β-Glucose-1,6-Bisphosphate stabilizes pathological phophomannomutase2 mutants in vitro and represents a lead compound to develop pharmacological chaperones for the most common disorder of glycosylation, PMM2-CDG

Article abstract of DOI:10.3390/ijms20174164

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, β-glucose-1,6-bisphosphate, was synthesized and characterized via ³¹P-NMR. β-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. β-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.

Full text of DOI:10.3390/ijms20174164

International Journal of
Molecular Sciences
Article
β-Glucose-1,6-Bisphosphate Stabilizes Pathological
Phophomannomutase2 Mutants In Vitro and Represents
a Lead Compound to Develop Pharmacological Chaperones
for the Most Common Disorder of Glycosylation, PMM2-CDG
Maria Monticelli 1 , Ludovica Liguori 2,3, Mariateresa Allocca 2,3, Giuseppina Andreotti 3,
and Maria Vittoria Cubellis 1,3
*
1
Dipartimento di Biologia, Università Federico II, 80126 Napoli, Italy
Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università degli Studi della
Campania “Luigi Vanvitelli”, 81100 Caserta, Italy
2
3
Istituto di Chimica Biomolecolare—CNR, 80078 Pozzuoli, Italy
*
Correspondence: gandreotti@icb.cnr.it; Tel.: +39-081-867-5241
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 July 2019; Accepted: 22 August 2019; Published: 26 August 2019
Abstract: A large number of mutations causing PMM2-CDG, which is the most frequent disorder
of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone
to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2,
α
-glucose-1,6-bisphosphate. The compound,
β-glucose-1,6-bisphosphate, was synthesized and
characterized via 31P-NMR.
β-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding
induces a large conformational change that was predicted by the program PELE and validated in vitro
by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2,
as well as frequently encountered pathogenic mutants, was measured using thermal shift assay.
β-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses
natural esose-bisphosphates.
Keywords: PMM2-CDG; pharmacological chaperone; glucose-1,6-bisphosphate
1. Introduction
In humans there exist two phosphomannomutases, PMM1 and PMM2, that interconvert
mannose-6-phosphate (M6P) into
into -glucose-1-phosphate ( G1P) [
or -glucose-1,6-bisphospate, for their mutase activity. Besides being a mutase, PMM1 is phosphatase and plays a
α-mannose-1-phosphate (αM1P) and to a minor extent, glucose-6-phosphate
α
α
1]. Both enzymes require a sugar bisphosphate,
α-mannose-1,6-bisphosphate
α
prevalent role in the degradation of esose bisphosphate in particular in the presence of inosine monophosphate
(IMP) [2,3].
The following scheme summarizes the activities of PMM1 and PMM2.
α-hexose-1,6-P2 + PMM ꢀ hexose-6P (or α-hexose-1P) + PMM-P
(1)
α-hexose-1P (or hexose-6P) + PMM-P ꢀ α-hexose-1,6-P2 + PMM
PMM-P + H2O ꢀ PMM + Pi
(2)
(3)
hexose = mannose, (1) + (2) = phosphomannomutase activity
hexose = glucose, (1) + (2) = phosphoglucomutase activity
hexose = mannose or glucose, (1) + (3) = phosphatase activity
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www.mdpi.com/journal/ijms

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Only PMM2 has been associated with human diseases and is responsible for the most common
congenital disorder of glycosylation (CDG) [ ]. PMM2-CDG, also known as CDG-1A or Jaeken
4
syndrome, is a rare autosomic recessive disease without a cure. The disease is particularly frequent
in Scandinavian countries but is pan-ethnically distributed. More than 110 pathological mutations
have been associated with the disease [
with life [ ]. Usually, patients are composite heterozygous with one inactivating mutation and one
hypomorphic mutation, more rarely they carry two different hypomorphic mutations in heterozygosis
or one in homozygosity [ ]. R141H, which is the most common allele, is inactive because the mutation
5,6]. A complete absence of PMM2 activity is not compatible
7
8,9
affects the active site [10], but has never been observed in homozygosity [6].
Pharmacological chaperones (PCs) are low molecular weight molecules that are able to rescue
the activity of hypomorphic mutants if the active site is integer [11]. These mutants are intrinsically
functional and the low residual activity in the cell is due to their instability. Usually [12], but not
necessarily, pharmacological chaperones are non-covalent competitive inhibitors of their target
enzymes [13–15]. They bind and stabilize mutant enzymes, but natural substrates can compete with
them for the active site. In fact, the ultimate test for PC is proving that after the administration to
cells, the activity of their target enzyme increases. The genotypes associated with PMM2-CDG retain
residual activity and are in principle amenable of treatment with PCs since they all retain residual
activity. Even a slight increase in PMM2 activity produced by PCs could alleviate the symptoms
of the patients since heterozygous carriers with just 50% normal phosphomannomutase activity in
fibroblasts are asymptomatic, patients with approximately 25% normal enzymatic activity exhibit a
moderate clinical picture whereas patients with less than 10% normal enzymatic activity present a
severe phenotype [16
,17]. Common hypomorphic PMM2 mutants are thermo-sensitive, but can be
stabilized by low molecular weight molecules [
5
,
10 18 21]. In general, this has been observed on single
,
–
mutants, but in some cases, it has been proved in heterozygosity [22].
Some attempts have been carried out to find PC for PMM2-CDG either starting from large
collections of commercially available products [19,23] or from known ligands of PMM2 [10,18,22],
yet no drug is available.
Perez and coworkers [19] screened 10,000 small molecules from the Myria screen diversity collection
and found four hits that did not resemble chemically the natural ligands of PMM2. These molecules
were able to stabilize PMM2 mutants in vitro and rescued the activity in human cells. One molecule,
1-(3-chlorophenyl)-3,3-bis(pyridin-2-yl) urea, appeared very appealing for drug development since it
did not inhibit PMM2 and did not possess predictable adverse features. Perlestein and co-workers [23
]
undertook drug repositioning testing 2560 compounds consisting of FDA approved drugs, bioactive
tool compounds, and natural products, on a yeast strain whose phophomannomutase gene is defective.
Drug repositioning is a very useful approach to look for medicines for rare diseases because, in case of
success, it decreases the gap from bench to bedside [24]. Three compounds, α-cyano-4-hydroxycinnamic
acid, suramin hexasodium, and 20,20-bisepigallocatechin digallate, suppressed growth deficiencies
of the defective yeast strain. Their mechanism of action is not known because they were not
tested on purified mutant proteins. None of these molecules resembles natural ligands of PMM2.
α
-cyano-4-hydroxycinnamic acid enhanced PMM2 enzymatic activity in fibroblasts derived from
patients and in a nematode model of PMM2-CDG [25]. Since -cyano-4-hydroxycinnamic acid
α
shares the carboxylic acid-containing pharmacophore of aldose reductase inhibitors, Perlestein and
co-workers [25] tested other commercially available inhibitors of the same enzyme in the nematode
model and fibroblasts. They found that epalrestat, which is a safe, orally bioavailable, and brain
penetrant aldose reductase inhibitor used to treat diabetic peripheral neuropathy, rescued PMM2
enzymatic activity in both species. Epalrest is a monocarboxylic acid and contains a phenyl and a
rhodanine group and does not resemble chemically PMM2 ligand. Direct binding and stabilization to
mutant PMM2 proteins is not among the modes of actions proposed by Perlestein and co-workers for
epalrest [25].

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α
-D-Glucose 1,6-bisphosphate (
α
G16P) could be considered an effective chaperone to rescue
unstable pathological variants because it binds PMM2, induces the closure of the enzyme, raises the
melting temperature, and activates catalysis [10,18,22]. However, it is rapidly hydrolyzed by PMM1 in
particular in the presence of IMP [2,3].
In this paper, we describe a novel approach. We synthesized a molecule, β-glucose-1,6-bisphosphate
(βG16P) that is an analog of a natural ligand of PMM2, αG16P. βG16P and αG16P were fully
characterized by 31P-NMR. This technique can be very useful to measure the activity of PMM1
and PMM2 directly without the aid of ancillary enzymes. In silico observations using the program
PELE [26] show that
closure of the active site.
As it is the case for the majority of PCs used so far for other diseases,
β
G16P induces a large conformational change in the structure of PMM2 and the
G16P can bind PMM2 and stabilize the wild type and hypomorphic PMM2.
G16P is an inhibitor of its target
β
β
enzyme [12]. Compared to αG16P, the β anomer is more resistant to the hydrolysis by PMM1.
2. Results
2.1. Synthesis and 31P-NMR Characterization of βG16P
βG1P was synthesized from maltose with bacterial phosphorylase and phosphorylated with
phosphofructokinase to generate the bisphosphate sugar (about 40 mg were obtained starting from 650
mg of maltose).
β αG16P.
G16P was analyzed by 31P-NMR spectroscopy and compared to commercial
1H-decoupled one-dimensional 31P spectrum shows two signals, one at 5.30 pmm and one at 7.45 ppm,
for the P nucleus in the position 1, P(1), and in position 6, P(6) respectively (Figure 1A). Both signals
are deshielded compared to those of the
recorded for both anomers. The signals of
α
anomer (5.06 and 7.36 ppm). 2D HSQC 1H-31P spectra were
G16P are clearly resolved and distinguishable from those
β
of αG16P (Figure 1B,C).
Figure 1. 31P-NMR characterization of
bisphosphate (βG16P). (A B,C) Selected regions of the
α
-D-Glucose 1,6-bisphosphate (
α
G16P) and
β-glucose-1,6-
) 1H-decoupled one-dimensional 31P spectrum. (
1H-31P HSQC spectrum. Both the spectra were acquired in H2O + D2O 10% in the presence of EDTA
50 mM; ppm were referred to creatine phosphate (0 ppm, 31P scale) and to trimethylsilylpropanoic
acid (0 ppm, 1H scale). P(6) and P(1) indicate the positions of phosphorous nuclei in the molecules;
§: inorganic phosphate.
2.2. βG16P Binds PMM2 and Induces a Conformational Change
In silico docking was carried out to test the binding of βG16P onto PMM2 with the program
PELE [26]. PMM2 is a homodimer and each subunit is made up by a core (res 1–81; 189–247) and a cap
(res 86–185) domain, connected by hinge peptides. The structure of the enzyme is deposited in the
PDB with the code 2AMY. Due to the absence of a few atoms and to some disordered regions in 2AMY,
the structure cannot be used as such for in silico docking. In a previous paper of ours, we described

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how 2AMY can be fixed and a native-like model can be generated [10]. The native-like model, which
is in open conformation as 2AMY, was used to carry out in silico docking of G16P. The ligand was
initially placed far from the active site (~30 Å) and was free to explore the protein surface with no bias.
After a global search, a refinement was carried out. The binding of G16P binding into the active site
β
β
induces a large backbone motion and the closing up of PMM2. Actually, we observed two binding
modes, in one case the phosphate at position 1, P(1), interacts with the catalytic site (Asp12, Asp14,
and Asp 217 that form an acidic triad coordinated with Mg2+), in the other case, it is the phosphate
at position 6, P(6) to interact with the catalytic site. We will refer to the first case as P1_Mg mode
and the second case as P6_Mg mode. In Figure 2 we show the initial unbound PMM2 structure and
the two bound structures. It can be observed that the closure is tighter in the P6_Mg mode than in
P1_Mg mode. In Figure 3A,B, we show the residues that interact with
salt bridges in P1_Mg mode and P6_Mg mode respectively.
βG16P by hydrogen bonds or
Figure 2. In silico binding models for PMM2 and
mode ( ), or the phosphate proximal to C1, P1_Mg mode (
in the initial state is in cyan ( ); the two closed conformations are in green (
red; Mg2+ are shown as spheres.
β
G16P: either the phosphate proximal to C6, P6_Mg
), can interact with the catalytic site. PMM2
) and pink ( ); G16P is
A
B
A
,
B
A
B β
For comparison, we show the interactions that were observed when
the same PMM2 native-like structure (Figure 3C,D) with the same protocol [10]. PMM2 forms fewer
contacts with G16P than with G16P, in particular in P1_Mg mode, and Asp14 of acidic triad does not
come in contact with the phosphate. Upon G16P binding, the core and the cap domain close up and
αG16P was docked onto
β
α
β
the distance between Arg21 and Gln138 alpha carbons experiences the largest change. The number of
contacts correlates with the tightness of the closure of PMM2. In fact, we measured that the distance
between Arg21 and Gln138 alpha carbons is 12.2 and 9.8 Å for
βG16P respectively in P1_Mg mode and
P6_Mg mode and 7.7 and 7.4 for αG16P in P1_Mg mode and P6_Mg mode respectively.

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Figure 3. Interactions between PMM2 and ligands: residues that interact with βG16P by hydrogen
bonds or salt bridges in P1_Mg mode (A) and P6_Mg mode (B) respectively; residues that interact with
αG16P by hydrogen bonds or salt bridges in P1_Mg (C) mode and P6_Mg mode (D) respectively.
When the core and cap domains close up, the solvent accessibility of some residues changes.
In particular, the exposure of Arg21 to solvent decreases dramatically passing from 98% in the open
model to 15% in the closed model. The conformational change that is observed in silico can be tested
by limited proteolysis with trypsin. Under the conditions used in the experiment, neutral pH NaCl 150
mM, it had been demonstrated that wt-PMM2 is a dimer [18]. In Figure 4 we show the results obtained
incubating wt-PMM2 with the protease in the absence or in the presence of ligands and demonstrate
that
βG16P binding protects PMM2 from the protease as well as αG16P possibly by rendering the
enzyme more compact. The results obtained by SDS-PAGE are qualitative but were observed in three
independent experiments. The conformational change induced by
in a previous paper of ours [10].
αG16P has already been discussed
Figure 4. Limited proteolysis of wild type PMM2 by trypsin. Wild type PMM2 was incubated at 37 ◦C
with trypsin in a 50:1 ratio in the presence or the absence of G16P or G16P 0.5 mM. Aliquots were
β
α
withdrawn at specified times (0, 20, 60, 120 min) and analyzed by SDS-PAGE and Coomassie staining.
2.3. βG16P Inhibits PMM2
PMM2 has two enzymatic activities since it acts as a phosphomannomutase (interconversion of
α
M1P and M6P) or as phosphoglucomutase (interconversion of
αG1P and G6P) and in both cases a
bis-phosphate sugar activator is needed [1,18].

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Standard enzymatic tests for PMM2 require the use of ancillary enzymes. Dosing phosphoglucomutase
activity is simpler and requires only glucose6P dehydrogenase to monitor the formation of G6P
measuring the production of NADPH spectrophotometrically or fluorimetrically. We carried out the
fluorimetric assay of phophoglucomutase activity using different pairs of substrates and activators.
In Figure 5A, it can be observed that
activator ( G1P + G16P). The controls were carried out employing only beta (
alpha anomers (αG1P + αG16P).
β
G1P is not a substrate (
β
G1P +
α
G16P) and that G16P is not an
β
α
β
βG1P + βG16P) or only
Figure 5. PMM2 phosphoglucomutase activity monitored by fluorescence spectroscopy (
phosphomannomutase activity monitored by 31P-NMR (
). (A) A total of 0.08 g PMM2 were incubated
at room temperature with different combinations of or G1P as substrates and or G16P as
activators, in the presence of G6PDH and NADP+. (B) A total of 0.12
g PMM2 were incubated at 32 ◦C
with 1 mM -mannose-1-phosphate ( M1P); G16P 5 M was used as an activator, in the absence or
the presence of βG16P 5 or 50 µM.
A) and
B
µ
α
β
α
β
µ
α
α
α
µ
Standard spectrophotometric tests of mannomutase activity are more complex and require three
ancillary enzymes to generate NADPH. Alternatively, the formation of M6P and the consumption of
αM1P can be monitored directly by recording 31P-NMR spectra.
These experiments require the accumulation of several scans for each NMR spectrum to have a good
S/N ratio. They cannot be used to measure initial velocities, but show clearly all the phosphorylated
species present in the solution and permit to avoid the usage of ancillary enzymes. These experiments
confirm that βG16P is not an activator and show that it inhibits PMM2 (Figure 5B).
2.4. βG16P Stabilizes PMM2
We carried out a thermal shift assay to test the ability of
to stabilize PMM2. The effects of G16P and G16P were compared (Figure 6A). It can be observed
that both anomers stabilize PMM2 although the extent of the stabilization is higher for G16P: the
0.3 to 58.5
βG16P to work as a chaperone, i.e.,
α
β
α
increase of the melting temperature was about 4 ◦C in the presence of
β
G16P (from 54.8
±
±
0.5 ◦C) and about 9 ◦C in the presence of αG16P (from 54.8 ± 0.3 to 63.4 ± 0.4 ◦C).
α
G1P is a stabilizer of PMM2 too. Its effect is strengthened by vandate, an inhibitor of PMM2 which
mimics phosphate and recreates a non-covalent complex with
in the active site. G1P does not stabilize PMM2 either in the absence or in the presence of vanadate
(Figure 6B). This observation and the lack of activity (Figure 4) suggests that G1P does not bind PMM2.
αG1P similar to sugar 1,6-bisphosphate
β
β

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Figure 6. Thermal stability in the presence of ligands. wt-PMM2 0.3 mg/mL was incubated with
different ligands 0.5 mM (( G16P and G16P; ( G1P and G1P, with and without vanadate);
A
)
α
β
B)
α
β
the melting curves were measured in the presence of dithiothreitol (DTT) 1 mM and Sypro Orange 2.4x,
from 20 to 90 ◦C with increments of 1 ◦C/min.
We also tested the effect
β
G16P on two pathological mutants, F119L and V129M. Overall,
β
G16P
0.5 mM caused an increase of about 4 ◦C, from 46.1
±
0.4 to 50.3
±
0.◦4 ◦C, while
αG16P at the same
0.8 C. As far as V129M-PMM2 is
◦
concentration produces an increase of about 8 C, up to 54.2
±
concerned the melting temperature in the presence of
53.9 ± 0.4 ◦C, and of about 10 ◦C in the presence of αG16P, up to 58.1 ± 1.3 ◦C (Figure 7B).
β
G16P increases of about 7 ◦C, from 47.3
±
0.4 to
Figure 7. Thermal stability of pathological mutants in the presence of ligands. F119L-PMM2 (
A
) and
V129M-PMM2 ( ) 0.3 mg/mL were incubated with different ligands, G16P (0.5 mM), and β
B
α
G16P
(0.5 mM). The melting curves were measured in the presence of DTT 1 mM and Sypro Orange 2.4x,
from 20 to 90 ◦C with increments of 1 ◦C/min.
Cell viability tests showed that
0.5 mM.
βG16P is well tolerated by cells when administered at 0.25 or

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2.5. βG16P is a Poor Substrate of PMM1
α
G16P is degraded rapidly by PMM1 in particular in the presence of IMP [
2,
3]. We tested the
stability of
β
G16P to the hydrolysis by PMM1 both in the absence and in the presence of IMP using
31P-NMR. It is very convenient that the signals of
α
G16P and G16P can be clearly distinguished in
β
the spectra. Hence, the experiment can be carried out by incubating both bisphosphates with PMM1
and examining whether or not only the alpha anomer is hydrolyzed.
The results are summarized in Table 1. PMM1 hydrolyses
αG16P preferentially and the different
susceptibilities of the two anomers are larger in the presence of IMP.
Table 1. Phosphatase activity monitored by 31P-NMR. A total of 40
min at 32 ◦C with 145
G16P or G16P or a combination of both, in the absence (
(+) of inosine monophosphate (IMP) 170 µM.
µ
g PMM1 were incubated for 90
µM
α
β
−) or the presence
Bisphosphate (145 µM)
IMP (170 µM)
Residual αG16P (%)
Residual βG16P (%)
αG16P
βG16P
αG16P + βG16P
αG16P
βG16P
αG16P + βG16P
-
-
-
+
+
+
54.2 ± 1.6
-
-
62.5 ± 7.0
93.6 ± 1.2
-
53.3 ± 1.3
0
-
0
65.7 ± 11.4
74.6 ± 3.1
It was also noticed that the hydrolysis of
α
G16P produces Pi,
α
G1P, or G6P conversely the
hydrolysis of G16P produces Pi and G1P only, suggesting that βG1P is not a substrate of PMM1
β
β
(data not shown).
3. Discussion
Although several therapeutic approaches for PMM2-CDG [27
,
28] have been proposed including
32], and the increase
mannose supplementation [29 30], membrane-permeable -mannose-1-P [31,
,
α
mannose-6-P flux into glycosylation pathways [33], metformin [34] and acetazolamide [35], a drug
addressing unstable PMM2 mutants is not yet available.
In many cases, the reduced activity of PMM2 hypomorphic mutants is a consequence of their
reduced intracellular concentration that in turn is the consequence of the clearance of unstable proteins
by the quality control systems of the cell. Using the program SDM [36] we could predict that 70% of
pathological mutations are destabilizing. In such cases, PCs represent a good choice for patients [19].
There are two approaches to find pharmacological chaperones. The first one requires the screening
of large libraries of molecules. The effort, which is big both in terms of time and budget, does not
guarantee finding a drug ready for clinical trials but may produce a lead compound that requires further
maturation. The other approach requires an educated guess. One can start from the structures of known
ligands of the enzyme and try to modify them to improve their potency, their safety, and their half-life.
Although the second approach may seem reductive, it has produced drugs for other pathologies that
have been approved and introduced into clinical practice [37
,38]. In this paper, we applied the second
approach. We synthesized an analog of G16P, which is a natural ligand of PMM2.
α
α
G16P itself could
be considered an effective chaperone to rescue unstable pathological variants because it binds PMM2,
induces the closure of the enzyme, raises the melting temperature, and activates catalysis. Regrettably,
α
G16P is hydrolyzed by PMM1 in particular in the presence of IMP [2,3]. Although data concerning
the half-life of G16P in vivo are not available, it can be predicted that its hydrolysis by PMM1 can
α
hinder its utility as a drug. For this reason, we explored the possibility of employing an analogous
bisphosphate sugar that would not be degraded as easily as αG16P by PMM1.
We synthesized the anomer
technique because it consents to monitor most phosphorylated metabolites and to distinguish
from G16P anomers. We proved that G16P binds PMM2 in silico. Incidentally, our experiments
β
G16P and characterized it by NMR. 31P-NMR proved to be a useful
α
G16P
β
β

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confirm the usefulness of PELE [26] as a program for in silico docking. In fact, the number of
contacts and the extent of closure predicted by PELE for the two anomers are fully in line with the
experimental results.
β
G16P is a “classical” PC since it is a mild non-covalent inhibitor of PMM2. Usually, the chaperoning
effect of drugs is tested in vitro on wild type enzymes. In the case of PMM2 some mutants can be
expressed in E. coli and purified. Hence the chaperoning effect of G16P can be tested on pathological
β
mutants as well as on the wild type enzyme. We have chosen two mutants, F119L and V129M.
Particularly, F119L is a very frequent variant and it has been observed both in association with R141H
and in homozygosis [8,9,39]. Since F119 does not occur in the active site, the mutant is active although
less stable than the wild type. V129M is relatively common in Italy where it has been observed in
association with R141H [40]. Position 129 is not located in the active site and the mutant retains more
than 50% activity but is less stable than wt-PMM2 [41]. F119L and V129M are amenable for the cure with
pharmacological chaperones in principle. We could prove that
G16P is a poor substrate of PMM1 and for this reason, it can be preferred to
confident that the pharmacokinetics of G16P can be superior to those of G16P. The next step of the
βG16P stabilizes pathogenic mutants.
β
αG16P as a PC. We are
β
α
process that starts from the known ligand, would be enhancing its bioavailability reducing the charges by
chemical modification with nontoxic acetoxymethyl groups as proposed for mannose-1 phosphate [31]
and using the hydrophobic derivative
would be hydrolyzed generating G16P. Another possibility to facilitate the entrance of
by liposomes as proposed by Glycomine [42]. The stabilizing effect of G16P (through the usage
βG16P as a prodrug. Once inside the cell, the modifying groups
β
βG16P is offered
β
of hydrophobic derivatives or with liposomes) could be tested on fibroblasts derived from patients
either measuring the increase of PMM2 activity or monitoring the normalization of N-glycosylated
biomarkers [28].
4. Materials and Methods
4.1. Materials
α
-D-Glucose 1,6-bisphosphate potassium salt hydrate,
α-D-Glucose 1-phosphate disodium salt
hydrate,
α
-D(+)Mannose 1-phosphate sodium salt hydrate, Inosine 50-monophosphate disodium
salt hydrate, Adenosine 50-triphosphate disodium salt hydrate, Maltose phosphorylase from
Enterococcus sp., Fructose-6-phosphate Kinase from Bacillus stearothermophilus, Trimethylamine,
3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich
(Sigma-Aldrich, Milan, Italy). Trypsin was purchased from ICN Pharmaceuticals (MP Biomedicals
Germany GmbH).
AG1x8 Resin 200-400 Mesh Hydroxide Form was purchased from Bio-Rad (Bio-Rad Laboratories
Srl, Milan, Italy).
Sypro Orange was from Invitrogen Molecular Probes (Invitrogen Molecular Probes, Monza, Italy),
StepOneTM Real Time PCR System from Applied Biosystems (Applied Biosystems, Foster City, CA,
USA), strips from Sarstedt (Multiply–µStrip Pro 8-strip low profile) (Sarstedt Srl, Milan, Italy).
D(+)Maltose monohydrate,
β-Nicotinamide adenine dinucleotide phosphate sodium salt,
Glucose-6-phosphate Dehydrogenase, Creatine phosphate disodium salt thetrahydrate were from Alfa
Aesar (Alfa Aesar, Thermo Scientific, Kandel, Germany).
4.2. Synthesis of α-Glucose-1-Phosphate and β-Glucose-1,6-Bisphosphate
β
-glucose-1-phosphate and
minor changes. Briefly, for -glucose-1-phosphate a solution containing maltose 1 M, potassium
phosphate 100 mM pH 7.5, MgCl2 2 mM was incubated with maltose phosphorylase 5 U/mL for 2 h
at room temperature. Synthesis of G1P
G1P was verified by 31P-NMR and the concentration of
was estimated by comparison with an internal standard. -glucose-1,6-bisphosphate was produced
β-glucose-1,6-bisphosphate were prepared as described by [43] with
β
β
β
β

Int. J. Mol. Sci. 2019, 20, 4164
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incubating
temperature in a buffer containing potassium phosphate 25 mM, Hepes 14 mM pH 7.5.
G1P and G16P were purified on an AG1x8 Hydroxide Form column equilibrated with 0.01 M
β
G1P and ATP-Mg2+ (1:1 ratio) with phosphofructokinase 15 U/mL overnight at room
β
β
triethylammonium bicarbonate pH 7.5. A step gradient of triethylammonium bicarbonate from 0.01 M
to 1 M was used to elute the sugars; fractions (lyophilized and dissolved in water) were analyzed
by 31P-NMR.
βG1P was eluted at 0.3–0.4 M, βG16 was eluted at 0.8–0.95 M. Triethylammonium
bicarbonate 1 M was prepared by bubbling CO2 gas into a triethylammine 1 M solution until reaching
the desired pH.
4.3. 31P-NMR Spectroscopy
A Bruker AVANCETMIII HD spectrometer 400 MHz, equipped with a BBO BB-H&F-D
CryoProbeTM Prodigy fitted with a gradient along the Z-axis, was used for the NMR analysis
(Bruker Italia Srl, Milan, Italy).
The 1H-decoupled one-dimensional 31P (zgpg) spectra were recorded at 161.976 MHz; spectral
width 120 ppm, delay time 1.2 s, pulse width of 12.0 µs were applied.
The phase-sensitive 2D HSQC using Echo/Antiecho-TPPI gradient selection, with decoupling
during acquisition, (hsqcetgp) 1H-31P spectra were recorded at 400.13/161.97 MHz; spectral width
32 ppm, delay time 1.2 sec, pulse width of 12.0
applied. Coupling constant was 120 Hz; function type was non-uniform sampling, with a NUS amount
of 70%.
µ
s, frequency offset of 2nd nucleus 4.0 ppm were
−
4.4. Docking and Structure Analysis
In silico docking of βG16P was carried out as described for αG16P [10].
Briefly two different exploration runs were performed: (a) a global free search and (b) a
local refinement.
(a) The global search was performed by combining a long (6 Å) and a short (1.5 Å) ligand perturbation
steps, with a 75%/25% probability, respectively. Rotations were kept in the [0◦–90◦] range.
A randomly chosen search direction was kept for two Monte Carlo steps, allowing a more
complete exploration of the entire protein surface. No information about the bound structure
was used to drive the search. Anisotropic normale mode perturbation included the lowest six
modes, with maximum displacements of the alpha carbon of 1 Å. Within the lowest six modes,
a randomly chosen mode was kept for six steps to facilitate large conformational exploration.
(b) The local search used translations of only 0.5 A and rotations in the [0◦–180◦] range. Furthermore,
to keep the ligand in the active site, random search direction was maintained to only one iteration.
Residue percent accessibility was calculated with PISA (PISA v1.48 European Bioinformatics
Institute, Hinxton, UK) [44]. Active site residues were identified with DrosteP [45]. The figure of
superimposed proteins was prepared with PyMOL (PyMOL 2.3.0 Schrödinger, LLC, New York, NY,
USA) [46]. Ligand protein interactions were drawn with Maestro (Maestro, Schrödinger Release 2015-2:
Maestro, Schrödinger, LLC, New York, NY, USA, 2015) [47].
4.5. Protein Expression and Purification
wt-PMM1 [3,48], wt-PMM2 and its mutants (F119L-PMM2 and V129M-PMM2) [18,41] were
expressed and purified as already described.
All the PMMs were expressed (using the vector Pet22b+) in E. coli BL21(DE3) strain grown at
37 ◦C in LB broth containing ampicillin 0.2 mg/mL. The expression of wt-PMM1 was performed by
adding IPTG 0.4 mM when the optical density was 0.5 and prolonging the incubation for 4 h after
induction. Bacteria were then harvested, washed with PBS, suspended in Hepes 50 mM pH 7.5
(containing 2-mercaptoethanol 0.1 mM, EDTA 1 mM, phenylmethylsulfonyl fluoride 0.1 mM), and

Int. J. Mol. Sci. 2019, 20, 4164
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enzymatically lysed with lysozyme 1 mg/mL, treated with Deoxyribonuclease I 0.005 mg/mL after
adding MgCl2 10 mM, and centrifuged. Ammonium sulphate was added to the clear homogenate
up to 50% saturation. The precipitate was recovered, dissolved, dialyzed (in Hepes 50 mM pH 7.1,
MgCl2 5 mM, 2-mercaptoethanol 1 mM), and fractionated on a DEAE-Sepharose ff with a salt gradient
(0–0.7 M NaCl in Hepes 50 mM pH 7.1, MgCl2 5 mM, 2-mercaptoethanol 1 mM). A subsequent
fractionation step was conducted on a Butyl-Sepharose ff column (equilibrated in Tris 50 mM pH 7.1
containing MgCl2 5 mM, 2-mercaptoethanol 1 mM, ammonium sulphate 20%). Ammonium sulphate
20% was added to the sample and then loaded onto the column, the proteins were eluted with a
gradient 20%–0% ammonium sulphate. The active fractions, judged pure by SDS-PAGE, were dialyzed
(in Hepes 20 mM pH 7.5, MgCl2 1 mM, NaCl 150 mM), concentrated, and stored at −20 ◦C.
The expression of PMM2s (wt and mutants) was performed by adding IPTG 0.4 mM when the
optical density was 0.5 (V129M) or 0.8 (F119L and wt) and prolonging the incubation for 4 h after
induction. Bacteria were harvested, washed with PBS, suspended in Tris 50 mM, pH 7.5 (containing
2-mercaptoethanol 1 mM, EDTA 5 mM, and phenylmethylsulfonyl fluoride 1 mM), and enzymatically
lysed with lysozyme 1 mg/mL, treated with Deoxyribonuclease I 0.005 mg/mL after adding MgCl2
10 mM, then centrifuged. Ammonium sulphate was added to the clear homogenate up to 60% saturation.
The precipitate was recovered, dissolved, and dialyzed (in Hepes 50 mM pH 7.1 containing 5 mM
MgCl2 and 2-mercaptoethanol 1 mM), then loaded on a DEAE-Sepharose ff column. The pass-through
was collected, concentrated, and subsequently fractionated on a Superdex 75 column (in Hepes 20 mM,
MgCl2 1 mM, NaCl 150 mM, pH 7.5). The active fractions, judged pure by SDS-PAGE, were pooled,
concentrated, and stored at −20 ◦C.
4.6. Limited Proteolysis
Limited proteolysis is a useful assay to test PCs [49]. wt-PMM2 to a final concentration 0.3 mg/mL
was incubated with trypsin in a 50:1 ratio, in Hepes 20 mM pH 7.5, MgCl2 1 mM, NaCl 150 mM,◦in the
presence or the absence of βG16P or αG16P 0.5 mM. The incubation was carried out at 37 C for
120 min, collecting samples at specified time intervals. The reaction was stopped by addition of sample
buffer up to Tris 50 mM pH 6.8, 10% glycerol, 2% SDS, dithiothreitol (DTT) 100 mM, 0.1 bromophenol
blue, then boiling for 5 min and quick cooling. Samples were analyzed by SDS-PAGE.
4.7. Enzyme Assay by Fluorescence Spectroscopy
Phosphoglucomutase activity was measured recording the reduction of NADP+ to NADPH.
0.08
G6PDH 2.6 U/mL, NADP+ 0.25 mM, BSA 0.1 mg/mL.
G16P or G16P 27 M were used as the activator. Fluorescence at 340/445 nm (ex/em) was recorded
µ
g PMM2 were incubated at 25 ◦C in a solution containing Hepes 20 mM pH 7.5, MgCl2 5 mM,
α
G1P or βG1P 40 µM were used as the substrate;
α
β
µ
for 30 min using a Varian Cary Eclipse Fluorescence Spectrophotometer equipped with a Microplate
Reader (Agilent Technologies Italia SpA, Milan, Italy).
4.8. Enzyme Assay by 31P-NMR
Enzymatic activities of phosphomannomutases (phosphomannomutase and phosphatase
activities) can be analyzed recording 31P-NMR spectra [
3]. Both these activities were measured
in Hepes 20 mM pH 7.3, MgCl2 1 mM, creatine phosphate (CP) 0.5 mM, D2O 10%. The reaction was
stopped by addition of EDTA 11 mM and heating for 5 min at 60 ◦C. The samples were then quickly
cooled and stored at −20 ◦C until NMR analysis.
31P-NMR spectra were acquired as described in the appropriate paragraph and the content of
the substrate and/or the product of the enzyme reaction was measured by integrating the area of the
signal. CP 0.5 mM was used as an internal standard. Quantitative results are the average of two
independent experiments.

Int. J. Mol. Sci. 2019, 20, 4164
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Phosphomannomutase activity was assayed by incubating 0.12
µ
g PMM2 at 32 ◦C with 1 mM
α
M1P as substrate and
were collected over a period of 30 min.
Phosphatase activity was assayed by incubating 42
G16P 145
M or a combination of both; at t0 (00) and at t1 (900) samples were collected. Each condition
αG16P or βG16P (5 or 50 µM) or a combination of both, as the activator. Samples
µ
g PMM1 at 32 ◦C for 90 min with
αG16P or
β
µ
was also tested in the presence of IMP 170 µM.
4.9. Thermal Shift Assay
Thermal shift assay was carried out as described [50]. In particular wt-PMM2, F119L-PMM2 or
V129M-PMM2 to a final concentration of 0.3 mg/mL were equilibrated in Hepes 20 mM pH 7.5, MgCl2
6.25 mM, NaCl 150 mM, DTT 1.25 mM, Sypro Orange 3x, then distributed in 0.2 mL PCR-strip (20
each). The appropriate ligand solution (5 L) was added and the strips were sealed and heated from
20 to 90 ◦C with temperature increments of 1 ◦C/min using a StepOne Real Time PCR System. Ligands
G1P, G1P, vanadate, G16P, and G16P were used to a final concentration of 0.5 mM. Four replicas
µL
µ
α
β
α
β
were run for each condition.
4.10. Miscellaneous
Cell viability assay was performed as described in [51] on Hek-293 and Caco2 cells using
0.25 and 0.5 mM for 18 h.
βG16P
Proteins were quantified with the Quick Start Bradford (Bio-Rad Laboratories Srl, Milan, Italy)
using BSA as the standard [52]. SDS-PAGE was performed as in the standard procedures [53].
Author Contributions: Conceptualization: M.V.C. and G.A.; Writing-Original Draft Preparation: M.V.C.;
Writing-Review & Editing: G.A., M.M., L.L., M.A.; Investigation: M.M, L.L, M.A.; Validation: M.M, L.L,
M.A.; Supervision, M.V.C. and G.A.
Funding: Publication costs for this manuscript were sponsored by a grant from MIUR PRIN 2015 2015JHLY35
(to MVC). The funding body had no role in the design of the study and collection, analysis, and interpretation of
data and in writing the manuscript.
Acknowledgments: We are grateful to Victor Guallar, Joint BSC-CRG-IRB Research Program in Computational
Biology, Barcelona Supercomputing Center, for his help with PELE. Technical support was provided by the
NMR Service of the Institute of Biomolecular Chemistry—National Research Council, Pozzuoli (NA) Italy.
We are grateful to Emilio P. Castelluccio for technical assistance. L.L., PhD program at the DISTABIF, Universit
degli Studi della Campania “Luigi Vanvitelli”, PhD fellowship PON RI 2014/2020 “Dottorati innovativi con
caratterizzazione industriale”. M.A., PhD program at the DISTABIF, Universit degli Studi della Campania
à
à
“Luigi Vanvitelli”, PhD fellowship POR Campania FSE 2014/2020 “Dottorati di Ricerca Con Caratterizzazione
Industriale”. G.A. acknowledges Short Term Mobility program 2018 (CNR). This work is dedicated to our friend
and colleague Maria Malanga.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CP
creatine phosphate
DTT
dithiothreitol
αG1P
αG16P
βG16P
IMP
αM1P
M6P
PC
α-glucose-1-phosphate
α-glucose-1,6-bisphosphate
β-glucose-1,6-bisphosphate
inosine monophosphate
α-mannose-1-phosphate
mannose-6-phosphate
pharmacological chaperone

Int. J. Mol. Sci. 2019, 20, 4164
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).