Macromolecular organization of saliva: identification of 'insoluble' MUC5Bassemblies and non-mucin proteins in the gel phase

Stimulated human submandibular/sublingual (HSMSL) and whole saliva were sep arated into sol and gel phases and mucins were isolated by density-gradient centrifugation in CsCl/4M guanidinium chloride. MUC5B and MUC7 were identi fied using anti-peptide antisera raised against sequences within the MUC5B and MUC7 apoproteins respectively. MUC7 was found mainly in the sol phase o f both HSMSL and whole saliva, but some MUC7 was consistently present in th e gel phase, suggesting that this mucin may interact with the salivary gel matrix. In HSMSL saliva, MUC5B was found in the gel phase; however, most of the material was 'insoluble' in guanidinium chloride and was only brought into solution by reduction. In whole saliva, the MUC5B mucin was present bo th in the sol and gel phases although some material was again 'insoluble'. Rate-zonal centrifugation of whole saliva showed that MUC5B mucins in the s ol phase were smaller than those in the gel phase, suggesting differences i n oligomerization and/or degradation. Antibodies against IgA, secretory com ponent, lysozyme and lactoferrin were used to study the distribution of non -gel-forming proteins in the different phases of saliva. The majority of th ese proteins was found in the sol phase of both HSMSL and whole saliva. How ever, a significant fraction was present in the gel phase of whole saliva, suggesting a post-secretory interaction with the salivary gel matrix. A mon oclonal antibody against a parotid salivary agglutinin was used to show tha t this protein is present mainly in the gel phase of both whole saliva and parotid secretion.