scholarly journals Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity.

1984 ◽  
Vol 98 (2) ◽  
pp. 407-416 ◽  
Author(s):  
S Hickman ◽  
J L Theodorakis ◽  
J M Greco ◽  
P H Brown
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Immunoglobulin A ◽  
Complex Type ◽  
Mannose Residue ◽  
Alpha Chain ◽  
Oligosaccharide Processing ◽  
Carbonyl Cyanide ◽  
High Mannose ◽  
Initial Processing

The processing of asparagine-linked oligosaccharides on the alpha-chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N-acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.

scholarly journals Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells.

1984 ◽  
Vol 99 (1) ◽  
pp. 296-305 ◽  
Author(s):  
C A Gabel ◽  
S Kornfeld
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Line ◽  
Golgi Complex ◽  
Proteolytic Processing ◽  
Acid Hydrolases ◽  
Complex Type ◽  
Processing Enzymes ◽  
Oligosaccharide Processing ◽  
Mannose 6 Phosphate ◽  
Ionophore Monensin

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes. At this time 30% of the pulse-labeled IgA was still intracellular. The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides. This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the beta-glucuronidase had reached lysosomes. Therefore, beta-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex. The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides. In contrast, monensin only slightly delays the transport of newly synthesized beta-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex. However, the labeled beta-glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex.

scholarly journals Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits

1986 ◽  
Vol 239 (3) ◽  
pp. 679-683 ◽  
Author(s):  
A McElduff ◽  
A Watkinson ◽  
J A Hedo ◽  
P Gorden
Keyword(s):  
Insulin Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Beta Subunits ◽  
Relative Distribution ◽  
Single Chain ◽  
Complex Type ◽  
Mannose Residue ◽  
Predominant Species ◽  
High Mannose

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

scholarly journals Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat α1-proteinase inhibitor and α1-acid glycoprotein

1986 ◽  
Vol 236 (3) ◽  
pp. 853-860 ◽  
Author(s):  
V Gross ◽  
T A Tran-Thi ◽  
R T Schwarz ◽  
A D Elbein ◽  
K Decker ◽  
...  
Keyword(s):  
Proteinase Inhibitor ◽  
De Novo ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Complex Type ◽  
Glucose Residue ◽  
Acid Glycoprotein ◽  
Glucosidase Inhibitors ◽  
Oligosaccharide Processing ◽  
High Mannose

The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted.

scholarly journals Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

1998 ◽  
Vol 337 (1) ◽  
pp. 133-140 ◽  
Author(s):  
Adrie J. M. VERHOEVEN ◽  
Bernadette P. NEVE ◽  
Hans JANSEN
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Trafficking ◽  
De Novo ◽  
Hepatic Lipase ◽  
Brefeldin A ◽  
Apparent Increase ◽  
Triacylglycerol Hydrolase ◽  
Oligosaccharide Processing ◽  
Carbonyl Cyanide ◽  
Catalytically Active

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of α1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8±2.1-fold higher (mean±S.D., n = 7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3±0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose–heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7±0.3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not α1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

scholarly journals Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

1987 ◽  
Vol 104 (3) ◽  
pp. 749-760 ◽  
Author(s):  
W E Balch ◽  
K R Wagner ◽  
D S Keller
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Free System ◽  
A Cell ◽  
Vesicular Stomatitis ◽  
High Mannose

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

scholarly journals Accessibility to proteases of the cytoplasmic G protein domain of vesicular stomatitis virus is increased during intracellular transport.

1989 ◽  
Vol 109 (5) ◽  
pp. 2057-2065 ◽  
Author(s):  
D Mack ◽  
B Kluxen ◽  
J Kruppa
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Gel Electrophoresis ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Protein Domain ◽  
Complex Type ◽  
Carbonyl Cyanide ◽  
Vesicular Stomatitis ◽  
High Mannose

G1 and G2 are two forms of the membrane-integrated G protein of vesicular stomatitis virus that migrate differently in gel electrophoresis because G1 is modified by high-mannose and G2 by complex-type oligosaccharide side chains. The cytoplasmic domain in G1 is less exposed to cleavage by several proteases than in G2 molecules. Acylation by palmitic acid as well as inhibition of carbohydrate processing by swainsonine and deoxynojirimycin resulted in the same pattern of proteolytic sensitivity of both glycoproteins as in untreated cells. In contrast, accessibility of the cytoplasmic domain to proteases did not change when the intracellular transport of the G protein was blocked in carbonyl cyanide m-chlorophenylhydrazone- or monensin-treated BHK-21 cells, respectively. The results suggest that the increase in accessibility of the cytoplasmic tail of the G protein occurs after the monensin block in the trans-Golgi and might reflect a conformational change of functional significance--i.e., making the cytoplasmic domain of the viral spike protein competent for its interaction with the viral core, inducing thereby the formation of the budding virus particle.

scholarly journals Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method

1989 ◽  
Vol 257 (1) ◽  
pp. 43-49 ◽  
Author(s):  
S Kijimoto-Ochiai ◽  
Y U Katagiri ◽  
T Hatae ◽  
H Okuyama
Keyword(s):  
Sialic Acid ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Complex Type ◽  
Type Analysis ◽  
Mannose Residue ◽  
Hybrid Type ◽  
Oligosaccharide Chain ◽  
Ricinus Communis Agglutinin ◽  
High Mannose

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a ‘bisecting’ acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.

scholarly journals Pharmacological Targeting of the ER-Resident Chaperones GRP94 or Cyclophilin B Induces Secretion of IL-22 Binding Protein Isoform-1 (IL-22BPi1)

2019 ◽  
Vol 20 (10) ◽  
pp. 2440
Author(s):  
Paloma Gómez-Fernández ◽  
Andoni Urtasun ◽  
Ianire Astobiza ◽  
Jorge Mena ◽  
Iraide Alloza ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Protein ◽  
Secreted Protein ◽  
Complex Type ◽  
Cyclophilin B ◽  
Interleukin 22 ◽  
Unfolded Protein ◽  
Protein Isoform ◽  
High Mannose ◽  
Protein Response

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.

scholarly journals Recycling glycoproteins do not return to the cis-Golgi.

1988 ◽  
Vol 107 (1) ◽  
pp. 79-87 ◽  
Author(s):  
J J Neefjes ◽  
J M Verkerk ◽  
H J Broxterman ◽  
G A van der Marel ◽  
J H van Boom ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Lines ◽  
Complex Type ◽  
Cell Biol ◽  
Site Of Action ◽  
High Mannose ◽  
Surface Glycoproteins

Recycling of a number of glycoproteins along the site of action of mannosidase I (the distal endoplasmic reticulum/cis-Golgi) was followed in several different cell lines. Treatment of cells with 1-deoxymannojirimycin (dMM) produced high mannose oligosaccharides at positions otherwise occupied by complex-type oligosaccharides in these glycoproteins. Conversion of high-mannose-type oligosaccharides to complex oligosaccharides of proteins initially synthesized in the presence of dMM was used as a marker for recycling of glycoproteins along the site of action of dMM. In contrast to findings reported by Snider and Rogers (Snider, M. D., and O. C. Rogers. 1986. J. Cell Biol. 103:265-275), removal of dMM did not result in reconversion of high-mannose oligosaccharides to complex-type sugars, even after prolonged periods of culture. We conclude that surface glycoproteins do not recycle through the cis-medial Golgi elements.

Biosynthesis, processing and transport of storage proteins and lectins in cotyledons of developing legume seeds

1984 ◽  
Vol 304 (1120) ◽  
pp. 309-322 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Storage Proteins ◽  
Proteolytic Processing ◽  
Protein Bodies ◽  
Side Chains ◽  
Transport Pathway ◽  
Legume Seeds ◽  
High Mannose ◽  
Processing Steps

The storage proteins and lectins that accumulate in the protein bodies of developing legume cotyledons undergo a number of processing steps along the transport pathway from their site of synthesis to their site of deposition. The polypeptides are synthesized on polysomes attached to the endoplasmic reticulum. Synthesis of the polypeptides is always accompanied by the co-translational removal of a signal peptide. Those proteins that are glycoproteins in their mature form are co-translationally glycosylated with high-mannose oligosaccharide side chains. Co-translational sequestration into the lumen of the endoplasmic reticulum is followed by the formation of oligomers. Transport of these oligomers to the Golgi complex may occur via tubular connections between the endoplasmic reticulum and the Golgi. In the Golgi complex some of the high-mannose side chains are modified by the removal of five to six mannosyl residues, and the addition of fucosyl and terminal A-acetylglucosaminyl residues. This phenomenon has so far been observed only for phytohaemagglutinin, the lectin of Phaseolus vulgaris . From the Golgi complex the storage proteins and lectins are transported to the protein bodies. This transport is mediated by small electron-dense vesicles. In the protein bodies two types of processing occur: proteolytic processing resulting in the formation of smaller polypeptides, and glycolytic processing resulting in the removal of the terminal N -acetylglucosaminyl residues from the modified carbohydrate side chains. All storage proteins and lectins undergo some of these processing steps, and specific examples are discussed in this paper.

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