| Abstract: | The major sulfated protein of the mouse pancreatic acinar cell, gp300, hsa been identified and characterized with monoclonal and polyclonal antibidies. gp300 is a glycoprotein of Mr = 300,000 which contains ～40% of metabolically incroporated 35S] sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis. demonstrating that the sulfate is carbohydrate-linked rather than tyrosine-linked. gp300 metabolically labeled with 3H]glucosamine and 35H]sulfate was chemically and enzymaticlly treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycan. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released ～17% of 3S]. Mild alkaline borohydride treatment after removal of N-linked sugar relased the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthetic studies and PNGase F digestion indicating the presence of sulfated O-linked oligosaccharides. Biosunynthetic studies and PNGase digestion F digestion indicate that the core protein is ～210 KDa, with apparent contrinution of ～35 KDa N-linked sugar, and ～55 KDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presece of Galβ(1–3)GalNAc and sialic acid α(2–3)Gal in O-linked oligosaccharide, and Galβ(1–4)GLcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral memberane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in reponse to hormone stimulation ofacini, so it is not a secertroy product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule bigeneses. |
