Human Mannose 6-Phosphate-uncovering Enzyme Is Synthesized as a Proenzyme That Is Activated by the Endoprotease Furin

Do, Hung; Lee, Wang-Sik; Ghosh, Pradipta; Hollowell, Tracy; Canfield, William M.; Kornfeld, Stuart

doi:10.1074/jbc.m202369200

Cited by 43 publications

(47 citation statements)

References 29 publications

(25 reference statements)

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“…Cleavage of Pro-CNP by Purified Recombinant Furin-LoVo cells were transfected with a pro-CNP-expressing plasmid or the control vector pcDNA and grown in Opti-MEM medium for 24 h. The cells (ϳ10 6 ) were washed once with phosphate-buffered saline and lysed in 1 ml of 100 mM HEPES, 1% Triton X-100, 1 mM 2-mercaptoethanol, and 1 mM calcium chloride (30). Purified recombinant human furin was added to the cell lysate, and the mixture was incubated at 30°C for 2 h. Cleavage of pro-CNP was analyzed by Western blotting using an anti-V5 antibody.…”

Section: Fig 2 Processing Of Pro-anp and Pro-cnp In Transfected Sw mentioning

confidence: 99%

Furin-mediated Processing of Pro-C-type Natriuretic Peptide

Pan

et al. 2003

Journal of Biological Chemistry

186

110

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C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family that is involved in a variety of homeostatic processes. Here we characterize the processing essential for the conversion of the precursor, human pro-CNP, to the biologically active hormone. In human embryonic kidney 293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a mature peptide intracellularly as detected by Western analysis. Expression of recombinant human corin, a proatrial natriuretic peptide convertase, did not enhance the processing of pro-CNP in these cells. The processing of pro-CNP was inhibited in the presence of an inhibitor of the endoprotease furin but was not affected by inhibitors of matrix metalloproteinases and tumor necrosis factor-␣ convertase. In furin-deficient human colon adenocarcinoma LoVo cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of recombinant human furin in LoVo cells restored the ability of these cells to process pro-CNP. Furthermore, incubation of purified recombinant human furin with LoVo cell lysate containing pro-CNP led to the conversion of the precursor to a mature peptide. The furin-processed CNP was shown to be biologically active in a cell-based cGMP assay. These results demonstrate that furin is a critical enzyme for the processing of human pro-CNP.The natriuretic peptide family consists of three structurally related peptides: atrial natriuretic peptide (ANP), 1 brain or B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (1-7). ANP and BNP are produced mainly in cardiomyocytes in the heart and are important in maintaining normal body fluid and sodium homeostasis. CNP is expressed in many tissues and cell types, including the brain, vascular endothelial cells, and chondrocytes (8 -13). The dominant receptor for CNP is natriuretic peptide receptor-B, whereas the receptor for ANP and BNP is natriuretic peptide receptor-A. The biological functions of CNP are apparently different from those of ANP and BNP. Studies have shown that CNP inhibits the proliferation of vascular smooth muscle cells in culture (14, 15) and prevents balloon injury-induced coronary artery restenosis in animal models (16 -18). Recent studies of CNPdeficient mice indicate that CNP plays an important role in chondrocyte differentiation and bone formation (11).The natriuretic peptides are synthesized as prepropeptides. The signal peptide is removed to form propeptides, but a further proteolytic cleavage of the propeptide is required to convert the precursor to a biologically active peptide. This activation mechanism is critical in regulation of the activity of the natriuretic peptides, but the enzyme(s) responsible for the conversion remained uncharacterized for many years. Recently, we identified a cardiac serine protease, corin (19), that is a member of the type II transmembrane serine protease family (20,21). In cell-based functional studies, we showed that corin converted pro-ANP to biologically active ANP in a highly sequence-specific manner (22...

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Section: Fig 2 Processing Of Pro-anp and Pro-cnp In Transfected Sw mentioning

confidence: 99%

Furin-mediated Processing of Pro-C-type Natriuretic Peptide

Pan

et al. 2003

Journal of Biological Chemistry

186

110

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“…This enzyme is well known for catalyzing the second step in the synthesis of mannose-6-phosphate tag on lysosomal hydrolases destined to ER from trans-Golgi [27]. There was no report of human diseases associated with NAGPA until Kang et al [14] found mutations in this gene from unrelated stuttering individuals.…”

Section: Lysosomal Enzyme-targeting Pathway and Stutteringmentioning

confidence: 99%

Recent advances in genetic studies of stuttering

Kang

2015

J Genet Med

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“…Amino terminus sequencing showed that the ␣-subunit begins at Asp 21 in the signal peptide, leaving an intact HPC4 epitope on the protein. The ␤-and ␥-subunits contained the amino terminus sequence identical to the predicted sequence (Fig.…”

Section: Expression Of Er-targeted Glcnac-phosphotransferase ␣/␤-Subumentioning

confidence: 99%

“…Interestingly, UCE, the second enzyme in Man-6-P biosynthesis, is one of the substrates of furin and activated by furin in the trans-Golgi network (21). Unlike UCE, GlcNAc-phosphotransferase does not contain a consensus cleavage sequence for furin.…”

mentioning

confidence: 99%

Structural Requirements for Efficient Processing and Activation of Recombinant Human UDP-N-acetylglucosamine:Lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

Kudo

Canfield

2006

Journal of Biological Chemistry

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Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomalenzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an ␣ 2 ␤ 2 ␥ 2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the ␣-and ␤-subunits are encoded by a single gene as a precursor, whereas the ␥-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the ␣-and ␤-subunits but not the ␥-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the ␣-and ␤-subunits. Because this modification prevented precursor processing to mature ␣-and ␤-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified ␣/␤-subunits (␣/␤-subunits) precursor and wild type ␥-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an ␣ 2 ␤ 2 ␥ 2 -subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAcphosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid ␣-glucosidase in vitro.Most lysosomal hydrolases are targeted to the lysosome via mannose 6-phosphate (Man-6-P) 2 -modified N-glycans. The initial step of Man-6-P biosynthesis is catalyzed by the membrane-bound enzyme UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17), which transfers GlcNAc 1-phosphate from UDP-GlcNAc to mannose in high mannose-type glycans (1-3). The second step is removal of GlcNAc by N-acetylglucosamine-1-phosphodiester ␣-GlcNAcase (UCE) to expose mannose 6-phosphate (4, 5).Previously, we reported that the bovine GlcNAc-phosphotransferase has an ␣ 2 ␤ 2 ␥ 2 -configuration (6). We also found that GlcNAc-phosphotransferase is encoded by two genes; one encoding the ␣/␤-subunits precursor (7,8) and the other, the ␥-subunit (9). Characterization of the cDNAs revealed that the ␣-and ␤-subunits contain amino and carboxyl terminus transmembrane domains, respectively, whereas the ␥-subunit does not. The mechanism responsible for the processing of the ␣/␤-subunits precursor into the mature ␣-and ␤-subunits is not yet known, but likely to be the result of protease(s) in the endoplasmic reticulum (ER) or cis-Golgi, where GlcNAc-phosphotransferase resides (24). Access to the cDNAs for all three subunits of GlcNAc-phosphotransferase made it possible to express recombinant GlcNAc-phos...

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Human Mannose 6-Phosphate-uncovering Enzyme Is Synthesized as a Proenzyme That Is Activated by the Endoprotease Furin

Cited by 43 publications

References 29 publications

Furin-mediated Processing of Pro-C-type Natriuretic Peptide

Furin-mediated Processing of Pro-C-type Natriuretic Peptide

Recent advances in genetic studies of stuttering

Structural Requirements for Efficient Processing and Activation of Recombinant Human UDP-N-acetylglucosamine:Lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

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