Glycomimetic affinity-enrichment proteomics identifies partners for a clinically-utilized iminosugar

Article abstract of DOI:10.1039/c3sc50826a

Widescale evaluation of interacting partners for carbohydrates is an underexploited area. Probing of the 'glyco-interactome' has particular relevance given the lack of direct genetic control of glycoconjugate biosynthesis. Here we design, create and utilize a natural product-derived glycomimetic iminosugar probe in a Glycomimetic Affinity-enrichment Proteomics (Glyco-A^eP) strategy to elucidate key interactions directly from mammalian tissue. The binding partners discovered here and the associated genomic analysis implicate a subset of chaperone and junctional proteins as important in male fertility. Such repurposing of existing therapeutics thus creates direct routes to probing in vivo function. The success of this strategy suggests a general approach to discovering 'carbohydrate-active' partners in biology.

Full text of DOI:10.1039/c3sc50826a

Chemical Science
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Glycomimetic affinity-enrichment proteomics identifies
partners for a clinically-utilized iminosugar†
Cite this: Chem. Sci., 2013, 4, 3442
a
b
c
d
e
Isa N. Cruz,‡ Conor S. Barry,‡ Holger B. Kramer, C. Celeste Chuang, Sarah Lloyd,
d
d
a
b
Aarnoud C. van der Spoel,§ Frances M. Platt, Min Yang* and Benjamin G. Davis*
Widescale evaluation of interacting partners for carbohydrates is an underexploited area. Probing of the
glyco-interactome’ has particular relevance given the lack of direct genetic control of glycoconjugate
‘
biosynthesis. Here we design, create and utilize a natural product-derived glycomimetic iminosugar
e
probe in
a
Glycomimetic Affinity-enrichment Proteomics (Glyco-A P) strategy to elucidate key
Received 26th March 2013
Accepted 4th July 2013
interactions directly from mammalian tissue. The binding partners discovered here and the associated
genomic analysis implicate a subset of chaperone and junctional proteins as important in male fertility.
Such repurposing of existing therapeutics thus creates direct routes to probing in vivo function. The
success of this strategy suggests a general approach to discovering ‘carbohydrate-active’ partners in biology.
DOI: 10.1039/c3sc50826a
www.rsc.org/chemicalscience
7
active proteome. One such conceivable strategy is to use clini-
cally-utilized compounds to identify novel binding partners; this
Introduction
Approximately 2–3% of most genomes are devoted to carbohy- advantageously would allow the re-purposing of pre-approved
1
drate-active proteins (CAP). These include lectins that bind drugs and hence facilitate rapid translation and application.
carbohydrates, glycosylhydrolases that degrade them and gly-
cosyltransferases that construct them (http://www.cazy.org).
One glycomimetic suitable for this purpose is the iminosu-
gar n-butyldeoxynojirimycin (NB-DNJ, FDA-approved in 2002 as
Zavescaꢀ (miglustat)). NB-DNJ is prescribed for the treatment
2
Deciencies of CAP can lead to pathological states, such as
3
congenital muscular dystrophies and lysosomal storage disor-
of type 1 Gaucher disease, an inherited lysosomal storage
4
8–10
ders; CAPs are implicated in a range of host–pathogen inter-
disorder and Niemann–Pick type C disease;
in humans, NB-
5,6
actions that lead to disease. Despite these vital roles, most DNJ is generally well-tolerated. This powerful glycomimetic is
current strategies for determining their interactions (e.g. arrays therefore an archetype of modulation of glycobiology by small
or assays), whilst powerfully allowing the determination of in molecules. One of its most remarkable properties is that in
11
vitro specicities, do not allow widescale probing of cellular or certain mouse strains (from the C57-lineage, e.g. C57BL/6,
organismal samples. Affinity strategies have rarely been used to AKR/J and BALB/c) it induces reversible, dose-dependent male
identify novel binding partners or prole the carbohydrate-
10,12
infertility at very low doses (15 mg per kg per day).
In
contrast, other strains (of the Swiss Castle lineage, such as FVB/
a
13
Department of Pharmaceutical & Biological Chemistry, UCL School of Pharmacy, N ) display a phenotype insensitive to NB-DNJ-induced infer-
University College London, 29/39 Brunswick Square, London, WC1N 1AX, UK.
12
tility. Studies with C57BL/6$FVB/N interstrain hybrid mice
E-mail: min.yang@ucl.ac.uk; Fax: +44(0)2077535964; Tel: +44(0)2077535876
b
have suggested multiple genes (and hence multiple protein
targets) contribute to this striking function (infertility) induced
Department of Chemistry, Chemistry Research Laboratory, Oxford University,
Manseld Road, Oxford, OX1 3TA, UK. E-mail: ben.davis@chem.ox.ac.uk; Fax:
12
by NB-DNJ. This raises the intriguing possibility that modu-
lation of the carbohydrate-active proteome may be intimately
linked to reproduction. There are some early indications of the
+
44(0)1865285002; Tel: +44(0)1865275652
c
Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks
Road, Oxford, OX1 3PT, UK
d
Department of Pharmacology, University of Oxford, Manseld Road, Oxford, OX1 origins of this exciting effect. Treated mice display lower sperm
3
QT, UK
counts and abnormal sperm morphologies (deformed or no
e
MRC Prion Unit, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK
10,12
acrosomes, non-falciform nuclei ) rendering them incapable
e
†
Electronic supplementary information (ESI) available: Synthesis of glyco-A P
14
of binding the zona pellucida to initiate fertilization. However,
matrix, protein interrogation, 2D-gel proteomics
&
data, pathway
&
the exact protein targets are unknown – providing a suitably
single-nucleotide polymorphism analysis. A separate data set le describing all
of the proteins identied is also available. No live animals or human subjects challenging test of our strategy. NB-DNJ inhibits b-glucosidase 2
were involved in this research. All work using animal tissues followed all local (GBA2), lysosomal acid b-glucosidase I (GBA) and glucosylcer-
ethics and legal requirements. See DOI: 10.1039/c3sc50826a
15–17
amide synthase (GCS/UGCG).
Knockout of GBA2 impairs
‡
These authors contributed equally to this work.
18
mouse fertility and creates sperm abnormalities.
The
§
Present address: Atlantic Research Centre, Dalhousie University, University
epididymal spermatozoa of NB-DNJ-insensitive strains only
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Avenue, Halifax, Nova Scotia B3H 4R2, Canada.
3
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display minor morphological imperfections; accordingly, these mice) to identify proteins contributing to inducible infertility.
12
mice are normally fertile. However, all mouse strains show Protein fraction from homogenized tissue was split and inter-
e
similar elevated level of glucosylceramide when treated with rogated (see ESI†) with either glyco-A P probe matrix or
NB-DNJ. This suggests no direct link to glycosphingolipid unmodied agarose (control); washing removed unbound
metabolizing enzymes GBA/GBA2 and/or GCS/UGCG and fractions. The many selected or enriched binding proteins were
implicates instead the differences in genetic background and eluted from respective matrices with NB-DNJ-containing buffer
other protein partners. In fact, to date, no comprehensive study and were concomitantly further fractionated and directly visu-
of the cellular targets of NB-DNJ has been conducted and the alized by comparative 2D-polyacrylamide gel electrophoretic
protein(s) involved in induced male infertility remain unknown. analysis (Scheme 1 and Fig. 1), detected by silver staining. These
We report here a comprehensive affinity-enrichment pro- were directly analyzed by in-gel digestion and proteomic LC-MS/
e
teomic (A P) study utilizing an immobilized glyco-affinity probe MS analysis. From 351 proteins identied, 64 that were also
e
to identify proteins that interact with NB-DNJ and potentially identied in the control were discounted from the glyco-A P
responsible for its contraceptive activity. Immobilized imino- screen (Fig. 2a). We applied the following strategy to minimize
sugars have previously been used for simple glycosidase affinity the chances of false-positive identication of contaminant
19–22
chromatography,
however, here we show in a proof-of- proteins: Only signicant (p-value cut-off < 0.05) protein iden-
concept study how this archetypal glycomimetic can allow tications were accepted throughout all searches. Protein hits
proteomics directed towards its interactome. Unlike designed in the enriched sample were only considered, if identied twice
purication methods, which intentionally exploit a known (in replicates 1 and 2). At the same time all protein hits which
ligand-protein partnership for affinity, we choose NB-DNJ here were found in either replicate of the control sample (replicate 1
as a clinically approved probe molecule that is known to induce or 2) were discarded and not considered as potential interacting
phenotypic changes but in the absence of any such clear part- proteins (see ESI and later for additional discussion†). This
e
nership(s). In this way, the ‘glyco-A P’ method has the potential generated a focused list of 18 proteins reliably identied
e
to identify unforeseen protein–ligand interactions that may be through glyco-A P probing (but not in controls) as strong
important in a therapeutically-relevant phenotype and so, as we carbohydrate-active candidates. Cross-validation of theoretical
show here, reveals a relevant, previously not considered, masses and isoelectric points allowed further narrowing of this
focused subset of the carbohydrate-active proteome.
focus and reduced the protein cohort to six (Table 1) with
plausible functional roles.
Two Hsp70 proteins (HSPA2 & Hypoxia up-regulated protein
Result and discussion
1
(HYOU1)) were identied. The testis functions on the brink of
26
23
hypoxia; low oxygen levels in the testis are required for sper-
A carboxyl-bearing probe ligand 1 derived from DNJ was
prepared in a two-step protecting-group-free synthesis employ-
ing methanolytic ozonolysis of cyclohexene followed by
reductive alkylation of DNJ (Scheme 1). The glyco-affinity probe
27
matogenesis. Proteins that are upregulated by low oxygen
concentrations, such as HYOU1 (Grp170), may play key roles in
spermatogenesis. Furthermore, HYOU1 directly regulates
24
28
(
(
Fig. 2b) insulin (INS) and vascular endothelial growth factor
1
was immobilized on amino-terminated agarose support
through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
EDAC)-mediated amide bond formation (Scheme 1). The
29
VEGF). Abnormal VEGF levels in seminal plasma correlate
30
with IVF pregnancy success. INS affects reproductive function
in humans and animals at multiple levels by effecting endocrine
control of spermatogenesis, as well as on mature ejaculated
(
e
binding ability of the resulting glyco-A P probe matrix was
validated with a clinically-utilized GBA preparation (ESI
31
16,25
spermatozoa. The second Hsp70 protein, HSPA2, is a testis-
Fig. 1†);
this importantly conrmed functional utility
specic form in mice, where it is regulated developmentally and
through extraction of a known protein partner.
Next, tissue was collected from a mammal with relevant
phenotype (mouse testis from NB-DNJ-sensitive male C57BL/6
32
expressed in spermatogenic cells. It has a unique role during
33
germ cell differentiation and is necessary for progression of
32
meiosis in mouse germ cells.
Junction plakoglobin (JUP, desmoplakin 3) is a junctional
plaque protein involved in the formation of desmosomes and
34
tight junctions. Mice with impaired ability to form tight
-
/-
junctions (e.g. Epas1 ) display higher testicular oxygen levels,
27
which interferes with spermatogenesis. It is conceivable that
desmosome disruption by NB-DNJ may affect spermatogenesis
by such a mechanism. Indeed reversible male infertility has
been demonstrated by unrelated small molecules that disrupt
35
adherens junctions in the testis.
Intriguingly, we have discovered here the protein SET (also
known as template activating factor 1b, a histone chaperone for
36
37
nucleosome assembly and as I2PP2A ). I2PP2A inhibits
multifunctional protein phosphatase PP2A. Notably, I2PP2A
binds sphingolipids, including ceramide and PTY720, a clinical
e
Scheme 1 Glyco-A P probe matrix and method.
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Fig. 1 Comparative 2D-polyacrylamide gel electrophoretic analysis of C57BL/6 mouse testis tissue interrogated with unmodified control (panel A) and glycomimetic
affinity (panel B) matrices. pI scale 3–10. Middle: molecular weight marker.
38
sphingosine analogue. Given that miglustat, in its inhibition
of GCS, competitively inhibits ceramide binding, it may be
39
miglustat binds SET/I2PP2A in a similar manner.
Interestingly, several of the cohort proteins are chaperones:
not only HYOU1, HSPA2, and SET but also T-complex protein 1
subunit zeta (CCT6A), a chaperone protein involved in the
40
folding of tubulin and actin and other proteins. Notably,
NB-DNJ sensitive strains (C57BL/6) show decreased levels of
acrosomal proteins, aer treatment with NB-DNJ, compared
12
with non-sensitive strains (FVB/N). It is possible that NB-DNJ
impairment of chaperone protein function may be responsible
for this phenotype. Some of the selectively identied partners
have no obvious potential role. The acidic leucine-rich nuclear
phosphoprotein 32 family members (ANP32) (spot 45) have
been implicated in a number of cellular processes including cell
41
cycle progression, differentiation and apoptosis. However,
gene disruption studies of ANP32A produced mouse strains
42
which were both viable and fertile suggesting that these
proteins are not directly implicated in NB-DNJ induced male
infertility and potentially highlight a functionally unrelated
interaction.
Together these data implicate a glycomimetic-interactome
(Hsps, junctional proteins, chaperones and ceramide binders)
that would not have been readily predicted but that now suggest
functionally plausible pathways (e.g., the effects of chaperones
and tight junction assembly on spermiogenesis) for investiga-
tion that could lead to validated drug targets for contraception.
Inspection of the genetic differences that code for this proteome
between sensitive and insensitive mice allows identication of
single-nucleotide polymorphisms (SNPs). SNP analysis (see ESI
for further details†) was applied to genes encoding for the
e
proteins identied using the glyco-A P strategy examining
e
differences between miglustat-sensitive strain C57BL/6 and
Fig. 2 (a) Venn analysis of glyco-A P proteins identified (here at score > 20 – see
e
13
ESI for other score Venn analyses†). (b) Pathway analysis of primary glyco-A P- insensitive strain FVB/N. Annotated gene sequences were
identified target HYOU1 (Pathway Studio 9). HYOU1 is influenced by Glc and
searched 5 kb both upstream and downstream. No SNPs were
found for Hyou1, Jup, Anp32b or Set but were for Cct6a (180),
directly regulates VEGFA & INS as possible drivers of temporary male infertility.
Hspa2 (57 total) and Anp32a (367) (see ESI†). In Cct6a
3
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e
b
Table 1 Significant proteins identified from mouse testis with putative NB-DNJ affinities using glyco-A P
Spot
Accession number
Protein name
Code
Score
M
r
(Da)
pI
% Coverage
a
7
5
7
4
3
4
7
5
Q9JKR6
Q02257
P17156
O35381
Hypoxia up-regulated protein 1
Junction plakoglobin
Heat shock-related 70 kDa protein 2
Acidic leucine-rich nuclear
phosphoprotein 32 family member A
Protein SET
HYOU1
JUP
HSPA2
ANP32A
291
65
92
111 340
82 490
69 884
28 691
5.12
5.75
5.51
3.99
68
37.3
51
86
23.9
4
6
9
0/61
Q9EQU5
P80317
SET
CCT6A
49
45
33 358
58 424
4.22
6.63
29.1
51.8
T-complex protein 1 subunit zeta
a
b
Bold italic indicates proteins unique to the enriched sample; bold indicates enriched. Membrane-bound CAP glycosidases & glycosyltransferases
7
e
require bespoke separation methods (e.g. sucrose gradient of golgi membranes) and this may lead to under-representation in glyco-A P.
4
5–48
(
NM_009838) the majority of the SNPs were found in non- logic,
the detailed examination of the modulation of glyco-
0
0
coding regions including the 3 untranslated region (3 UTR). biological function has, until now, not examined such valuable
However, six changes were found in the coding region: ve of interactomes. Here using a glyco-A P strategy that exploits
e
which do not change the corresponding amino acid. One in the archetypal iminosugar mimetic and therapeutic NB-DNJ/
exon 9, Chr5 position 130299586 has the SNP designation miglustat/Zavesca we have elucidated a focused subsection
rs13470985 and changes amino acid 348 from E (C57BL/6) to G of the proteome hypothetically relevant to mammalian
in (FVB/N). In Hspa2 (NM_001002012) again non-coding region reproduction.
0
SNPs were the majority (including the 3 UTR); three SNPs were
These data suggest new interactions (Hsps, junctional
identied in the coding region but these do not change the proteins, chaperones, ceramide binders) that would not have
corresponding amino acids. In Anp32 (NM_009672) only SNPs been readily predicted and that might help dene the mecha-
0
in non-coding regions (including 3 UTR) were found. As well as nisms by which NB-DNJ causes male infertility. Notably, the
the single CCT6A-E348G difference identied, it cannot be primary probe used here (1) is a derivative of the natural
49
discounted that the identied non-coding changes may affect product nojirimycin. The increasing utility of both function-
50
gene regulation. However, in the absence of microarray analysis alized natural products and therapeutics, as two classes of
of FVB/N testis tissue it is not yet possible to compare expres- small molecules that have been selected in contrasting manners
sion levels.
for protein interaction, suggests that future affinity strategies
It should be noted that there might be inherent variability in might also be usefully based on their exploitation. To this end
e
tissue sample, phenotypic state and through handling varia- we are currently exploring extension of the glyco-A P strategy to
tions. However, it is noteworthy that the method showed other small molecule, natural product glycomimetics.
e
apparent robustness in this regard; an additional glyco-A P
experimental round using testis tissue successfully identied
with high signicance ve out of the six proteins identied in
Acknowledgements
the primary rounds (see ESI†). Preliminary experiments using We thank the Computational Biology Research Group, Nuffield
brain tissue did not identify these proteins.
Department of Clinical Medicine, Oxford for maintenance of
In addition, the methods of data analysis, performed here the Mascot server, the Fundaç~
ao para a Ci ˆe ncia e a Tecnologia,
using the MOWSE scoring system as implemented by MASCOT Portugal (INC) and BBSRC (CSB) for nancial support and Dr
43
(
see ESI†) will have a profound effect upon initial agreements Andreas Schatzlein, Holger Hummer, Benjamin Thomas and
on identied proteins (see the ESI† for extended discussion and Svenja Hester for technical assistance. BGD is a Royal Society
analysis of the effect of scoring thresholds). Notably, at low Wolfson Research Merit Award recipient.
thresholds (Fig. 2a, score > 20) there is lower initial overlap
between enriched sets, whereas at higher thresholds (ESI, >50†) References
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