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Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells.

Kuliawat R, Klumperman J, Ludwig T, Arvan P - J. Cell Biol. (1997)

Bottom Line: By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent release of procathepsin B.Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules.Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 beta-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature beta-granules, while immunolabeling for cathepsin L, but not B, persists in mature beta-granules. By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

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The occurence of cathepsin L in IGs and mature granules of mouse islet β-cells. Ultrathin cryosections were double immunolabeled for proinsulin (5-nm gold) and cathepsin L (10-nm gold). (A) Overview of the Golgi (G) region. Cathepsin L labeling (small arrowheads) was clearly seen over proinsulin-positive IGs (i). The arrows point to coated buds on IGs. (B and C) Cathepsin L label was  abundantly observed over both immature (i) and mature granule profiles (M), although some granules were more extensively labeled  (large arrowheads) than others. mito, mitochondrion; P, plasmalemma. Bars, 200 nm.
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Figure 10: The occurence of cathepsin L in IGs and mature granules of mouse islet β-cells. Ultrathin cryosections were double immunolabeled for proinsulin (5-nm gold) and cathepsin L (10-nm gold). (A) Overview of the Golgi (G) region. Cathepsin L labeling (small arrowheads) was clearly seen over proinsulin-positive IGs (i). The arrows point to coated buds on IGs. (B and C) Cathepsin L label was abundantly observed over both immature (i) and mature granule profiles (M), although some granules were more extensively labeled (large arrowheads) than others. mito, mitochondrion; P, plasmalemma. Bars, 200 nm.

Mentions: Similar double labelings confirmed the presence of cathepsin L immunoreactivity in IGs, while substantial persistence of this labeling was observed in many granules that no longer contained proinsulin immunoreactivity (Fig. 10). Indeed, mature β-granules (identified with antiinsulin) were strongly, albeit heterogeneously, immunolabeled for cathepsin L (Fig. 11, A and B). Moreover, in triple immunolabeling, mature β-granules that could be strongly labeled for cathepsin L contained cathepsin B labeling at only low levels (Fig. 11 C). A quantitative analysis of β-cells immunolabeled with antiproinsulin was then performed to estimate the relative concentration of cathepsin B between IGs and mature granules. The mean labeling density fell an order of magnitude from 2.3 ± 0.35 (SEM) over IGs to 0.23 ± 0.06 over mature granules, and this decline was statistically significant (P < 0.005). In similar double labelings with anti–cathepsin L, the mean labeling density over IGs (2.5 ± 0.25) was not significantly different from that over mature granules (2.5 ± 0.68). These data clearly indicate that in mouse islets, both proenzymes can enter β-cell IGs, but ProL tends to remain to a much higher degree than ProB in mature storage granules.


Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells.

Kuliawat R, Klumperman J, Ludwig T, Arvan P - J. Cell Biol. (1997)

The occurence of cathepsin L in IGs and mature granules of mouse islet β-cells. Ultrathin cryosections were double immunolabeled for proinsulin (5-nm gold) and cathepsin L (10-nm gold). (A) Overview of the Golgi (G) region. Cathepsin L labeling (small arrowheads) was clearly seen over proinsulin-positive IGs (i). The arrows point to coated buds on IGs. (B and C) Cathepsin L label was  abundantly observed over both immature (i) and mature granule profiles (M), although some granules were more extensively labeled  (large arrowheads) than others. mito, mitochondrion; P, plasmalemma. Bars, 200 nm.
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Related In: Results  -  Collection

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Figure 10: The occurence of cathepsin L in IGs and mature granules of mouse islet β-cells. Ultrathin cryosections were double immunolabeled for proinsulin (5-nm gold) and cathepsin L (10-nm gold). (A) Overview of the Golgi (G) region. Cathepsin L labeling (small arrowheads) was clearly seen over proinsulin-positive IGs (i). The arrows point to coated buds on IGs. (B and C) Cathepsin L label was abundantly observed over both immature (i) and mature granule profiles (M), although some granules were more extensively labeled (large arrowheads) than others. mito, mitochondrion; P, plasmalemma. Bars, 200 nm.
Mentions: Similar double labelings confirmed the presence of cathepsin L immunoreactivity in IGs, while substantial persistence of this labeling was observed in many granules that no longer contained proinsulin immunoreactivity (Fig. 10). Indeed, mature β-granules (identified with antiinsulin) were strongly, albeit heterogeneously, immunolabeled for cathepsin L (Fig. 11, A and B). Moreover, in triple immunolabeling, mature β-granules that could be strongly labeled for cathepsin L contained cathepsin B labeling at only low levels (Fig. 11 C). A quantitative analysis of β-cells immunolabeled with antiproinsulin was then performed to estimate the relative concentration of cathepsin B between IGs and mature granules. The mean labeling density fell an order of magnitude from 2.3 ± 0.35 (SEM) over IGs to 0.23 ± 0.06 over mature granules, and this decline was statistically significant (P < 0.005). In similar double labelings with anti–cathepsin L, the mean labeling density over IGs (2.5 ± 0.25) was not significantly different from that over mature granules (2.5 ± 0.68). These data clearly indicate that in mouse islets, both proenzymes can enter β-cell IGs, but ProL tends to remain to a much higher degree than ProB in mature storage granules.

Bottom Line: By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent release of procathepsin B.Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules.Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 beta-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature beta-granules, while immunolabeling for cathepsin L, but not B, persists in mature beta-granules. By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

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