J Periodontal Implant Sci.  2016 Oct;46(5):320-328. 10.5051/jpis.2016.46.5.320.

Levels of common salivary protein 1 in healthy subjects and periodontal patients

• Heo SM
• Lee S
• Wang H
• Jeong JH
• Oh SW

• KMID: 2354930
• DOI: http://doi.org/10.5051/jpis.2016.46.5.320

Abstract

PURPOSE
Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients.
METHODS
This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student's t-test.
RESULTS
Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434-10,139 ng/mL) and 8,598 ng/mL (range, 7,421-9,877 ng/mL), respectively. The Student's t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024).
CONCLUSIONS
The presence of a significant difference in CSP1 levels between healthy subjects and periodontal patients suggests that CSP1 may be a potential biomarker for the detection or screening of periodontitis patients.

Keyword

Common salivary protein 1; Enzyme-linked immunosorbent assay; Monoclonal antibody; Periodontitis; Saliva

MeSH Terms

Bacteria
Chronic Periodontitis
Electrophoresis
Enzyme-Linked Immunosorbent Assay
Ethics Committees, Research
Healthy Volunteers*
Humans
Jeollabuk-do
Mass Screening
Molecular Weight
Peptides
Periodontitis
Saliva
Sodium
Peptides
Sodium

Figure

• 

  Figure 1 Flow diagram of the study. This controlled study was conducted in periodontally healthy subjects and in patients with chronic periodontitis. All participants between 20 and 70 years of age were asked to take part in the study, agreed, and signed a written consent form. After providing informed consent, patients received a periodontal examination. The periodontal examinations were used to determine whether each patient belonged to the healthy group or to the group of patients with periodontitis. CSP1, common salivary protein 1; ELISA, enzyme-linked immunosorbent assay; n, number.

• 

  Figure 2 Detection of CSP1 in human saliva with a mAb. CB-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates molecular size markers (lane M), purified GST-tagged recombinant hCSP1 as a positive control (lane 1), and human saliva (lane 2). The WB is an immunoblot analysis of the CB probed with primary mAb-hCSP1#4. mAb recognized a single protein band at approximately 27-kDa in the blot. CSP1, common salivary protein 1; mAb, monoclonal antibody; CB, Coomassie blue; GST, glutathione S-transferase; WB, western blot.

• 

  Figure 3 Profiles of human saliva CSP1 concentrations in the healthy group and periodontal group determined using an enzyme-linked immunosorbent assay system. The profiles showed that a greater amount of CSP1 was present in the saliva of the periodontal patients than in the saliva of healthy individuals. The median values (25th to 75th percentiles) of the healthy subjects and periodontal patients were 8,595 ng/mL (range, 7,421–9,877 ng/mL) and 9,474 ng/mL (range, 8,434–10,139 ng/mL), respectively. The Student’s t-test results indicated that a statistically significant difference was present between the 2 groups (P=0.024). The central box and the middle line of each graph represent the values from the lower to upper quartiles (25th to 75th percentiles) and median, respectively. The horizontal line extends from the minimum to the maximum values, excluding outlier values that are displayed as separate points. Outlier values were defined as those smaller than the 10th percentile or larger than the 90th percentile. CSP1, common salivary protein 1.

Reference

1. Savage A, Eaton KA, Moles DR, Needleman I. A systematic review of definitions of periodontitis and methods that have been used to identify this disease. J Clin Periodontol. 2009; 36:458–467.

2. Baser U, Oztekin G, Ademoglu E, Isik G, Yalcin F. Is the severity of periodontitis related to gingival crevicular fluid and serum high-sensitivity C-reactive protein concentrations? Clin Lab. 2014; 60:1653–1658.

3. Wilmarth PA, Riviere MA, Rustvold DL, Lauten JD, Madden TE, David LL. Two-dimensional liquid chromatography study of the human whole saliva proteome. J Proteome Res. 2004; 3:1017–1023.

4. Yao Y, Berg EA, Costello CE, Troxler RF, Oppenheim FG. Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches. J Biol Chem. 2003; 278:5300–5308.

5. Mullins JJ, Mullins LJ, Dunbar DR, Brammar WJ, Gross KW, Morley SD. Identification of a human ortholog of the mouse Dcpp gene locus, encoding a novel member of the CSP-1/Dcpp salivary protein family. Physiol Genomics. 2006; 28:129–140.

6. Girard LR, Castle AM, Hand AR, Castle JD, Mirels L. Characterization of common salivary protein 1, a product of rat submandibular, sublingual, and parotid glands. J Biol Chem. 1993; 268:26592–26601.

7. Kim SA, Lee Y, Jung DE, Park KH, Park JY, Gang J, et al. Pancreatic adenocarcinoma up-regulated factor (PAUF), a novel up-regulated secretory protein in pancreatic ductal adenocarcinoma. Cancer Sci. 2009; 100:828–836.

8. D’Aiuto F, Parkar M, Andreou G, Suvan J, Brett PM, Ready D, et al. Periodontitis and systemic inflammation: control of the local infection is associated with a reduction in serum inflammatory markers. J Dent Res. 2004; 83:156–160.

9. Mealey BL, Oates TW; American Academy of Periodontology. Diabetes mellitus and periodontal diseases. J Periodontol. 2006; 77:1289–1303.

10. Wu YY, Xiao E, Graves DT. Diabetes mellitus related bone metabolism and periodontal disease. Int J Oral Sci. 2015; 7:63–72.

11. Ambatipudi KS, Hagen FK, Delahunty CM, Han X, Shafi R, Hryhorenko J, et al. Human common salivary protein 1 (CSP-1) promotes binding of Streptococcus mutans to experimental salivary pellicle and glucans formed on hydroxyapatite surface. J Proteome Res. 2010; 9:6605–6614.

12. Koo H, Vacca Smith AM, Bowen WH, Rosalen PL, Cury JA, Park YK. Effects of Apis mellifera propolis on the activities of streptococcal glucosyltransferases in solution and adsorbed onto saliva-coated hydroxyapatite. Caries Res. 2000; 34:418–426.

13. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol. 1999; 4:1–6.

14. Wang H, Heo SM, Jin HY, Choi EY, Oh SW. Common salivary protein 1 in serum of diabetes patients. J Clin Lab Anal. Forthcoming. 2016.

15. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979; 76:4350–4354.

16. Raval S, Gowda SB, Singh DD, Chandra NR. A database analysis of jacalin-like lectins: sequence-structure-function relationships. Glycobiology. 2004; 14:1247–1263.

17. Marsh PD. Host defense and microbial homeostasis: role of microbial interactions. J Dent Res. 1989; 68:1567–1575.

18. Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009; 73:407–450.

19. Kaufman E, Lamster IB. The diagnostic applications of saliva--a review. Crit Rev Oral Biol Med. 2002; 13:197–212.

20. Hansen TV, Simonsen MK, Nielsen FC, Hundrup YA. Collection of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort: comparison of the response rate and quality of genomic DNA. Cancer Epidemiol Biomarkers Prev. 2007; 16:2072–2076.