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

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Abstract
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.