Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.