Bactericidal activity and poly-L-proline II conformation of the tandem repeat sequence of human salivary mucin glycoprotein (MG2).
The tandem repeat 23-residue sequence [TRS23 (145-167): T-T-A-A-P-P-T-P-S-A-T-T-P-A-P-P-S-S-S-A-P-P-E] of human salivary mucin glycoprotein MG2 was examined for its in vitro bactericidal activity against four oral microorganisms, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus gordonii, and Streptococcus mutans. The conformational features of the proline-rich peptide were determined by circular dichroism (CD) and 600 MHz two-dimensional (2D) nuclear magnetic resonance (NMR) in aqueous solution. The strains of P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are highly sensitive to this peptide at 1.5-3.0 microM concentrations, suggesting that the proline-rich repeat sequence is a potent bactericidal agent for oral pathogens. The assignment of backbone and side-chain proton resonances was accomplished by the combined analysis of 2D total correlated spectroscopy and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and the 1H-2H exchange effect on amide NH resonances suggest the absence of intramolecularly hydrogen-bonded NH groups. The coupling constant (JNH-CalphaH) values, conformational restriction offered by the proline residues (phi = -60 degrees +/- 15 degrees), the set of medium- and short-range nuclear Overhauser effects observed for this sequence, and the results of restrained structure calculation using DIANA, the distance geometry algorithm for NMR applications, provide evidence for the existence of a significant population of poly-L-proline II-type helices in aqueous solution. The CD spectra of the peptide in phosphate buffer (pH 7.2) and in methanol are reminiscent of the CD spectrum of the poly-L-proline II helical conformation and are consistent with the NMR data. The bactericidal activity of the proline-rich repeat sequence suggests that bacterial colonization, facilitated by the adsorbed salivary mucins on tooth surface, could be partly controlled and cleared by proteolytically degraded proline-rich peptides of MG2 in saliva before the colonized organisms turn into pathogens. It appears that the poly-L-proline II helix is the biologically active backbone conformation for bactericidal activity of the tandem repeat sequences of salivary MG2.
Periodontal Disease Research Center, State University of New York at Buffalo, 311 Foster Hall, Buffalo, New York, 14214, USA. antony@mit.edu
K J
KJ
Antonyraj
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T
Karunakaran
P A
PA
Raj
D000595:Amino Acid Sequence; D001419:Bacteria / Q000187:drug effects* / Q000254:growth & development; D002942:Circular Dichroism; D006801:Humans; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry* / Q000494:pharmacology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012091:Repetitive Sequences, Nucleic Acid; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000494:pharmacology*; D012996:Solutions
C083238:MUC7 protein, human; D009077:Mucins; D010446:Peptide Fragments; D010455:Peptides; D012471:Salivary Proteins and Peptides; D012996:Solutions; C011083:polyproline