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. 2002 Mar;22(5):1545-54.
doi: 10.1128/MCB.22.5.1545-1554.2002.

Loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion

Wolfram Antonin  1 , Martin Wagner, Dietmar Riedel, Nils Brose, Reinhard Jahn
Affiliations

Loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion

Wolfram Antonin et al. Mol Cell Biol. 2002 Mar.

Abstract

Syncollin is a small protein that is abundantly expressed in pancreatic acinar cells and that is tightly associated with the lumenal side of the zymogen granule membrane. To shed light on the hitherto unknown function of syncollin, we have generated syncollin-deficient mice. The mice are viable and show a normal pancreatic morphology as well as normal release kinetics in response to secretagogue stimulation. Although syncollin is highly enriched in zymogen granules, no change was found in the overall protein content and in the levels of chymotrypsin, trypsin, and amylase. However, syncollin-deficient mice reacted to caerulein hyperstimulation with a more severe pancreatitis. Furthermore, the rates of both protein synthesis and intracellular transport of secretory proteins were reduced. We conclude that syncollin plays a role in maturation and/or concentration of zymogens in zymogen granules.

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Figures

FIG. 1.
FIG. 1.
(A) Syncollin is conserved in mammals. A sequence comparison of syncollin derived from rat, mouse, and human is shown. Sequence alignment was performed using the programs ClustalW and Boxshade. Identical and conserved amino acids are darkly and lightly shaded, respectively. (B) Gene targeting of syncollin. Maps of the wild-type syncollin gene, the respective targeting vector, and the resulting mutant gene are shown. Positions of exons (black boxes with base pairs of corresponding cDNA) and restriction enzyme sites are indicated. The positions of the probe to identify wild-type and mutant alleles are indicated by open bars. Neo, neomycin resistance gene; TK, thymidine kinase gene. (C) Southern blot analysis of deletion mutations. Mouse tail DNA from adult wild-type (+/+) mice and mice heterozygous (+/−) or homozygous (−/−) for the mutation in syncollin genes were analyzed as described in Materials and Methods. Positions of wild-type (WT) and mutant (KO) alleles are indicated. (D) Syncollin expression in mice. Zymogen granules (20 μg/lane) from adult wild-type (+/+) mice and heterozygous (+/−) or homozygous (−/−) mice derived from three independent stem cell clones (syl6, syl8, and syl7) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for SCAMP as a marker for zymogen granules and syncollin (syc).
FIG. 2.
FIG. 2.
The structure of the pancreas at the light and electron microscopy level is normal in syncollin-deficient mice. (A and B) Micrographs of pancreatic sections, stained with hematoxylin and eosin, that were obtained from 90-day-old wild-type (A) and syncollin-deficient (B) mice. The pancreatic morphology is normal. Bars, 50 μm. (C to F) Electron micrographs obtained from thin sections of pancreas derived from wild-type (C and E) and syncollin-deficient mice (D and F) show no difference in acinar cell differentiation and zymogen granules (C and D; bars, 1 μm) or in morphology of the endoplasmic reticulum (E and F; bars, 250 nm).
FIG. 3.
FIG. 3.
Damage of the pancreas after induction of acute pancreatitis is more severe in syncollin-deficient mice than in wild-type littermates. The figure shows micrographs of pancreatic sections, stained with hematoxylin and eosin, obtained from wild-type (A, C, E, and G) and syncollin-deficient (B, D, F, and H) mice 6 h (A and B), 12 h (C and D), 18 h (E and F), and 36 h (G and H) after induction. Bar, 100 μm.
FIG. 4.
FIG. 4.
Time course of serum (A) and tissue (B) amylase levels of wild-type (triangles) and knockout (squares) animals after induction of acute pancreatitis. The values are means for six (A) and four (B) animals, and the bars indicate the range of the data points. Tissue amylase was normalized to DNA content.
FIG. 5.
FIG. 5.
Amylase release is normal in the pancreas of syncollin-deficient mice. Amylase release was measured using isolated pancreatic lobules prepared from wild-type (triangle) and knockout (squares) mice (four mice per group). Secretion is expressed as the percentage of amylase released into the medium with respect to the total amylase content (sum of amylase discharged into the medium and amylase retained in the tissue). Each value is the mean for the four independent experiments; bars indicate the range. (A) Dose dependence of carbachol-induced amylase release. (B) Discharge kinetics of amylase in response to 0.5 and 0.1 μM carbachol.
FIG. 6.
FIG. 6.
Protein synthesis is reduced in the pancreas of syncollin-deficient mice. Pancreatic lobules derived from wild-type (white bars) and knockout (gray bars) mice (four mice per group) were incubated in medium containing 3H-labeled amino acids. At the indicated time points an aliquot of the lobules was removed, washed, and homogenized. Trichloroacetic acid-precipitable (prec.) radioactivity and DNA concentration were determined. Protein synthesis is normalized to DNA content. Values (expressed in arbitrary units [a.u.]) are means for the four independent experiments; bars indicate the range.
FIG. 7.
FIG. 7.
Intracellular transport of proteins is slowed in syncollin-deficient mice. Pancreatic lobules derived from wild-type (triangle) and knockout (square) mice (two mice per group) were incubated for 5 min in medium containing 3H-labeled amino acids. After a chase to cold medium containing either no stimulants (open symbols) or 0.5 μM carbachol (filled symbols), aliquots of the medium (A) and the lobules (B) were removed as indicated, and trichloroacetic acid-precipitable radioactivity was determined. In panel B, the values were normalized to the DNA content of the homogenates (see the legend to Fig. 6). Values are means for the two independent experiments; bars indicate the range.
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