Proteomic analysis of polymeric salivary mucins: no evidence for MUC19 in human saliva

  • Rousseau K
  • Kirkham S
  • Johnson L
 et al. 
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Abstract

MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293-302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155-165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated. Identification of mucins in the saliva of cow, rat and pig To determine whether Muc19 and Muc5b were present in cow, rat and pig saliva, we employed a less lengthy procedure to isolate the mucins. This was because we only wished to determine whether the mucin was present as opposed to obtaining maximum coverage of the polypeptide. For this approach, our aim was to enrich rather than purify the mucins. In brief, saliva from cow, rat and pig was dissolved with 8 M GdmCl and then dialysed into 6 M urea. An aliquot of this non-purified saliva was reduced and subjected to SDS/PAGE using NuPAGE 4–12% bisacrylamide gels (Invitrogen, Paisley, Renfrewshire, Scotland, U.K.). Gels were run for 2 h at 150 VinNuPAGE Mes/SDS running buffer and NuPAGE LDS sample buffer (Invitrogen). After electrophoresis, the gels were stained with PAS [29] and the high-molecular-mass smears at the top of the gel were excised, in-gel-digested with trypsin [34] and analysed by MS/MS as described above. While this is the first study that specifically demonstrates that Muc19 is a polymeric glycoprotein, earlier studies on recombinant N- and C-terminal fragments of PSM (pig submaxillary mucin; now known to be Muc19) showed that they were able to assemble in a disulfide-bond dependent manner [47–49], further confirming the polymeric nature of Muc19/PSM.

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Authors

  • Karine Rousseau

  • Sara Kirkham

  • Lindsay Johnson

  • Brian Fitzpatrick

  • Marj Howard

  • Emily J. Adams

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