ⅠINTRODUCTION

In a previous study the salivary complexes found in the acrylamide gel-filtration chromatography were characteristic functional elements in human whole saliva^1-3). Although many salivary proteins were assembled into several complexes, their molecular conformation were relatively stable for a while to achieve the important roles of each salivary protein. Among several peaks of SPCs two dominant SPC peak fractions were examined for their compositions of salivary proteins through IP-HPLC analysis in this study.

IP-HPLC is one of the protein detection methods, which is based on the real antigen-antibody reaction in PBS buffer solution, followed by purification using protein A/G -conjugated agarose beads. Although its procedures are simple and easy to apply most of biological samples, IP-HPLC may give minimum error range by using micro-beads instead of small wells in the mimicry to the enzyme-linked immunodorbent assay (ELISA)^4-6).

The two dominant SPCs in gel filtration chromatography are major functional groups in whole saliva, but their molecular structures have been hardly investigated so far^7,8), the present study is aimed to elucidate the salivary protein composition in the two dominant SPCs of human whole saliva.

ⅡMATERIALS AND METHODS

Normal whole saliva was collected from five adult volunteers who were in a good health. These saliva samples were not specific to any ethnic group and not pathologically complicated. The whole saliva was immediately centrifuged at 1600g for 20 min, and its supernatant was analyzed by gel-filtration chromatography using agarose column (Superose^TM 12, 10/300 GL, Sweden) and HPLC machine (Hewlett Packard 1050, USA). In this study the HPLC-based gel filtration was performed using K-buffer (20mM KH₂PO₄, 20mM K₂HPO₄) at 0.5 mL/min mobile speed for 60 min. 100 mL of whole saliva was injected, and then it was separated into multiple native SPCs. Among them the first dominant SPC (fraction 1) and the second dominant SPC (fraction 2) were collected through liquid fraction collector (FC204, Gilson, USA) (Fig. 1). The fractionated samples were mixed well with protein lysis buffer (0.3% SDS, 1% β-mercaptoethanol, 0.05M Tris-HCl pH 8.0, for 100 ml solution) and kept in refrigerator until usage.

In order to know the changes of SPC pattern between unstimulated whole saliva and stimulated whole saliva, the unstimulated whole saliva was obtained in the unstimulated rest state before meal, while the stimulated whole saliva was obtained at stimulated state using paraffin wax after meal. And the samples were immediately applied to the gel filtration chromatography. The first and second dominant SPCs were simultaneously fractionated, and the native whole saliva was used as a positive control.

The protein concentrations of SPC samples were measured using a protein assay solution (Bio-Rad Laboratories, Inc., USA) at 595 nm using a UV spectrophotometer (Optizen 3220UV, Mecasys, Korea), and about 1 mg of protein was applied to each immunoprecipitation procedure using a protein A/G agarose bead column (Amicogen, Korea). About 1 μg of each different antibody, including ^@α-amylase, ^*mucin-1, PRPs9), ^*histatin, ^*cystatin, ^$LL-37, ^#lysozyme, ^@lactoferrin, ^*β-defensin-1, -2, -3, ^$IgK (to detect IgA), ^*TGase 4, mucocidin^10,11), ^*α1-antitrypsin, ^*cathepsin G (^*Santa Cruz Biotech, USA; ^#DAKO, Denmark; ^@Abcam, Cambridge, UK; ^$ZYMED, USA), was pre-incubated with the protein A/G agarose beads (Amicogen, Korea) separately.

Briefly, the saliva fraction samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, 0.5% NP-40), and incubated in the protein A/G agarose column bound with different antisera at room temperature for one hour. The columns were placed on a rotating stirrer. After washing each column with a sufficient amount of PBS solution (pH 7.3, 137 mM NaCl, 2.7 mM KCl, 43 mM Na₂HPO₄-7H₂O, 1.4 mM KH₂PO₄), target proteins were eluted with 250 μL of IgG elution buffer (Pierce, USA). The immunoprecipitated proteins were analyzed by HPLC using a reverse phase column (YMC-Pack SIL, SL12S05-2506WT, USA) and 0.05M NaCl/5% acetonitrile solution as isocratic running buffer at 0.6 mL/min for 30 min^1-3,6). IP-HPLC was performed on control and experimental groups simultaneously. The SPC peak areas (mAU*s) obtained by HPLC were calculated after subtracting control antibody peak areas. The negative control IP-HPLC was performed using each antibody only except SPC sample. The value of IP-HPLC (mAU*s) was obtained and its square root value was calculated in each IP-HPLC analysis. For the comparison of salivary protein expression level between the first dominant SPC and the second dominant SPC all the IP-HPLC results of three groups, the first dominant SPC, the second dominant SPC, and whole saliva were plotted into a graph.

ⅢRESULTS

1.SPC peak pattern in the unstimulated and the stimulated whole saliva

The SPC peak pattern in gel filtration chromatography was compared between the unstimulated and the stimulated whole saliva. The chromatography products exhibiting multiple SPC peaks clearly disclosed that both the unstimulated and stimulated whole saliva showed almost similar SPC peak patterns, but the amount of salivary proteins was much reduced in the stimulated whole saliva compared to that of the unstimulated whole saliva (Fig. 1).

2.Salivary protein composition in two dominant SPCs

In the IP-HPLC analysis the two dominant SPCs showed variable expressions of the essential salivary proteins including α-amylase, mucin-1, PRPs, histatin, cystatin, LL-37, lysozyme, lactoferrin, β-defensin-1, -2, -3, IgA, TGase 4, mucocidin, α1-antitrypsin, and cathepsin G. However, the first dominant SPC (fraction 1) contained more amount of mucin-1, PRPs, α-amylase, lysozyme, cathepsin G than the second dominant SPC (fraction 2), while the latter contained more amount of cystatin, lactoferrin, β-defensin-1, -2, -3, mucocidin, IgA, TGase 4, α1-antitrypsin than the former. However, lactoferrin, mucocidin, and α1-antitrypsin were predominant in the second dominant SPC but minimum in the first dominant SPC (Fig. 2).

The whole saliva, which was compared as a normal positive control, consistently expressed the essential salivary proteins, but among them β-defensin-1 was more abundant in the whole saliva than in the two dominant SPCs. And more, α1-antitrypsin and cathepsin G which were mostly derived from gingival crevicular fluid were also consistently found in the two dominant SPCs. Cathepsin G was abundant in the two dominant SPCs but a little more in the first dominant SPC than the second dominant SPC, while α1-antitrypsin was predominantly found in the second dominant SPC compared to the first dominant SPC (Fig. 2).

ⅣDISCUSSION

The SPCs found in the gel filtration chromatography are native salivary materials mostly aggregated by salivary proteins, which are severely modified by glycosylation, sulfation, cross-linking, etc. The molecular interactions between salivary proteins are somehow specific and finally produce the characteristic SPC peaks in the native gel filtration chromatography. Due to the random and strong protein aggregation of SPCs the salivary investigation becomes much problematical and retarded compared to the other biological research³⁾. However, reversely the SPCs in whole saliva can carry out versatile salivary functions in oral cavity, in which numerous micro-organisms are inhabited and different chemical and physical injuries may be frequently happened. Therefore, it is highly suggested that the native SPCs are real functional units of salivary proteins in order to perform the different salivary protein functions simultaneously and to prolong their salivary protein functions in whole saliva. The present study demonstrated the salivary protein composition in two dominant SPCs fractionated from whole saliva through native gel filtration chromatography.

In the experiment for SPC changes in the unstimulated whole saliva and the stimulated whole saliva the major patterns of SPC were not changed, but the amount of SPC peak was much reduced in the stimulated whole saliva compared to the unstimulated whole saliva. Therefore, it may indicate that the SPCs are consistently produced independently from the neurologic stimulation, and also suggest that the salivary protein complexes are basically stable in their structures.

IP-HPLC analysis has been developed to detect the protein expression in biological samples quantitatively. It is designed to use protein A/G-conjugated microbeads for immunoprecipitation and to be followed by UV-spectrum analysis through HPLC. The IP-HPLC can analyze most of biological materials in easy and automatic ways^2,3,6,12). Therefore, it would be suggested that the quantitative measuring of IP-HPLC is quite accurate similar to that of ELISA, which usually use small wells conjugated with antibody and fluorescence spectroscopy^13,14). However, the present study disclosed that the first dominant SPC is rich in α-amylase, mucin-1, PRPs, lysozyme, and cathepsin G, while the second dominant SPC is rich in cystatin, lactoferrin, β-defensin-1, -2, -3, IgA, mucocidin, TGase 4, and α1-antitrypsin. Therefore, it is presumed that the first dominant SPC may play more prominent roles in the food digestion, protein degradation, and mucosa lubrication, while the second dominant SPC may play roles in mucosa protection and antimicrobial defense.

Mucocidin is a novel antimicrobial protein cloned as a name of salvic from human submandibular gland^10,11). Although the genomic locus of mucocidin has not been identified, mucocidin is consistently expressed in saliva as well as in salivary glands. Mucocidin has strong antimicrobial potential both in vivo and in vitro experiments^10,15). However, in the present study it was clear that the mucocidin was intensely found in the second dominant SPC but much weak in the first dominant SPC. These findings also support that the second dominant SPC may play an important role in the antimicrobial action in human whole saliva.

Particularly, β-defensin-1 was predominantly expressed in whole saliva compared to its expression in the first and second dominant SPCs. But β-defensin-2 and -3 showed relatively even distribution in the two dominant SPCs and whole saliva, however, they were a little more abundant in the second dominant SPC than in the first dominant SPC. These findings might imply that the large amount of β -defensin-1 was included in the other SPCs except the first and second dominant SPCs. Therefore, it is presumed that the predominant expression of β-defensin-1 in whole saliva might indicate its essential first line innate defense reaction in oral environment.

As the whole saliva contains not only the salivary secretions but also the gingival crevicular fluid from periodontal tissue, the present study also explored the protein expression of α1-antitrypsin and cathepsin G, which are known to be mostly released from neutrophils in the gingival crevicular fluid environment^16,17). Resultantly, cathepsin G was strongly expressed in the first and second dominant SPCs, but a little more in the first dominant SPC than the second dominant SPC. Whereas α1-antitrypsin expression was exclusively strong in the second dominant SPC, but it was minimum in the first dominant SPC. These findings may indicate that the cathepsin G which is a strong proteolytic enzyme may help the degradation of glycoprotein materials together with the digestive reaction of α-amylase and lysozyme in the first dominant SPC, and that the α1-antitrypsin which can function as an anti-inflammatory agent may be closely associated with the innate immunity-related salivary proteins, i.e., lactoferrin, β-defensin-1, -2, -3, IgA, mucocidin, in the second dominant SPC.

In summary the present study demonstrated the two dominant SPCs in human whole saliva as important functional units in oral cavity. The chromatography SPC pattern of the unstimulated whole saliva was almost not changed in the stimulated whole saliva, and the protein composition of each SPC was variable and might directly affect its salivary functions. The first dominant SPC, rich in α-amylase, mucin-1, PRPs, and cathepsin G, may play a role in food digestion, protein degradation, and mucosa lubrication, while the second dominant SPC, rich in cystatin, lactoferrin, β-defensin-1, -2, -3, mucocidin, IgA, TGase 4, and α1-antitrypsin, may play a role in mucosa protection and antimicrobial defense. Furthermore, the SPCs of whole saliva may have more significant biological roles than we thought, therefore, it is suggested that the SPCs of whole saliva should be investigated more in detail with fundamental molecular biological methods.