leupeptins and castanospermine

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted

Topics: Amino Acid Sequence; Animals; Cell Line; CHO Cells; Cricetulus; Glycosylation; Humans; Indolizines; Leupeptins; Monocytes; Mutagenesis, Site-Directed; Mutation; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Sorting Signals; Protein Stability; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sphingomyelin Phosphodiesterase; Swainsonine; Tunicamycin

The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP/MXR/ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation.

Topics: 1-Deoxynojirimycin; Amino Acid Sequence; Amino Acid Substitution; Asparagine; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Glucosamine; Glycosylation; Humans; Indolizines; Leupeptins; Macrolides; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Sequence Data; Neoplasm Proteins; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Proteasome Endopeptidase Complex; Sequence Alignment; Tunicamycin; Ubiquitin

It is now well known that the addition and trimming of oligosaccharide side chains during post-translational modification play an important role in determining the fate of secretory, membrane, and lysosomal glycoproteins. Recent studies have suggested that trimming of oligosaccharide side chains also plays a role in the degradation of misfolded glycoproteins as a part of the quality control mechanism of the endoplasmic reticulum (ER). In this study, we examined the effect of several inhibitors of carbohydrate processing on the fate of the misfolded secretory protein alpha1 antitrypsin Z. Retention of this misfolded glycoprotein in the ER of liver cells in the classical form of alpha1 antitrypsin (alpha1-AT) deficiency is associated with severe liver injury and hepatocellular carcinoma and lack of its secretion is associated with destructive lung disease/emphysema. The results show marked alterations in the fate of alpha1 antitrypsin Z (alpha1-ATZ). Indeed, one glucosidase inhibitor, castanospermine (CST), and two mannosidase inhibitors, kifunensine (KIF) and deoxymannojirimycin (DMJ), mediate marked increases in secretion of alpha1-ATZ by distinct mechanisms. The effects of these inhibitors on secretion have interesting implications for our understanding of the quality control apparatus of the ER. These inhibitors may also constitute models for development of additional drugs for chemoprophylaxis of liver injury and emphysema in patients with alpha1-AT deficiency.

Topics: 1-Deoxynojirimycin; Alkaloids; alpha 1-Antitrypsin; Cell Line; Cell-Free System; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Glucosidases; Humans; Indolizines; Leupeptins; Mannosidases; Microsomes; Mutagenesis; Time Factors

To identify factors involved in the expression of ligand-gated ion channels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of delta-alpha-gamma-alpha-beta to enclose the ion channel. We previously demonstrated that calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermine to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the alpha-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway.

Topics: Acetylcysteine; Biopolymers; Calcium-Binding Proteins; Calnexin; Calreticulin; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dimerization; DNA, Complementary; Glucose; Heat-Shock Proteins; Humans; Hydrolysis; Indolizines; Isomerases; Leupeptins; Multienzyme Complexes; Polyubiquitin; Proteasome Endopeptidase Complex; Protein Binding; Protein Disulfide-Isomerases; Receptors, Nicotinic; Ribonucleoproteins; Transfection; Ubiquitins

The nonfusing muscle cell line BC3H1 expresses a family of muscle-specific proteins when the fetal bovine serum (FBS) concentration is reduced from 20 to 1%. We have used a series of glycosylation inhibitors to assess the role played by glycoproteins in the initiation of differentiation in this cell line. Tunicamycin (TNM) and 2-deoxy-D-glucose, added to cells when the FBS concentration was reduced, blocked creatine phosphokinase (CPK) induction by 70-95%. These effects were dose dependent and reversible. TNM and 2-deoxy-D-glucose also reversed CPK induction in differentiated cells. Leupeptin and N-acetylglucosamine did not reverse these effects. 1-Deoxynojirimycin, 1-deoxymannojirimycin, and swainsonine have no effect on induced CPK expression, whereas castanospermine, a glucosidase I inhibitor, blocked its induction completely. As attempts to use conditioned medium from cells grown in 1 or 20% FBS have no effect on this differentiation process we conclude that high mannose structures, but not complex form glycoproteins, bound to the surface of BC3H1 cells play a role in transducing signals for differentiation and are probable mediators of cell/cell contact.

Topics: 1-Deoxynojirimycin; Acetylglucosamine; Alkaloids; Animals; Cell Differentiation; Cell Line; Creatine Kinase; Deoxy Sugars; Deoxyglucose; Enzyme Induction; Glucosamine; Glycoproteins; Indolizines; Leupeptins; Membrane Glycoproteins; Membrane Proteins; Mice; Muscles; Oligosaccharides; Swainsonine; Tunicamycin

Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20), castanospermine caused the formation of novel transient forms of higher Mr than corresponding controls, indicating a blocked removal of glucose residues. For the first three enzymes, the 'mature' (Golgi-processed) forms were similar in size to or slightly smaller than corresponding controls and were, as shown for aminopeptidase N, endoglycosidase-H-sensitive, evidence of a blocked attachment of complex sugars. Maltase-glucoamylase did not undergo conversion into a 'mature' form, suggesting that, unlike other microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression. The proteinase inhibitor leupeptin partially restored the suppressed synthesis, indicating that the majority of the wrongly processed enzymes, probably because of conformational instability, become degraded soon after synthesis rather than being transported to the microvillar membrane.

Topics: Alkaloids; Aminopeptidases; Animals; Carbohydrate Metabolism; CD13 Antigens; Electrophoresis, Polyacrylamide Gel; Indolizines; Intestinal Mucosa; Leupeptins; Membrane Proteins; Microvilli; Organ Culture Techniques; Swine