Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

Interaction of human salivary mucin MG2, its recombinant N-terminal region and a synthetic peptide with Actinobacillus actinomycetemcomitans.

The antimicrobial properties of human salivary mucin MG2 against the periodontal pathogen, Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), were investigated using purified MG2, rNMUC7 (a recombinant polypeptide containing residue 1-144 of MG2) and synthetic peptides PEP1 (residue 1-17) and PEP2 (residue 47-63). MG2 and rNMUC7 bound to A. actinomycetemcomitans strains SUNY75, SUNY465, SUNY523, 652 and JP2 in a liquid phase binding assay. The bactericidal activities of rNMUC7, PEP1 and PEP2 against A. actinomycetemcomitans SUNY523 were examined in a colony forming unit killing assay. The LD50 for rNMUC7 was 9 microM, for PEP2 was 20 microM and PEP1 did not exhibit bactericidal activity. The primary structure of these polypeptides was analyzed and a direct relationship between net positive charge and bactericidal activity was found. Screening of saliva samples from 60 individuals on Western blots probed with an anti-MG2 antibody against PEP2 revealed that a 20 kDa MG2 fragment was present in 66% of subjects and that this fragment was not present in glandular secretions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides derived from the 20 kDa fragment confirmed that this fragment contained a portion of the amino terminal region of MG2. The present study showed that the N-terminal region of MG2 and a subdomain within this region are microbicidal against A. actinomycetemcomitans and that a 20 kDa fragment of MG2 occurs in whole saliva. This suggests that cleavage of MG2 in vivo may produce fragments with microbicidal properties and that this may represent a novel mechanism of host defense.