title: Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.
abstract: Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.
authors.affiliation: Department of Oral Microbiology and Immunology, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul, Korea.
authors.forename: J E
authors.initials: JE
authors.lastname: Baik
authors.forename: S W
authors.initials: SW
authors.lastname: Hong
authors.forename: S
authors.initials: S
authors.lastname: Choi
authors.forename: J H
authors.initials: JH
authors.lastname: Jeon
authors.forename: O-J
authors.initials: OJ
authors.lastname: Park
authors.forename: K
authors.initials: K
authors.lastname: Cho
authors.forename: D-G
authors.initials: DG
authors.lastname: Seo
authors.forename: K-Y
authors.initials: KY
authors.lastname: Kum
authors.forename: C-H
authors.initials: CH
authors.lastname: Yun
authors.forename: S H
authors.initials: SH
authors.lastname: Han
mesh_terms: D000209:Acute-Phase Proteins / Q000494:pharmacology / Q000502:physiology*; D016976:Aggregatibacter actinomycetemcomitans / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology; D018441:Biofilms / Q000187:drug effects; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000032:analysis / Q000494:pharmacology / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007147:Immunoglobulin Light Chains / Q000032:analysis; D018836:Inflammation Mediators / Q000032:analysis; D054835:Lipocalin 1 / Q000032:analysis; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000502:physiology; D008264:Macrophages / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D026901:Membrane Transport Proteins; D051379:Mice; D009113:Muramidase / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis; D019056:Receptors, Polymeric Immunoglobulin / Q000032:analysis; D055333:Salivary Cystatins / Q000032:analysis; D055273:Salivary Proline-Rich Proteins / Q000032:analysis; D012471:Salivary Proteins and Peptides / Q000032:analysis / Q000494:pharmacology / Q000502:physiology*; D012709:Serum Albumin / Q000032:analysis; D017550:Spectroscopy, Fourier Transform Infrared; D051197:Toll-Like Receptor 4 / Q000187:drug effects; D025521:Tumor Suppressor Proteins; D037521:Virulence Factors / Q000378:metabolism; D000516:alpha-Amylases / Q000494:pharmacology / Q000502:physiology*; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D018836:Inflammation Mediators; D054835:Lipocalin 1; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009113:Muramidase; D011956:Receptors, Cell Surface; D019056:Receptors, Polymeric Immunoglobulin; D055333:Salivary Cystatins; D055273:Salivary Proline-Rich Proteins; D012471:Salivary Proteins and Peptides; D012709:Serum Albumin; D051197:Toll-Like Receptor 4; D025521:Tumor Suppressor Proteins; D037521:Virulence Factors; D000516:alpha-Amylases; D000099231:Lipopolysaccharide-Binding Protein; C108174:DMBT1 protein, human; C488892:PIP protein, human; C524745:PRB2 protein, human; C408143:SMR3B protein, human; C514728:lysozyme C, human