Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

Neefjes, Jacques; Lindhout, Josephine E; Broxterman, H. J. G.; Marel, Gijs A. van der; Boom, Jacques H. van; Ploegh, Hidde L.

doi:10.1016/s0021-9258(18)81795-1

Cited by 16 publications

(3 citation statements)

References 29 publications

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“…5 b, incubation of T. cruzi cells with DNJ concentrations up to 23-fold higher than those of [14C]glucose for 2 min did not affect incorporation of label into hot 10% trichloroacetic acid insoluble material (mainly amino acids in proteins). This last result also strongly suggested that the inhibitors entered T. cruzi cells by facilitated diffusion and not through an hexose transporter, the same as has been reported to occur for the mannose analog 1-deoxymannojirimycin in mammalian cells (27). Nevertheless, the cruzipain total content of lysosomes was partially affected by the drug: as shown in Fig.…”

Section: N J Does Not Affect T Cruzi Cell Growth Rate or The Parasite General Metabolism But Partially Affects Cruzipain Synthesis And Thsupporting

confidence: 79%

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.

Labriola

Cazzulo

Parodi

1995

The Journal of Cell Biology

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Abstract. It has been proposed that the UDPGlc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked GlclMan7.9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin-like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP-Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides.

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Section: N J Does Not Affect T Cruzi Cell Growth Rate or The Parasite General Metabolism But Partially Affects Cruzipain Synthesis And Thsupporting

confidence: 79%

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.

Labriola

Cazzulo

Parodi

1995

The Journal of Cell Biology

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“…Because MVBs continuously receive input of new membranes and sort cargoes toward diverse fates, notably including lysosomal proteolysis, we tested whether biosynthesis or degradation of GFP-CD63 contributes to the GFP decay measured during the time frame of our microscopy assay. A pulse-chase experiment with 1-deoxymannosidase I (DMM), an inhibitor of mannosidase I, 12 was used to assess the relative proportion of old GFP-CD63 bearing long polylactosaminoglycans versus newly synthesized GFP-CD63 carrying high-mannose N-linked glycans (migrating at a lower molecular weight on the gel) over time ( Figure 2 G), yielding a half-life longer than 24 h ( Figure 2 H), which was expected because CD63 is known to be highly stable. 13 These data indicate that the natural turnover of GFP-CD63 does not significantly contribute to the decay of endolysosomal GFP fluorescence following dimerizer addition.…”

Section: Resultsmentioning

confidence: 99%

Retrofusion of intralumenal MVB membranes parallels viral infection and coexists with exosome release

Perrin

Janssen

et al. 2021

Current Biology

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“…The concentrations of inhibitors in the growth medium were, therefore, 300-1500-fold higher than the I50 values. It is unknown whether the concentrations found in the lumen of the endoplasmic reticulum (where presumably glucosidase II is located) are similar to those of the external milieu, but it has been determined that deoxymannojirimycin (and probably DNJ) does not penetrate into mammalian cells through the hexose transporter but penetrates through facilitated diffusion (Neefjes et al, 1989). The fact that a 20-25-fold molar excess of DNJ or CNS over that of glucose did not interfere with the incorporation of label into trichloroacetic acid insoluble material suggests that the inhibitors and glucose penetrate into T. cruzi cells by different mechanisms.…”

Section: Discussionmentioning

confidence: 99%

A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum

Gañan¹,

Cazzulo²,

Parodi³

1991

Biochemistry

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N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.

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Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

Cited by 16 publications

References 29 publications

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.

Retrofusion of intralumenal MVB membranes parallels viral infection and coexists with exosome release

A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum

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