Ⅰ.INTRODUCTION

In the previous study the salivary protein complexes (SPCs) was identified in human whole saliva. SPCs were found in chromatography using Superose 12 column (GE Healthcare Bio-Science AB, Sweden)^1-4). Especially it was considered that SPCs are important structures in saliva to protect from aggressive proteolytic challenge of oral environment, and to prolong their functions in whole saliva ^1,3,4). With the complex salivary protein structures, SPCs can be stable for a long time to achieve their important roles in the bacteria-rich and strong proteolytic environment of whole saliva¹⁾.

The SPCs was found as the characteristic early and late dominant peaks in the gel filtration chromatography, the early and late dominant peaks appeared at 23-28 minutes and at 45-50 minutes in 0.6mL/min running buffer. Several small peaks are also found in the intermediate zone between the early and late dominant peaks. As the Superose 12 column is made for reverse phase molecular running, the early dominant peak may contain heavier SPC than the late dominant peak. In the previous studies, the protein components of two dominant SPCs were explored with IP-HPLC methods using different antisera. And it turned out that both SPCs contained almost every salivary proteins but in different amount. Particularly, the early (first) dominant SPC contained more amount of amylase, mucin-1, PRPs, lysozyme, and cathepsin G than the late (second) dominant SPC, while the late dominant SPC contained more amount of cystatin, lactoferrin, β-defensin-1, -2, -3, IgA, mucocidin, TGase 4, and α1-antitrypsin than the early dominant SPC²⁾. Therefore, it was presumed that both SPCs were made of different amount of salivary proteins immediately after excretion from salivary glands, and that the distribution pattern of salivary proteins might be relevant to the important functions of each SPC in whole saliva.

Although the SPCs are supposed to be composed of multiple salivary proteins and carbohydrates in complicate and complex structures through molecular cross-linking and aggregation, the chromatography of whole saliva disclosed characteristic SPCs pattern exhibiting the early and late dominant peaks, and variable small intermediate peaks. This chromatography pattern is similarly found in the whole saliva of every people, but the size of SPC peaks are variable depending on the salivary secretion status of individual^1,3,4).

The two dominant SPCs in gel filtration chromatography are highly considered as important functional groups in whole saliva, but their molecular structures have been not clearly elucidated so far^5,6). In the present study a new method of direct immunoreaction to salivary proteins was performed to give the conformational change of SPCs before gel filtration HPLC. It was supposed that if the SPCs in whole saliva were directly bound by each specific antibody, and then the involved SPCs would be deformed. Consequently the deformed SPCs may migrate differently in gel filtration HPLC, and result in the change of SPC peak patterns in chromatography.

As it is coincidently agreed that the salivary antimicrobial and protective proteins may play important roles in the bacteria-rich oral environment, the present study is aimed to explore the distribution of several antimicrobial and protective proteins in salivary chromatography in order to define which SPC contains the antimicrobial and protective proteins more abundantly. In this study the biological roles of salivary SPCs which contain specific antimicrobial and protective proteins were also discussed with the review of the literatures.

Ⅱ.Materials and methods

Whole saliva was collected from five adult volunteers who were good in health. These saliva samples were not specific to any ethnic group and not pathologically complicated. The whole saliva was immediately centrifuged at 1600g, and its supernatant (100 μL) was analyzed by gel filtration HPLC. For the direct immunoreaction to salivary proteins, 200 μL whole saliva was incubated with different antibody (2μg) at room temperature for 30 min before gel filtration HPLC. Total 12 antibodies were used, including ^*mucin-1, proline rich proteins (PRPs)⁷⁾, ^*histatin, ^$LL-37, #lysozyme, ^@lactoferrin, ^*β-defensin-1, -2, -3, ^$IgK (to detect IgA), mucocidin⁸⁾, and ^*α1-antitrypsin (^*Santa Cruz Biotech, USA; #DAKO, Denmark; ^@Abcam, Cambridge, UK; ^$ZYMED, USA).

After antibody incubation 100 μL whole saliva was injected into gel filtration HPLC using a Superpose 12 column (Superose^TM 12, 10/300 GL, GE Healthcare Bio-Science AB, Sweden) and HPLC machine (Hewlett Packard 1050, USA), running at 0.6 mL/min mobile buffer (20mM KH₂PO₄, 20mM K₂HPO₄) for 60 min. The whole saliva was separated into several SPC peaks in reverse phase by the Superpose 12 column. The salivary chromatography of each experimental group was overlapped with that of control group in the graphic program (HP ChemStation, USA), and followed by defining the salivary protein distribution in each SPC.

Ⅲ.Results

The SPC peaks in salivary chromatography were changed into four different patterns (A, B, C, and D patterns) by the direct antibody binding to salivary antimicrobial and protective proteins, including mucin-1, PRPs, lysozyme, LL-37, lactoferrin, histatin, β-defensin-1, -2, -3, IgA, mucocidin, and α1-antitrypsin. The A pattern of SPC peak showed increased SPC peak height, indicating that the antibody binding to target salivary proteins resulted the increase of SPC without conformational change of SPC. The B pattern of SPC peak showed decreased SPC peak height, indicating that the antibody binding to target salivary proteins resulted the decrease of SPC without conformational change of SPC. The C pattern of SPC peak showed shorter retention time of SPC peak, indicating that the antibody binding to target salivary protein resulted the increase the volume of SPC conformation, which can be migrated rapidly in the reverse phase column of HPLC. The D pattern of SPC peak showed longer retention time of SPC peak, indicating that the antibody binding to target salivary protein resulted the decrease the volume of SPC conformation, which can be migrated slowly in the reverse phase column of HPLC (Table 1).

The whole saliva interacted with mucin 1 antibody produced slight decrease of all SPC peaks evenly in the salivary chromatography (Fig. 1A). The whole saliva interacted with PRPs antibody produced marked decrease of the late dominant SPC and marked increase of intermediate peaks near the early dominant SPC peak in the salivary chromatography (Fig. 1B). The whole saliva interacted with lysozyme antibody produced marked decrease in the late dominant SPC peak more than the early dominant SPC peak in the salivary chromatography (Fig. 1C). The whole saliva interacted with LL-37 antibody produced slight decrease of all SPC peaks evenly in the salivary chromatography (Fig. 1D). The whole saliva interacted with lactoferrin antibody produced slight decrease of the early dominant SPC peak and marked deformation of the late dominant SPC (Fig. 1E). The whole saliva interacted with histatin antibody produced slight decrease of all SPC peaks evenly in the salivary chromatography (Fig. 1F) (Table 1).

The whole saliva interacted with β-defensin-1 antibody produced marked decrease of the late dominant SPC peak in the salivary chromatography (Fig. 2A). The whole saliva interacted with β-defensin-2 antibody produced slight decrease of the late dominant SPC peak in the salivary chromatography (Fig. 2B). The whole saliva interacted with β-defensin-3 antibody produced slight deformation of the late dominant SPC peak in the salivary chromatography (Fig. 2C). The whole saliva interacted with β-defensin-3 antibody produced marked increase of the late dominant SPC peak in the salivary chromatography (Fig. 2D). The whole saliva interacted with mucocidin antibody produced marked increase of all SPC peak in the salivary chromatography (Fig. 2E). The whole saliva interacted with α1-antitrypsin antibody produced slight rapid migration of the early dominant SPC peak, marked increase of the late dominant SPC peak height, and an extra-peak before the early dominant SPC peak (Fig. 2F) (Table 1).

From the above results, it was found that mucin 1 is evenly distributed in all SPCs, while PRPs are more abundant in the late dominant SPC than the early dominant SPC and also in the intermediate SPCs. Most of antimicrobial proteins including lysozyme, LL-37, lactoferrin, β-defensin-1, -2, -3, IgA, mucocidin, and α1-antitrypsin were more abundantly distributed in the late dominant SPC than the early dominant SPC, while histatin showed relatively even distribution in all SPCs.

Ⅳ.Discussion

The SPCs found in the salivary chromatography are native salivary materials in whole saliva, which are mostly aggregated by salivary proteins via glycosylation, sulfation, cross-linking, etc^9,10). The molecular interactions between salivary proteins are complicate but specific, and finally produce the characteristic SPC peaks in gel filtration chromatography. Due to the random and strong aggregation of SPCs in whole saliva, the salivary investigation becomes more difficult and problematic compared to the other fields of biological research¹¹⁾.

However, the SPCs in whole saliva can carry out versatile salivary functions in oral cavity, in which numerous micro-organisms are inhabited and different toxic 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 multiple salivary protein functions simultaneously and also to prolong their salivary protein functions in whole saliva. The present study demonstrated the salivary antimicrobial and protective protein distribution in SPC peaks through gel filtration HPLC.

Direct antibody interaction to native whole saliva can detect the distribution of target protein in the SPCs through gel filtration HPLC. Among different salivary proteins, the present study explored twelve salivary antimicrobial and protective proteins to know their distribution in the SPCs of salivary chromatography. Mucin 1 and PRPs are known as major protective proteins in whole saliva, which also have bacteriostatic and antiviral functions^12,13). The present study clearly demonstrated that mucin 1 was evenly distributed in all SPCs of salivary chromatography, while PRPs were more abundant in the late dominant SPC than the early dominant SPC. These findings directly indicate that the late dominant SPC is closely relevant to the protective function of saliva in oral mucosa.

It was reported that the major salivary antimicrobial proteins, including lysozyme, LL-37, lactoferrin, histatin, IgA, and mucocidin were abundantly found in the fraction of the late dominant SPC more than the early dominant SPC through IP-HPLC in the previous study²⁾. The present study also defined the localization of those proteins in the late dominant SPC using direct antibody binding to the target proteins. The major salivary antimicrobial proteins were specifically bound to the late dominant SPC compared to the early dominant SPC, resulted in the changes of target SPC shape and migration speed.

In the previous study it was reported that β-defensin-1 might be mostly expressed in other SPCs rather than in the early and late dominant SPCs, and that β-defensin-2 and -3 showed relatively even distribution in the two dominant SPCs of whole saliva, and a little more abundant in the late dominant SPC than in the early dominant SPC²⁾. But the present study clearly disclosed the most of β-defensin-1, -2, -3 were involved with the late dominant SPC rather than the early dominant SPC. At this time it is thought that the present findings are more believable than the previous results, because the direct antibody binding to the target protein in native whole saliva is supposed to be more specific and sensitive than the IP-HPLC procedures.

Mucocidin is a novel antimicrobial protein cloned as a name of salvic from human submandibular gland⁸⁾. Although the genomic locus of mucocidin has not been clearly identified, mucocidin is consistently expressed in human salivary glands as well as in human saliva. Mucocidin has strong antimicrobial potential both in vivo and in vitro experiments^8,14). However, in the present study it was clear that the mucocidin was intensely found in the late dominant SPC and also diffusely found in other SPCs. These findings were similar to the previous report analyzed by IP-HPLC²⁾.

α1-antitrypsin which functions as an anti-inflammatory agent may be closely associated with the innate immunity-related salivary proteins¹⁵⁾. As its protective roles are critical in the procedures of PMN functions, it was supposed that α1-antitrypsin could be derived from not only from saliva but also from gingival fluid¹⁵⁾. The direct antibody reaction to whole saliva produced remarkable changes of both the early and the late dominant SPC peaks by increasing their migration speed, and also produced an extra-peak before the early dominant SPC peak. This extra-peak should be analyzed more in detail in the following investigation, however, it was presumed that α 1-antitrypsin might be an important molecule distributed widely throughout the SPCs of whole saliva.

The present study clearly demonstrated that most of antimicrobial proteins including lysozyme, LL-37, lactoferrin, β-defensin-1, -2, -3, IgA, mucocidin, and α1-antitrypsin were more abundantly distributed in the late dominant SPC than in the early dominant SPC, while histatin showed relatively even distribution in all SPCs. Therefore, it was presumed that the late dominant SPC containing abundant antimicrobial and protective proteins could be applied as a biomarker to measure the defensive potential of whole saliva in oral diseases.