dictyNews
Electronic Edition
Volume 40, number 7
February 28, 2014
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Abstracts
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Properties of a non-bioactive fluorescent derivative of differentiation-
inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum
Yuzuru Kubohara 1, Haruhisa Kikuchi 2, Yusuke Matsuo 2, Yoshiteru
Oshima 2 and Yoshimi Homma 3
1 Department of Molecular and Cellular Biology, Institute for Molecular
and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
2 Laboratory of Natural Product Chemistry, Tohoku University Graduate
School of Pharmaceutical Sciences, Sendai 980-8578, Japan
3 Department of Biomolecular Science, Institute of Biomedical Sciences,
Fukushima Medical University School of Medicine, Fukushima
960-1295, Japan
Biology Open, in press
Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold
Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3
(Bu-DIF-3) are potent anti-tumor agents. To investigate the activity of DIF-
like molecules in tumor cells, we recently synthesized a green fluorescent
DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular
localization. In this study, we synthesized a red (orange) fluorescent DIF-3
derivative, BODIPY-DIF-3R, and compared the cellular localization and
bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer
cells. Both fluorescent compounds penetrated the extracellular membrane
within 0.5 h and localized mainly to the mitochondria. In formalin-fixed cells,
the two BODIPY-DIF-3s also localized to the mitochondria, indicating that
the BODIPY-DIF-3s were incorporated into mitochondria independently of
the mitochondrial membrane potential. After treatment for 3 days, BODIPY-
DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and
suppressed cell proliferation. Interestingly, the swollen mitochondria were
stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added
to isolated mitochondria in vitro, BOIDPY-DIF-3G dose-dependently
increased the rate of O2 consumption, but BODIPY-DIF-3R did not. These
results suggest that the bioactive BODIPY-DIF-3G suppresses cell
proliferation at least in part by altering mitochondrial activity, whereas the
non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not
affect mitochondrial activity or cell proliferation.
Submitted by Yuzuru Kubohara [[log in to unmask]]
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N-glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum
by 'off-line' liquid chromatography and mass spectrometry.
Hykollari A, Dragosits M, Rendic D, Wilson IBH, Paschinger K.
Electrophoresis. 2014 Feb 26. doi: 10.1002/elps.201300612.
[Epub ahead of print]
In this study, we have performed the first mass spectrometric analysis of
N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium
discoideum, previously shown to have a defect in glucosidase II. Together
with glucosidase I, this enzyme mediates part of the initial processing of
N-glycans; defects in either glucosidase are associated with human diseases
and result in an accumulation of incorrectly-processed oligosaccharides
which are not, or only poor, substrates for a range of downstream enzymes.
To examine the effect of the glucosidase II mutation in Dictyostelium, we
employed off-line LC-MALDI-TOF-MS in combination with chemical and
enzymatic treatments and MS/MS to analyse the neutral and anionic
N-glycans of the mutant as compared to the wild-type. The major neutral
species were, as expected, of the composition Hex10-11 HexNAc2-3 with
one or two terminal glucose residues. Consistent with the block in processing
of neutral N-glycans caused by the absence of glucosidase II, fucose was
apparently absent from the N-glycans and bisecting N-acetylglucosamine
was rare. The major anionic oligosaccharides were sulphated and/or
methylphosphorylated forms of Hex8-11 HexNAc2-3 , many of which
surprisingly lacked glucose residues entirely. As anionic N-glycans are
considered to be mostly associated with lysosomal enzymes in Dictyostelium,
we hypothesise that glycosidases present in the acidic compartments may
act on the oligosaccharides attached to such slime mould proteins.
Furthermore, our chosen analytical approach enabled us, via observation of
diagnostic negative-mode MS/MS fragments, to determine the fine structure
of the methylphosphorylated and sulphated N-glycans of the M31
glucosidase mutant in their native state.
This article is protected by copyright. All rights reserved.
Submitted by Iain Wilson [[log in to unmask]]
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[End dictyNews, volume 40, number 7]
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