CHARACTERIZATION OF THE MAJOR SULFATED PROTEIN OF MOUSE PANCREATIC ACINAR-CELLS - A HIGH-MOLECULAR-WEIGHT PERIPHERAL MEMBRANE GLYCOPROTEIN OF ZYMOGEN GRANULES

The major sulfated protein of the mouse pancreatic acinar cell, gp300, has been identified and characterized with monoclonal and polyclonal antibodies. gp300 is a glycoprotein of M(r) = 300,000 which contains similar to 40% of metabolically incorporated [S-35]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 [H-3]glucosamine and [S-35]sulfate was chemically and enzymatically treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycans. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released similar to 17% of [H-3] and little [S-35]. Mild alkaline borohydride treatment after removal of N-linked sugar released the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthetic studies and PNGase F digestion indicate that the core protein is similar to 210 kDa, with apparent contributions of similar to 35 kDa N-linked sugar, and similar to 55 kDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presence of Gal beta(1-3)GalNAc and sialic acid alpha(2-3)Gal in O-linked oligosaccharide, and Gal beta(1-4)GlcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral membrane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in response to hormone stimulation of acini, so it is not a secretory 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 biogenesis. (C) 1994 Wiley-Liss, Inc.