Sequential Proteolytic Processing of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) Protein by Ectodomain Shedding and γ-Secretase-dependent Intramembranous Cleavage

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

Download Full-text

Calreticulin Biosynthesis and Processing in Human Myeloid Cells: Demonstration of Signal Peptide Cleavage and N-Glycosylation

Blood ◽

10.1182/blood.v90.1.372 ◽

1997 ◽

Vol 90 (1) ◽

pp. 372-381 ◽

Cited By ~ 28

Author(s):

Gerene M. Denning ◽

Kevin G. Leidal ◽

Valerie A. Holst ◽

Shankar S. Iyer ◽

Doran W. Pearson ◽

...

Keyword(s):

Signal Peptide ◽

Human Lymphocytes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Myeloid Cells ◽

Myeloid Cell ◽

Proteolytic Processing ◽

Peptide Cleavage ◽

Wide Range

Abstract Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

Download Full-text

Endosomal Localization of TLR8 Confers Distinctive Proteolytic Processing on Human Myeloid Cells

The Journal of Immunology ◽

10.4049/jimmunol.1401375 ◽

2014 ◽

Vol 193 (10) ◽

pp. 5118-5128 ◽

Cited By ~ 37

Author(s):

Noriko Ishii ◽

Kenji Funami ◽

Megumi Tatematsu ◽

Tsukasa Seya ◽

Misako Matsumoto

Keyword(s):

Myeloid Cells ◽

Proteolytic Processing

Download Full-text

Proteolytic Processing of Neuregulin 2

Molecular Neurobiology ◽

10.1007/s12035-019-01846-9 ◽

2019 ◽

Vol 57 (4) ◽

pp. 1799-1813 ◽

Cited By ~ 2

Author(s):

Maria Czarnek ◽

Joanna Bereta

Keyword(s):

Immunohistochemical Staining ◽

Hippocampal Neurons ◽

Neuronal Cell ◽

Brain Structures ◽

Proteolytic Processing ◽

Erbb Receptors ◽

Ectodomain Shedding ◽

Post Translational Modifications ◽

Neuronal Cell Lines ◽

Egf Family

AbstractNeuregulin 2 (NRG2) belongs to the EGF family of growth factors. Most of this family members require proteolytic cleavage to liberate their ectodomains capable of binding and activating their cognate ErbB receptors. To date, most of the studies investigating proteolytic processing of neuregulins focused on NRG1, which was shown to undergo ectodomain shedding by several ADAM proteases and BACE1 and the remaining fragment was further cleaved by γ-secretase. Recently, NRG2 attracted more attention due to its role in the neurogenesis and modulation of behaviors associated with psychiatric disorders. In this study, we used genetic engineering methods to identify proteases involved in proteolytic processing of murine NRG2. Using non-neuronal cell lines as well as cultures of primary hippocampal neurons, we demonstrated that the major proteases responsible for releasing NRG2 ectodomain are ADAM10 and BACE2. Co-expression of NRG2 and BACE2 in neurons of certain brain structures including medulla oblongata and cerebellar deep nuclei was confirmed via immunohistochemical staining. The cleavage of NRG2 by ADAM10 or BACE2 generates a C-terminal fragment that serves as a substrate for γ-secretase. We also showed that murine NRG2 is subject to post-translational modifications, substantial glycosylation of its extracellular part, and phosphorylation of the cytoplasmic tail.

Download Full-text

A Disintegrin and Metalloproteinase 9 Is Involved in Ectodomain Shedding of Receptor-Binding Cancer Antigen Expressed on SiSo Cells

BioMed Research International ◽

10.1155/2014/482396 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 3

Author(s):

Kenzo Sonoda ◽

Kiyoko Kato

Keyword(s):

Receptor Binding ◽

Uterine Cancer ◽

Proteolytic Processing ◽

Soluble Form ◽

Ectodomain Shedding ◽

Cancer Antigen ◽

Poor Overall Survival ◽

A Disintegrin And Metalloproteinase ◽

Metalloproteinase 9 ◽

Protease Expression

In several human malignancies, the expression of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is associated with aggressive characteristics and poor overall survival. RCAS1 alters the tumor microenvironment by inducing peripheral lymphocyte apoptosis and angiogenesis, while reducing the vimentin-positive cell population. Although proteolytic processing, referred to as “ectodomain shedding,” is pivotal for induction of apoptosis by RCAS1, the proteases involved in RCAS1-dependent shedding remain unclear. Here we investigated proteases involved in RCAS1 shedding and the association between tumor protease expression and serum RCAS1 concentration in uterine cancer patients. A disintegrin and metalloproteinase (ADAM) 9 was shown to be involved in the ectodomain shedding of RCAS1. Given the significant correlation between tumor ADAM9 expression and serum RCAS1 concentration in both cervical and endometrial cancer as well as the role for ADAM9 in RCAS1 shedding, further exploration of the regulatory mechanisms by which ADAM9 converts membrane-anchored RCAS1 into its soluble form should aid the development of novel RCAS1-targeting therapeutic strategies to treat human malignancies.

Download Full-text

Proteolytic processing of the amyloid-beta protein precursor of Alzheimer's disease

Essays in Biochemistry ◽

10.1042/bse0380037 ◽

2002 ◽

Vol 38 ◽

pp. 37-49 ◽

Cited By ~ 27

Author(s):

Janelle Nunan ◽

David H Small

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Amyloid Beta ◽

Alternative Pathway ◽

Protein A ◽

Proteolytic Processing ◽

Gamma Secretase ◽

Amyloid Beta Protein ◽

Protein Precursor ◽

Amyloid Beta Protein Precursor

The proteolytic processing of the amyloid-beta protein precursor plays a key role in the development of Alzheimer's disease. Cleavage of the amyloid-beta protein precursor may occur via two pathways, both of which involve the action of proteases called secretases. One pathway, involving beta- and gamma-secretase, liberates amyloid-beta protein, a protein associated with the neurodegeneration seen in Alzheimer's disease. The alternative pathway, involving alpha-secretase, precludes amyloid-beta protein formation. In this review, we describe the progress that has been made in identifying the secretases and their potential as therapeutic targets in the treatment or prevention of Alzheimer's disease.

Download Full-text