A novel pancreas-specific serpin (ZG-46p) localizes to the soluble and membrane fraction of the Golgi complex and the zymogen granules of acinar cells.
By immunoscreening a cDNA expression library of rat pancreas using a polyspecific antiserum raised against purified pancreatic zymogen granule membranes, we have identified a cDNA clone coding for a novel protein, named ZG-46p. The cDNA contains an ORF of 1215 bp coding for a protein of 405 amino acids with a calculated molecular mass of 46 kDa. Sequence analysis revealed high homologies to known serine protease inhibitors (serpins), e.g. human anti-thrombin III (47.2%) or human and rat anti-trypsin (44%). The highest homology is present in the serpin signature, a consensus sequence common to all serpins, as well as its flanking hinge region. Northern blot analysis reveals the exclusive expression of the novel serpin mRNA in the pancreas, both during embryonic development and in the adult gland but not in the acinar carcinoma cell line AR4-2J. In vitro translation experiments demonstrate that the protein is N-glycosylated, but in vivo and in vitro phosphorylation was not found in spite of multiple phosphorylation sites. By immunofluorescence studies pancreatic serpin was localized predominantly to the Golgi complex in a similar distribution as the marker protein TGN38. Western blot analysis of various subcellular fractions showed ZG-46p mainly as soluble protein in the Golgi but also in zymogen granule content. A minor but significant portion of the protein was firmly attached to both the zymogen granule and Golgi membranes as Triton X-114 extraction indicates. The cellular localization, the distribution in the soluble and membrane fraction of Golgi complex and zymogen granules, and the finding that pancreatic serpin is associated with aggregated secretory proteins suggest a role in the sorting of pancreatic enzymes during granule formation.
Chen
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CY
Department of Cell Biology and Cell Pathology, Philipps-University, Marburg/Germany.
Cronshagen
U
U
Kern
H F
HF
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000201:enzymology; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000378:metabolism; D005333:Fetus / Q000166:cytology; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D006031:Glycosylation; D006056:Golgi Apparatus / Q000737:chemistry* / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry* / Q000166:cytology / Q000196:embryology; D010190:Pancreatic Neoplasms; D011485:Protein Binding / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017422:Sequence Analysis, DNA; D015843:Serpins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000737:chemistry; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000502:physiology
D018076:DNA, Complementary; D008565:Membrane Proteins; D012333:RNA, Messenger; D015843:Serpins; D000681:Amylases; D010450:Endopeptidases