Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Aug 1;381(Pt 3):867-75.
doi: 10.1042/BJ20031824.

Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides

Howard R Mellor  1 , David C A Neville, David J Harvey, Frances M Platt, Raymond A Dwek, Terry D Butters
Affiliations

Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides

Howard R Mellor et al. Biochem J. .

Abstract

In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861-866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes a-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1-3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER a-glucosidase inhibition to date.

PubMed Disclaimer

Figures

Figure 1
Figure 1. HPLC chromatograms illustrating the identification of Glcα3Man, Glcα3Glcα3Man and Glcα2Glcα3Glcα3Man structures (small FOS) by enzyme digestion with α-glucosidases I and II
The oligosaccharides marked X, Y and Z, which were found in the extracts of HL60 cells treated with NB-DNJ and NN-DNJ, were identified by incubating the 2-AB-labelled carbohydrates with purified ER carbohydrate-processing enzymes α-glucosidases I and II. The samples were subsequently analysed by normal-phase HPLC, as described in the text, and the peaks were found to be Glcα3Man-2AB (X), Glcα3Glcα3Man-2AB (Y) and Glcα2Glcα3Glcα3Man-2AB (Z). The structures of the relevant peaks and the positions of the products of each digestion are shown. The other peaks in the chromatogram are GSL-derived oligosaccharides as described in the accompanying paper [2a]. (A) NB-DNJ (1 mM), no enzyme; (B) NB-DNJ (1 mM), α-glucosidase I; (C) NB-DNJ (1 mM), α-glucosidases I and II. ○, mannose; □, glucose.
Figure 2
Figure 2. HPLC chromatograms showing optimized recovery of large FOS derived from HL60 cells treated with N-alkylated DNJs
FOS were extracted from HL60 cells treated for 16 h with or without imino sugar analogues, as described in the text, using optimized methods for recovery. Samples were prepared in 4 mM MgCl2 to which methanol and chloroform were added to obtain upper and lower phases. Carbohydrates were purified from the upper aqueous phase using an Oasis™ cartridge and de-salted using a GlycoClean S cleanup cartridge. The sugars were 2-AB labelled and analysed by normal-phase HPLC as described in the text. Chromatograms shown are representative of those obtained from three HL60 cell extracts obtained after treatment with or without imino sugar analogues. (A) DNJ (2 mM); (B) NB-DNJ (1 mM); (C) NN-DNJ (100 μM); (D) C18-DNJ (10 μM); (E) NB-DGJ (1 mM); (F) control, no imino sugar. The relationships between peak number, GU values, structure and abundance are given in Table 2.
Figure 3
Figure 3. HPLC chromatograms showing digestion of large FOS from NB-DNJ-treated and control HL60 cells with α-glucosidases I and II
FOS were extracted from HL60 cells and fluorescently labelled as described in the text. 2-AB-labelled carbohydrates were dried under vacuum and treated with α-glucosidase I and/or α-glucosidase II for 16 h at 37 °C before HPLC analysis, as described in the text. Note the α-glucosidase susceptible peaks 9 and 37 in the undigested control chromatogram (E). (A) NB-DNJ (1 mM), no enzyme; (B) NB-DNJ (1 mM), α-glucosidase I; (C) NB-DNJ (1 mM), α-glucosidase II; (D) NB-DNJ (1 mM), α-glucosidases I and II; (E) control, no enzyme; (F) control, α-glucosidase I; (G) control, α-glucosidase II; (H) control, α-glucosidases I and II. In (B), peaks labelled ‘n’ show new species generated following α-glucosidase I digestion. The relationships between peak number, GU values, structure and abundance are given in Table 2.
Figure 4
Figure 4. HPLC chromatograms showing glucosidase-I- and II-digested large FOS from NB-DNJ-treated and control HL60 cells following treatment with jack—bean α-mannosidase
2-AB-labelled carbohydrates were prepared in 100 mM sodium acetate buffer, pH 5.0, to which jack-bean α-mannosidase was added at a final enzyme concentration of 25 units/ml. Samples were mixed and incubated at 37 °C for 24 h. The products of the enzyme digestions are shown after incubation with α-mannosidase and the peaks are labelled with their structures. (A) NB-DNJ (1 mM); (B) control, no imino sugar; (C) Glc3Man7GlcNAc2. *, contaminant present prior to enzyme digest; ▪, GlcNAc; ○, mannose.
Figure 5
Figure 5. HPLC chromatograms showing digestion of large FOS from NB-DNJ-treated and control HL60 cells with Endo H
2-AB-labelled carbohydrates were prepared in 10 μl of sodium citrate buffer, pH 5.0, as described in the text and incubated with 1000 units of Endo H for 24 h at 37 °C. (A) NB-DNJ (1 mM), no enzyme; (B) NB-DNJ (1 mM)+Endo H; (C) control, no imino sugar, no enzyme; (D) control, no imino sugar+Endo H. Peak numbers 19, 25, 35 and 37 in (A) and 14 and 37 in (C) represent oligosaccharide structures which were susceptible to Endo H digestion and, therefore, contained a chitobiose core. The relationships between peak number, GU values, structure and abundance are given in Table 2.
Scheme 1
Scheme 1. Overview of the effects of N-alkylated DNJ inhibition of carbohydrate processing in HL60 cells
N-alkylated DNJ analogues rapidly enter the cell (<1 min). The N-butyl/N-nonyl derivatives gain access to the lumen of the ER and inhibit N-linked oligosaccharide trimming by α-glucosidases I and II. For glycoproteins that require calnexin/calreticulin for correct folding, retention of two or three glucose residues prevents the interaction of the glycoprotein with the chaperone(s). The misfolded protein may still be processed to the final destination or may be translocated to the cytosol via the Sec61 channel. Prior to proteasomal degradation of the polypeptide, the glucosylated FOS-GlcNAc2 are released by a cytosolic PNGase. A cytosolic chitobiase generates FOS-GlcNAc1 from FOS-GlcNAc2. For glycoproteins that reach the Golgi, endomannosidase cleaves the α2 linkage between the glucose-substituted mannose residue and the remaining oligosaccharide, allowing circumvention of the block in oligosaccharide processing. Glucosylated FOS are found in cellular secretions, suggesting export from the Golgi. The sites of action of N-alkylated DNJs are shown, together with the relative rates of intracellular access. No information regarding the rate of ER entry is available. N-Alkyl-DNJ compounds also inhibit CGT that synthesizes glucosylceramide, the precursor for most GSLs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Butters T. D., van den Broek L., Fleet G. W. J., Krulle T. M., Wormald M. R., Dwek R. A., Platt F. M. Molecular requirements of imino sugars for the selective control of N-linked glycosylation and glycosphingolipid biosynthesis. Tetrahedron: Asymmetry. 2000;11:113–124.
    1. Platt F. M., Neises G. R., Dwek R. A., Butters T. D. N-butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. J. Biol. Chem. 1994;269:8362–8365. - PubMed
    1. Mellor H. R., Neville D. C. A., Harvey D. J., Platt F. M., Dwek R. A., Butters T. D. Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis. Biochem. J. 2004;381:861–866. - PMC - PubMed
    1. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 1985;54:631–664. - PubMed
    1. Helenius A., Trombetta E. S., Herbert D. N., Simons J. F. Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 1997;7:193–200. - PubMed

Publication types

MeSH terms