Structural characterisation of cysteines in a bacterial-binding motif of human salivary mucin MG2.
Human salivary mucin MG2 is a 180 kDa glycoprotein secreted by submandibular/sublingual and minor salivary glands. Secreted MG2 contains a domain with the only two cysteines (Cys(45) and Cys(50)) present in the polypeptide backbone; in native and recombinant MG2 this domain is involved in mucin binding to oral microbes. As the reduction and alkylation of MG2 has been shown to abolish binding, the present study was undertaken to determine whether the cysteine residues exist in the dithiol or disulphide form. Electrophoretic analysis under reducing and non-reducing conditions showed that intermolecular disulphide bonds do not occur between MG2 molecules. The same incorporation of radiolabelled iodoacetamide into MG2 was obtained with or without prior reduction. When radiolabelled alkylated MG2 was digested with Endoproteinase Lys-C and the derived peptides were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), radioactivity was found in two fractions. Mass spectral analyses of these fractions showed the presence of peptides Cys-Leu-His-Lys and Arg-Cys-Arg-Pro-Lys, both containing carboxymethylated cysteines. These results show that the cysteines in the structural motif associated with bacterial binding exist in the dithiol form, and suggest the potential use of cysteine-containing peptides as agents to modify interactions of MG2 with microbes and oral surfaces.
Soares
Rodrigo V
RV
Department of Periodontology and Oral Biology, Boston University Medical Center, Boston, MA 02118, USA.
Liu
Bing
B
Oppenheim
Frank G
FG
Offner
Gwynneth D
GD
Troxler
Robert F
RF
D020816:Amino Acid Motifs; D000596:Amino Acids / Q000032:analysis; D001419:Bacteria / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; C083238:MUC7 protein, human; D009077:Mucins; D012471:Salivary Proteins and Peptides; D003545:Cysteine