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Proteins in Whole Saliva during the First Year of Infancy
S. Ruhl1,*,
S.A. Rayment2,
G. Schmalz1,
K.-A. Hiller1 and
R.F. Troxler2
1 Department of Operative Dentistry and Periodontology, Dental School, University of Regensburg, D-93042 Regensburg, Germany; and
2 Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA;
Correspondence: * corresponding author, stefan.ruhl{at}klinik.uni-regensburg.de
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ABSTRACT
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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.
Key Words: saliva • proteins • mucins • infants • development
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INTRODUCTION
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During the first year of life, the infant undergoes a rapid development that is accompanied by drastic changes within the oral environment. Some of these changes may be related to the emergence of the first teeth during this time period. The presence of teeth facilitates a shift in nutrition from fluid to solid food, and, concomitantly, the oral microbial community changes, because the teeth provide new ecological niches for bacterial colonization (Gibbons, 1989; Wan et al., 2003; Haraldsson et al., 2004). Since salivary proteins play important roles in the mastication and digestion of food, the maintenance of tooth integrity, and adhesion of oral bacteria (Mandel, 1987; Scannapieco, 1994; Tabak, 1995), it is of interest whether the above-described changes in the oral cavity during infant development might be accompanied by developmental alterations in the composition of salivary proteins.
Whereas in rodents the ontogeny of salivary proteins, including their regulation at the transcriptional level, has been well-studied (Kousvelari and Tabak, 1991; Ann et al., 1997), there are few reports on the expression of salivary proteins during human infant development, particularly in relation to tooth eruption. An exception is the development of mucosal immunity provided by secretory immunoglobulin A (S-IgA), which has been studied in great detail, mainly regarding antibody activity and concentration (Tenovuo et al., 1987; Gleeson et al., 1991; Smith and Taubman, 1993; Fitzsimmons et al., 1994; Russell et al., 1999). Less is known about other non-immune salivary proteins. The activity of salivary  -amylase was found to increase from birth to later ages (Sevenhuysen et al., 1984; Dezan et al., 2002). Also, certain antibacterial components in saliva were shown to be differently expressed between children and adults (Tenovuo et al., 1987). Although, in infant saliva, the concentrations of certain electrolytes, S-IgA, and -amylase were different from adult levels, no differences before and after tooth eruption were detected (Ben-Aryeh et al., 1984).
To prove the hypothesis that the expression of certain proteins changes during infant development, and might be associated with tooth eruption, we monitored the general composition of salivary proteins and glycoproteins, as well as the expression of serum albumin, salivary -amylase, S-IgA, and the mucins MUC5B and MUC7 in whole saliva, in individual infants at monthly intervals from the first month after birth until the end of the first year of life.
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MATERIALS & METHODS
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Saliva
The study was approved by the Ethics Committee of the Medical Faculty of the University of Regensburg, and parents had provided informed consent for the participation of their infants. Whole unstimulated saliva from ten infants (three females, seven males) was collected at monthly intervals, beginning with the first month after birth, by manual suction with the use of disposable Pasteur pipettes (Plastibrand®, Brand GmbH & Co., Wertheim, Germany). Samples (from 50 to 500 µL) taken from healthy infants (who had not been fed for at least 2 hrs) were collected on ice and stored at –80°C. Total protein concentrations were determined with the use of the bicinchoninic acid (BCA) Protein Assay Reagent (Pierce, Rockford, IL, USA), with bovine serum albumin (BSA) as the standard.
SDS-PAGE, Transfer and Detection of Proteins and Glycoproteins
Salivary proteins were denatured under reducing conditions, separated by SDS-PAGE (0.75 µg per lane) on 4-20% gradient gels (Invitrogen, Karlsruhe, Germany), and visualized by means of an ammoniacal silver stain kit (SilverXpress®, Invitrogen) or transferred to nitrocellulose (10 µg per lane), as previously described (Ruhl et al., 2000). Glycosylated components were labeled by a modification of the hydrazide method (Ruhl et al., 1996), and lectin blotting was performed as previously described (Ruhl et al., 2004), with lectins from Canavalia ensiformis, Arachis hypogaea, and Limax flavus (EY Laboratories, San Mateo, CA, USA).
Western Blotting
Western blotting was performed as previously described (Ruhl et al., 2000). Nitrocellulose blots were blocked in 2% non-fat dry milk in Tris-buffered saline (TBS-milk) and incubated for 1 hr with either rabbit anti-human -amylase, rabbit anti-human IgA--chain (Sigma Chemical Co., St. Louis, MO, USA), rabbit anti-human MUC5B, or rabbit anti-human MUC7 antisera, each diluted 1:1000 in TBS-milk. Blots were washed with TBS, incubated for 1 hr with horseradish peroxidase-conjugated goat anti-rabbit IgG second antibody (Biorad) diluted 1:3000 in TBS-milk, washed, and developed.
Quantification of Albumin and Mucins by ELISA
Albumin was quantified by a sandwich ELISA as previously described (Hoek et al., 2002). In brief, high-affinity microtiter wells (Nunc, Roskilde, Denmark) were coated overnight with 100 µL of rabbit anti-human albumin IgG (Dako Cytomatation GmbH, Hamburg, Germany), diluted 1:8000 in sodium bicarbonate buffer, pH 9.6. After wells were washed with PBS containing 0.1% Tween 20 (PBST), 100-µL quantities of salivary samples, diluted 1:100 and 1:1000 in PBST, were added in duplicate. Human serum albumin, fraction V (Sigma, Taufkirchen, Germany), was used as the standard in serial dilutions, starting from 100 ng/mL. After incubation for 3 hrs at 37°C and being washed with PBST, a 100-µL quantity of horseradish peroxidase-conjugated rabbit anti-human serum albumin antiserum (Dako), diluted 1:4000 in PBST, was added for 1 hr at 37°C. After samples were washed again, color reaction was developed with TMB (Sigma) and stopped after 10 min with 2 N H2SO4. Photometric readings were taken at 450 nm with a THERMOmax multiplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA).
The capture ELISAs for quantification of MUC5B and MUC7 and the preparation of purified MUC5B and MUC7, as well as of specific antisera, have been described in detail previously (Troxler et al., 1995; Liu et al., 1999; Rayment et al., 2000).
Statistics
Median concentrations for experimental groups, together with the 25 and 75% quantiles and 5%-trimmed intra-individual ranges, were calculated. Statistical evaluation was performed with the non-parametric Mann-Whitney U Test (SPSS PC+, v5.01, SPSS Inc., Chicago, IL, USA) at the 0.05 level of significance. Correlation analyses were performed with TableCurve 2Dv4 software (SPSS Inc.).
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RESULTS
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Salivary Protein Profiles
When whole saliva collected longitudinally during the first 12 mos of life was separated by SDS-PAGE, and proteins were visualized by silver stain, the overall profile of bands showed a remarkable constancy in each individual infant (Figs. 1A –1D). Moreover, around the time of tooth eruption, no significant changes could be observed. Also, when the general composition of glycoproteins was examined by the hydrazide method, or by lectin blotting with ConA, PNA, or LFA, the overall profile of bands remained constant for each infant during the first year (data not shown).
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Figure 1. Protein profile during the first year of infancy. Whole saliva from four infants (A-D) was collected at monthly intervals and examined by SDS-PAGE; gels were stained with silver. The arrows indicate the age of first tooth emergence.
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Total Protein and Albumin
The median concentrations of total protein in whole saliva samples varied between 1.0 and 2.7 mg/mL (Fig. 2A). In single infants, the concentrations of total protein varied at different time points during the collection period, within an intra-individual range of 0.8 to 5.1 mg/mL. Median protein concentrations, with the exception of the first month (about two-fold higher value), remained constant during the first year of life and were not influenced by tooth eruption.
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Figure 2. Median concentrations of total protein (A) and serum albumin (B) from 10 infants determined at each month of age (closed circles). Error bars indicate the 25% and 75% quantiles. The curves (solid lines) were deduced by regression analysis of median concentrations of protein (r2 = 0.88) and albumin (r2 = 0.93) vs. age, respectively. Arrows indicate first tooth emergence of each respective infant.
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The median concentrations of serum albumin in whole saliva increased from 1 to 12 µg/mL during the first year of infancy, within an intra-individual range from 10.4 to 105 µg/mL. A shift in median concentrations was observed prior to the eruption of the first teeth (Fig. 2B ). In individual infants, the albumin concentration started to rise about a month before eruption of the first tooth.
Expression of S-IgA, -Amylase, and Mucins
The S-IgA Western blot contained a 60-kDa band with a molecular mass consistent with that of the -subunit of S-IgA and 2 additional bands of less intensity in the ~ 40- and ~90-kDa regions (Fig. 3A). These additional bands were not observed in all infants and were absent on blots of adult saliva (Ruhl, unpublished). The median signal intensity of the S-IgA bands among individuals was independent of age and tooth eruption (Fig. 3B) within an intra-individual range from 7.7e+4 to 2.4e+5.
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Figure 3. Expression of S-IgA, salivary -amylase, MUC5B, and MUC7 during the first year of infancy. Whole saliva was collected at monthly intervals, and expression patterns were determined by Western blotting. Sample blots from single infants are depicted for S-IgA -chain (A), -amylase (C), MUC5B (E), and MUC7 (G). The arrow indicates the age of first tooth emergence. Blots from different infants were analyzed by densitometry, and signal intensities are depicted for S-IgA blots (six infants) (B), -amylase blots (seven infants) (D), MUC5B blots (four infants) (F), and MUC7 blots (four infants) (H). Closed circles represent median signal intensities. Error bars indicate the 25% and 75% quantiles. The curves (solid lines) were deduced by regression analysis of median signal intensities of S-IgA (r2 = 0.42), -amylase (r2 = 0.71), and MUC7 (r2 = 0.94) vs. age, respectively. Arrows indicate first tooth emergence of each respective infant.
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Salivary -amylase was identified by Western blotting as a doublet of bands of approximately 60 kDa (Fig. 3C). The median signal intensity of these bands increased with the age of infants within an intra-individual range from 7.8e+3 to 2.0e+4 (Fig. 3D). At the first month of age in two individuals, no -amylase could be detected.
MUC5B was visualized by Western blotting as a smear band at the origin of the gel and could be detected in samples taken at all time points of collection (Fig. 3E). Not all of the MUC5B entered the separating gel, making it impossible for the intensity of bands to be reliably quantified by densitometry. Nevertheless, a signal could be detected in each lane, and a median signal intensity for each month of age was estimated (Fig. 3F) within an intra-individual range from 2.8e+4 to 6.9e+4. Because of the uncertainties of quantification, a regression analysis of age vs. signal intensity was not performed. MUC7, in contrast, was detected as a strong band with a molecular mass of about 150 kDa. The intensity of this band decreased considerably, from high expression at the beginning to low expression toward the end of the collection period (Fig. 3G), within an intra-individual range from 8.5e+4 to 2.9e+5. This observation was verified by densitometry showing a highly correlated (r2 = 0.94) significant decrease in median signal intensity of the MUC7 band with age (Fig. 3H).
The results of Western blotting experiments were confirmed and extended by measurement of MUC5B and MUC7 in quantitative ELISAs. Analysis of the data showed a significant increase in the quantity of MUC5B and a significant decrease in the quantity of MUC7 in all subjects between 1 mo and 12 mos after birth (Fig. 4), within an intra-individual range from 17 to 77 mg% for MUC5B and 31 to 64 mg% for MUC7. It is noteworthy that the MUC5B and MUC7 curves intersected after 6 mos at, or prior to, the time of eruption of the first tooth in most infants.
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Figure 4. Concentrations of MUC5B and MUC7 during the first year of infancy. Median concentrations of MUC5B (circles) and MUC7 (triangles) from nine infants were determined by ELISA after each month of age. Error bars indicate the 25% and 75% quantiles. Curves for MUC5B (solid line, r2 = 0.95) and MUC7 (dotted line, r2 = 0.86) were deduced by regression analysis of median concentrations vs. age. Arrows indicate first tooth emergence of each respective infant.
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DISCUSSION
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Little is known about changes in the composition of proteins in human saliva during the first year of an infant’s life. This question is of considerable interest, since a developmentally determined adaptation to the changing demands of food processing and a concomitant shift in bacterial ecology occur during this time period in the oral cavity. In the present investigation, the overall protein and glycoprotein compositions, along with the expression of serum albumin, S-IgA, salivary -amylase, MUC5B, and MUC7, were monitored longitudinally in individual infants. The results showed that, although the overall composition of proteins in infant saliva remained constant over the course of the first year of life, salivary mucins were differentially expressed, such that MUC7 was more abundant at the beginning and MUC5B more abundant toward the end of this time period. Furthermore, a close association with the eruption of the infants’ first teeth was found for the concentrations of albumin in whole saliva.
Earlier studies have shown that certain components in saliva are regulated during infancy. In this respect, little or no S-IgA was detected in saliva at birth (Seidel et al., 2001; Weemaes et al., 2003), but concentrations were shown to rise soon after birth (Alaluusua, 1983; Smith and Taubman, 1993; Fitzsimmons et al., 1994; Weemaes et al., 2003). Also, the activity of salivary -amylase was found to increase from low values at birth to approximately two-thirds of adult values by 3 mos (Ben-Aryeh et al., 1984; Sevenhuysen et al., 1984). In the present study, an increase in the amount of -amylase with age was shown, but no significant changes in signal intensity of bands in the Western blots for the heavy chain of S-IgA could be detected. This could be due, in part, to the fact that Western blotting provides a qualitative indication of protein levels but not of the biological activity of a protein. Another explanation could be that the greatest changes in S-IgA concentration take place mainly between birth and the first month of age (Smith and Taubman, 1993; Weemaes et al., 2003). Noticeably, in the present study, at the first month in some infants, -amylase and S-IgA were low or absent. Thus, in a follow-up study, it would be interesting to focus on changes in the profile of salivary proteins between birth and the first month of age.
It is well-established that certain proteins and glycoproteins in saliva serve as receptors for bacterial adhesins, and thus may be a prerequisite for bacterial colonization of teeth (Gibbons, 1989; Scannapieco, 1994). In this regard, the natural acquisition of bacteria such as Streptococcus mutans, S. sanguinis, and Actinomyces naeslundii, as well as oral anaerobes such as Fusobacterium nucleatum, was shown to be correlated with tooth emergence (Smith and Taubman, 1993; Cole et al., 1998; Könönen et al., 1999; Caufield et al., 2000; Wan et al., 2003). Furthermore, it is known that certain salivary proteins exert functions both in innate mucosal immunity and for the protection of mineralized tooth surfaces (Mandel, 1987; Tabak, 1995). Thus, for the present study, it was hypothesized that certain proteins may be developmentally up- or down-regulated before emergence of teeth. However, for most components monitored, no alterations or quantitative changes could be found coinciding with tooth eruption. The only exceptions were serum albumin and the salivary mucins. Albumin in whole saliva was found to shift to significantly higher concentrations about one month before eruption of the infants’ first teeth, thus being an indicator of forthcoming tooth eruption. This may be due to an increased permeability of the epithelium covering the erupting teeth, or to microlesions caused by the infants’ increased chewing activities. Mucins were clearly differentially expressed, with MUC7 more abundant at the beginning and MUC5B more abundant at the end of the first year. Since mucins can serve as receptors for oral bacterial adhesins (Scannapieco, 1994; Tabak, 1995; Ruhl et al., 2004), the shift in mucin concentrations may have implications for bacterial colonization in the infant’s oral cavity. Noticeably, the shift in mucin concentrations took place prior to tooth eruption in most subjects, and it would be interesting to investigate whether this shift is a developmentally induced phenomenon. This question is complex, because MUC5B and MUC7 are expressed in different glandular cells (Piludu et al., 2003) and can be ultimately resolved in well-established animal models, where gene expression can be monitored (Kousvelari and Tabak, 1991; Ann et al., 1997). The present investigation has shown that the majority of salivary proteins are expressed as early as a month after birth. The possible implications of the observed changes in albumin and mucin expression for oral microbial ecology during infancy deserve further investigation.
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ACKNOWLEDGMENTS
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We are grateful to Verena Wittmann, Erika Treml, and Andreas Eidt for excellent technical assistance. We are indebted to the parents of Alicia-Patricia, Jakob, Jannik, Kilian, Leonhard, Linus, Lukas, Natalie, Niklas, and Sarah for their great support in monthly collecting saliva from their infants. This investigation was supported by DFG (Deutsche Forschungsgemeinschaft) grants ru409/4-1 and SFB 585/B5 (SR), and by NIH grants DE 11691 (RFT) and T32 DE 07206 (SAR).
Received for publication December 22, 2003.
Revision received October 8, 2004.
Accepted for publication October 13, 2004.
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Journal of Dental Research, Vol. 84, No. 1,
29-34 (2005)
DOI: 10.1177/154405910508400104
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ABSTRACT
INTRODUCTION