The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL

A Dragonetti, M Baldassarre, R Castino, M Démoz, A Luini, R Buccione, C Isidoro

Research output: Contribution to journalArticle

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Abstract

Basophils and mast cells contain a peculiar class of inflammatory granules that discharge their content upon antigen-mediated crosslinking of IgE-membrane receptors. The pathways for granule biogenesis and exocytosis in these cells are still largely obscure. In this study we employed the rat basophilic leukemia (RBL)/mast cell line to verify the hypothesis that inflammatory granules share common bioactive molecules and functional properties with lysosomes. We demonstrate that inflammatory granules, as identified by the monoclonal 5G10 antibody (which recognises an integral membrane protein) or by Toluidine Blue staining, have an intralumenal acidic pH, possess lysosomal enzymes and are accessible by fluid-phase and membrane endocytosis markers. In addition, we studied the targeting, subcellular localisation and regulated secretion of the lysosomal aspartic protease cathepsin D (CD) as affected by IgE receptor stimulation in order to obtain information on the pathways for granule biogenesis and exocytosis. Stimulation with DNP-BSA of specific IgE-primed RBL cells led to a prompt release of processed forms of CD, along with other mature lysosomal hydrolases. This release could be prevented by addition of EGTA, indicating that it was dependent on extracellular calcium influx. Antigen stimulation also induced exocytosis of immature CD forms accumulated by ammonium chloride, suggesting the existence of an intermediate station in the pathway for granule biogenesis still sensitive to regulated exocytosis. The targeting of molecules to secretory granules may occur via either a mannose-6-phosphate-dependent or mannose-6-phosphate-independent pathway. We conclude that endosomes and lysosomes in basophils/mast cells can act as regulated secretory granules or actually identify with them.

Original languageEnglish
Pages (from-to)3289-98
Number of pages10
JournalJournal of Cell Science
Volume113 ( Pt 18)
Publication statusPublished - Sep 2000

Fingerprint

Cathepsin D
Exocytosis
Mast Cells
Leukemia
Peptide Hydrolases
IgE Receptors
Cell Line
Basophils
Secretory Vesicles
Lysosomes
Antigens
Ammonium Chloride
Tolonium Chloride
Membranes
Endosomes
Egtazic Acid
Hydrolases
Endocytosis
Immunoglobulin E
Membrane Proteins

Keywords

  • Animals
  • Antigens
  • Basophils
  • Calcium
  • Cathepsin D
  • Dinitrophenols
  • Endocytosis
  • Endosomes
  • Exocytosis
  • Lysosomes
  • Mannosephosphates
  • Mast Cells
  • Rats
  • Serum Albumin, Bovine
  • Tumor Cells, Cultured

Cite this

The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL. / Dragonetti, A; Baldassarre, M; Castino, R; Démoz, M; Luini, A; Buccione, R; Isidoro, C.

In: Journal of Cell Science, Vol. 113 ( Pt 18), 09.2000, p. 3289-98.

Research output: Contribution to journalArticle

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T1 - The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL

AU - Dragonetti, A

AU - Baldassarre, M

AU - Castino, R

AU - Démoz, M

AU - Luini, A

AU - Buccione, R

AU - Isidoro, C

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N2 - Basophils and mast cells contain a peculiar class of inflammatory granules that discharge their content upon antigen-mediated crosslinking of IgE-membrane receptors. The pathways for granule biogenesis and exocytosis in these cells are still largely obscure. In this study we employed the rat basophilic leukemia (RBL)/mast cell line to verify the hypothesis that inflammatory granules share common bioactive molecules and functional properties with lysosomes. We demonstrate that inflammatory granules, as identified by the monoclonal 5G10 antibody (which recognises an integral membrane protein) or by Toluidine Blue staining, have an intralumenal acidic pH, possess lysosomal enzymes and are accessible by fluid-phase and membrane endocytosis markers. In addition, we studied the targeting, subcellular localisation and regulated secretion of the lysosomal aspartic protease cathepsin D (CD) as affected by IgE receptor stimulation in order to obtain information on the pathways for granule biogenesis and exocytosis. Stimulation with DNP-BSA of specific IgE-primed RBL cells led to a prompt release of processed forms of CD, along with other mature lysosomal hydrolases. This release could be prevented by addition of EGTA, indicating that it was dependent on extracellular calcium influx. Antigen stimulation also induced exocytosis of immature CD forms accumulated by ammonium chloride, suggesting the existence of an intermediate station in the pathway for granule biogenesis still sensitive to regulated exocytosis. The targeting of molecules to secretory granules may occur via either a mannose-6-phosphate-dependent or mannose-6-phosphate-independent pathway. We conclude that endosomes and lysosomes in basophils/mast cells can act as regulated secretory granules or actually identify with them.

AB - Basophils and mast cells contain a peculiar class of inflammatory granules that discharge their content upon antigen-mediated crosslinking of IgE-membrane receptors. The pathways for granule biogenesis and exocytosis in these cells are still largely obscure. In this study we employed the rat basophilic leukemia (RBL)/mast cell line to verify the hypothesis that inflammatory granules share common bioactive molecules and functional properties with lysosomes. We demonstrate that inflammatory granules, as identified by the monoclonal 5G10 antibody (which recognises an integral membrane protein) or by Toluidine Blue staining, have an intralumenal acidic pH, possess lysosomal enzymes and are accessible by fluid-phase and membrane endocytosis markers. In addition, we studied the targeting, subcellular localisation and regulated secretion of the lysosomal aspartic protease cathepsin D (CD) as affected by IgE receptor stimulation in order to obtain information on the pathways for granule biogenesis and exocytosis. Stimulation with DNP-BSA of specific IgE-primed RBL cells led to a prompt release of processed forms of CD, along with other mature lysosomal hydrolases. This release could be prevented by addition of EGTA, indicating that it was dependent on extracellular calcium influx. Antigen stimulation also induced exocytosis of immature CD forms accumulated by ammonium chloride, suggesting the existence of an intermediate station in the pathway for granule biogenesis still sensitive to regulated exocytosis. The targeting of molecules to secretory granules may occur via either a mannose-6-phosphate-dependent or mannose-6-phosphate-independent pathway. We conclude that endosomes and lysosomes in basophils/mast cells can act as regulated secretory granules or actually identify with them.

KW - Animals

KW - Antigens

KW - Basophils

KW - Calcium

KW - Cathepsin D

KW - Dinitrophenols

KW - Endocytosis

KW - Endosomes

KW - Exocytosis

KW - Lysosomes

KW - Mannosephosphates

KW - Mast Cells

KW - Rats

KW - Serum Albumin, Bovine

KW - Tumor Cells, Cultured

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VL - 113 ( Pt 18)

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JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

ER -