Proteins in whole saliva during the first year of infancy.

During the first year of an infant's life, the oral environment is subject to drastic changes that coincide with the eruption of teeth. Proteins in saliva are important for protecting oral surfaces and provide receptors for bacterial adhesins. The objective of this longitudinal study was to monitor the general composition and expression of proteins in whole saliva of infants, to prove the hypothesis that expression of certain proteins changes during infant development, and might be associated with tooth eruption. The results showed a remarkable constancy in the overall pattern of salivary proteins and glycoproteins during infancy. Exceptions were the mucins and albumin. The mucins are expressed differentially, with first MUC7 and later MUC5B being predominant. Albumin, a marker of serum leakage, started to rise in whole saliva preceding tooth eruption. Thus, the expression of only few proteins appears to be changed during infant development.

The effects of duration and intensity of stimulation on total protein and mucin concentrations in resting and stimulated whole saliva.

The present investigation has characterized the influence of the duration and intensity of stimulation on the secretion pattern of total protein and salivary mucins MG1 and MG2 in whole saliva. Resting and stimulated whole saliva was collected from six healthy subjects on 2 consecutive days. Whole saliva was collected for 2 five-minute intervals under resting conditions followed by collection under masticatory stimulation induced by the chewing of parafilm (1 g) at 10 or 60 strokes/min for 15 min. Flow rates were different under the 2 levels of stimulation. The concentration of total protein was different in resting and stimulated whole saliva but was not affected by the duration or intensity of stimulation. Analysis of mucin concentrations determined by capture ELISAs revealed that the pattern of MG1 secretion was similar to that of total protein. The pattern of MG2 secretion was unique in that no differences were observed in the concentration of this mucin under resting and stimulated conditions. This study shows that the pattern of protein secretion in whole saliva does not reflect the combined pattern observed for protein secretion in parotid and submandibular/sublingual glands, and that the secretion patterns of MG1 and MG2 in whole saliva are quite different from one another.

Salivary mucin: a factor in the lower prevalence of gastroesophageal reflux disease in African-Americans?

OBJECTIVES: Organic and inorganic constituents of saliva have been implicated as protective components in the esophagus, and deficiencies in one or more of these factors in different races may be an important element in the prevalence of gastroesophageal reflux disease (GERD). To determine whether there are differences in the concentration of salivary mucins between different racial groups, we measured the concentration of mucous glycoprotein MG1 and mucous glycoprotein MG2 in whole saliva of African-Americans and Caucasians. METHODS: Whole saliva was collected from 19 African-American (four male, 15 female; mean age 34 yr, range 19-53 yr) and 25 Caucasian (11 male, 14 female; mean age 31 yr, range 20-51 yr) volunteers under masticatory stimulation (1 g Parafilm, 60 strokes/min) between 11:00 AM and 12:00 noon. Total salivary carbohydrate was measured with a periodic acid-Schiff assay and total protein by absorbance at 215 nm. Immunological reagents were employed to quantify MG2 in a combined enzyme-linked immunosorbent assay/enzyme linked lectin assay (ELISA/ELLA) and to quantify MG1 in a capture ELISA. RESULTS: The total carbohydrate, protein, MG1 and MG2 values were 24.4 +/- 11.9, 243.5 +/- 62.7, 21.8 +/- 13.4, and 11.6 +/- 9.5 mg% for African-Americans, and the corresponding values were 23.3 +/- 9.3, 221.7 +/- 39.7, 25.7 +/- 16.2, and 10.9 +/- 8.7 mg% for Caucasians. There was no statistical difference for any of the parameters measured between the two groups. Furthermore, it was shown that no correlation existed between salivary flow rate and the concentration of carbohydrate, protein, or salivary mucins in African-Americans and in Caucasians. These results show that flow rate did not influence the measured values for salivary parameters in the two groups. CONCLUSIONS: No differences were found in the concentration of salivary mucins MG1 and MG2 in whole saliva of African-Americans and Caucasians, and it seems unlikely that variations in mucin levels influence the prevalence of GERD in these groups.

Immunoquantification of human salivary mucins MG1 and MG2 in stimulated whole saliva: factors influencing mucin levels.

While more and more is known about the structure and function of human salivary mucins, there is relatively little information on quantification of these glycoconjugates in whole saliva and on factors influencing their secretion. The goal of the present work was to develop capture ELISAs that would allow for rapid, inexpensive, and reliable measurement of the salivary mucins MG1 and MG2, and to use these immunological procedures to investigate the significance of age, gender, flow rate, and protein concentration on mucin levels in whole saliva. Previously, we described a rabbit polyclonal antibody against MG1 (Troxler et al., 1995) and a rabbit polyclonal peptide antibody against an epitope in the N-terminal region of MG2 (Liu et al., 1999) which were used to develop the capture ELISAs. We verified the accuracy and specificity of these assays by showing correct measurement of known quantities of purified MG1 or MG2 added to whole saliva and lack of cross-reactivity between mucins and heterologous antisera on Western blots or in ELISAs. Whole saliva was collected from 60 subjects under conditions of masticatory stimulation, flow rates were recorded, and mucin concentrations were determined. The results showed that the mean concentration of MG1 and MG2 was 23.3 +/- 14.6 mg% and 13.3 +/- 11.6 mg%, respectively, and that mucins constitute approximately 16% of the total protein in whole saliva. No significant correlations were found between mucin levels and age or flow rate; however, a significant correlation was found between MG2 levels and total protein concentration. Furthermore, there were statistically significant gender differences in flow rate and MG1 levels, but not in MG2 levels. The availability of these immunoassays for quantification of MG1 and MG2 will help to elucidate the role of mucin in oral health and disease.

The recombinant N-terminal region of human salivary mucin MG2 (MUC7) contains a binding domain for oral Streptococci and exhibits candidacidal activity.

MG2 (the MUC7 gene product) is a low-molecular-mass mucin found in human submandibular/sublingual secretions. This mucin is believed to agglutinate a variety of microbes and thus is considered an important component of the non-immune host defence system in the oral cavity. We have shown that MUC7 can bind to cariogenic strains of Streptococcus mutans and that this binding requires a structural determinant in the N-terminal region. In the present study an expression construct, pNMuc7, encoding the N-terminal 144 amino acids of MUC7 was generated, and the recombinant protein rNMUC7 was expressed in Escherichia coli. Purified rNMUC7 was characterized and the binding of this protein to oral bacteria was investigated in an established assay. The results showed that the recombinant protein bound to S. mutans ATCC 25175 and ATCC 33402, and that alkylation of the two cysteine residues (Cys(45) and Cys(50)) resulted in the complete loss of bacterial binding. This suggests that binding of MUC7 to S. mutans occurs between the N-terminal region of the mucin molecule and the bacterial surface, and that this interaction is dependent on a cysteine-containing domain within this region of MUC7. In addition, the killing activity of rNMUC7 was compared with that of the candidacidal salivary protein histatin 5 in an established Candida albicans (ATCC 44505) blastoconidia killing assay. It was found that the LD(50) values of rNMUC7 and histatin 5 were comparable, and that the recombinant protein displayed significant killing activity at the physiological concentration range of MUC7 in whole saliva. This study is the first to show that the N-terminal region of MUC7 contains a structural determinant for bacterial binding and that this region exhibits candidacidal activity.