Isolation of human salivary mucin MG2 by a novel method and characterization of its interactions with oral bacteria.
Human salivary mucin MG2 was purified from submandibular/sublingual gland secretion by ultrafiltration and sequential gel filtration chromatography on Sephadex G-200, Superose 6 (prepgrade), and Superose 6. This method differs from earlier procedures in that all steps are performed in the presence of 4 M guanidine hydrochloride and do not involve covalent modification of the mucin molecule. Electrophoretic analyses and Western blotting showed that purified MG2 did not contain detectable levels of other salivary proteins. Amino acid analysis showed that the composition of purified MG2 was in excellent agreement with the deduced sequence of MG2 apomucin encoded in the MUC7 gene. The yield of purified MG2 was 10-15 mg from 750 ml of salivary secretion. Binding of purified MG2 to Streptococcus mutans in vitro was not significantly affected by reductive methylation, but was nearly abolished by reduction and alkylation. These data identified a functional determinant for mucin-bacterial interactions in the N-terminal region where the only two cysteines (Cys45 and Cys50) in the MG2 apomucin occur. Additionally, purified MG2 bound to four strains of oral Streptococci, indicating that the binding is not dependent on complexing with other salivary proteins, such as secretory immunoglobulin A. The purification procedure described in this work will facilitate investigation of the role of MG2 in the oral environment.
Liu
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Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Rayment
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Oppenheim
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Troxler
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D000328:Adult; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D008875:Middle Aged; D009055:Mouth / Q000382:microbiology; D009077:Mucins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013295:Streptococcus mutans / Q000378:metabolism*
D000596:Amino Acids; C083238:MUC7 protein, human; D009077:Mucins; D012471:Salivary Proteins and Peptides