A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.
GP-2, a 75-kDa glycoprotein, was isolated from dog pancreatic zymogen granule membranes (ZGMs). In a carbohydrate-shift strategy, N-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP-2, respectively, by gas-phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library. The sequence of these clones revealed an open reading frame that encodes a protein of 509 amino acids, eight N-linked oligosaccharide attachment sites, and an N-terminal signal sequence absent from the mature form of GP-2 associated with ZGMs. The C terminus shows a 20-residue hydrophobic transmembrane domain preceded by a decapeptide containing potential phosphatidylinositol-glycan attachment sites. GP-2 completely released from ZGMs by exogenous phospholipase C showed similar immunochemical properties and electrophoretic mobilities compared to the form associated with ZGMs. A similar form of GP-2 was released from zymogen granules permeabilized with saponin and incubated in the absence of added phospholipase C. Kinetic analysis of GP-2 release at 0 degrees C and 37 degrees C suggested the presence of a granule enzyme responsible for endogenous release of GP-2 to granule contents and into the apical medium. The data indicate that GP-2 is a phosphatidylinositol-glycan-linked membrane protein released from the membrane of mature zymogen granules by an enzymatic mechanism. The cDNA structure presented here thus encodes both membrane-bound and free forms of GP-2.
Fukuoka
S
S
Laboratory of Cell and Molecular Biology, Dana Research Institute, Boston, MA.
Freedman
S D
SD
Scheele
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000502:physiology*; D004285:Dogs; D015723:Gene Library; D007425:Intracellular Membranes / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000502:physiology*; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes