidases: antibacterial effects on cariogenic bacteria and clinical applications in preventive dentistry
Proceedings of the Finnish Dental Society
Antimicrobial function of human saliva—how important is it for oral health?
Acta Odontologica Scandinavica
Cited by (4)
The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities
2015, Archives of Oral BiologyCitation Excerpt :Accordingly, the interactions on surfaces may behave in a distinct manner from those in solution. There have also been reports of interactions between antimicrobial supplements and candidate substances of artificial saliva such as animal mucins,12,13 hyaluronic acid,14–16 and yam tuber mucilage.17,18 The results of interactions could be additive, synergistic, or inhibitory, and surface interactions may differ from those in solution.
Mouthwashes in the 21<sup>st</sup> century: a narrative review about active molecules and effectiveness on the periodontal outcomes
2017, Expert Opinion on Drug DeliveryDose-dependent effect of lysozyme upon Candida albicans biofilm
2017, Molecular Medicine Reports