Neurosporaside, a tetraglycosylated sphingolipid from neurospora crassa

Article abstract of DOI:10.1021/np1009493

The new tetraglycosylceramide neurosporaside (1a) has been isolated from the fungus Neurospora crassa. Neurosporaside is a tetraglycosylated glycosphingolipid characterized by a sugar chain unprecedented among natural glycoconjugates. The structure of neurosporaside was elucidated by extensive spectroscopic analysis and microscale degradation analysis, which allowed full structure elucidation using less than 1 mg of compound.

Full text of DOI:10.1021/np1009493

ARTICLE
pubs.acs.org/jnp
Neurosporaside, a Tetraglycosylated Sphingolipid from Neurospora
crassa
Valeria Costantino,*,† Alfonso Mangoni,† Roberta Teta,† Galia Kra-Oz,‡ and Oded Yarden‡
†Dipartimento di Chimica delle Sostanze Naturali, Universitꢀa degli Studi di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy
‡Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew
University of Jerusalem, Rehovot 76100, Israel
S Supporting Information
b
ABSTRACT: The new tetraglycosylceramide neurosporaside
(1a) has been isolated from the fungus Neurospora crassa.
Neurosporaside is a tetraglycosylated glycosphingolipid char-
acterized by a sugar chain unprecedented among natural
glycoconjugates. The structure of neurosporaside was eluci-
dated by extensive spectroscopic analysis and microscale de-
gradation analysis, which allowed full structure elucidation
using less than 1 mg of compound.
ungi are known to produce a wide array of compounds, many
of them unique to the members of this kingdom. Some of
report5 on N. crassa showed that neutral GSLs were characterized
as β-glucopyranosylceramides.
F
these compounds, their polymers, or their derivatives are an
integral part of the barriers and gateways between the fungal
cytoplasm and the environment. The significance of the integrity
of the plasma membrane with regard to maintaining the fungal
cell intact cannot be overestimated. In addition to serving as a
barrier between the internal cellular components and the envir-
onment, the plasma membrane provides an architectural setting
for proteins involved in cell wall biosynthesis (chitin and glucan
synthases), transport, and signaling. Sphingolipids, sterols, and
glycophospholipids are the prime lipid components of eukaryotic
membranes and are particularly abundant in the plasma membrane.
Although all eukaryotic organisms produce sphingolipids,
fungi use phytoceramide (a more hydroxylated sphingolipid than
the mammalian ceramide counterpart) as the hydrophobic core
for the synthesis of more complex sphingolipids,1 namely, glyco-
sphingolipids (GSLs). Although GSLs may have important roles
in the cell, there are only few systematic studies of fungal GSLs.
Since evidence2 has been accumulating that GSLs may serve as
key ligands for some endogenous lectins in animals, possible
similar roles for fungal GSLs could be considered.
In the course of our study to assess the sphingolipid profile and
their relevance to the biology of N. crassa, we found a novel
tetraglycosylated glycosphingolipid, neurosporaside (1a). In this
paper, we report on the isolation and structure elucidation of
neurosporaside, whose structure is unique in that the sugar
moiety linked to the ceramide is formed by a chain of three
β-galactopyranoside residues, with a glucopyranoside R-linked
to the outer galactopyranoside.
Two subclasses of GSLs appear to be widely expressed in the
fungal kingdom: (i) the inositolphosphoceramides, in which the
1-hydroxyl group of ceramide is linked to myo-inositol and
derivatives by a phosphodiester link, and (ii) the glycosylcer-
amides, such as glucosylceramides (GlcCer) and galactosylcer-
amides (GalCer), also called neutral GSLs.
’ RESULTS AND DISCUSSION
Neurospora crassa’s long history as an excellent genetic, biochem-
ical, and cell biological model of fungal and higher eukaryotic biology,
together with the recent availability of a fully sequenced and annotated
genome,3,4 makes it a convenient model ascomycete for studying
almost any aspect of fungal growth and development. A previous
Lyophilized N. crassa mycelium obtained from liquid medium
cultures was extracted with CHCl3 and MeOH, and the combined
Received: May 20, 2010
Published: March 22, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
extracts were partitioned between H2O and n-BuOH. Using a
standard procedure we developed to obtain a fraction composed
mainly of glycolipids,6 the organic phase was subjected to reversed-
phase and normal-phase column chromatography. The glycolipid
fraction was treated with acetic anhydride, and the peracetylated
glycolipids were subjected to repeated HPLC on SiO2 columns.
Only two GSLs were found in the mixture: the major component
was β-glucosylceramide (4.8 mg), known to be present in N. crassa,
but significant amounts of a new peracetylated GSL (1b, 1.2 mg)
were also isolated. While the sample appeared pure upon 1H NMR
analysis, mass spectral data showed that the sample was not
chemically homogeneous, containing minor amounts of homolo-
gues with alkyl chains of different length. A final separation was
therefore performed on reversed-phase HPLC, which gave pure
neurosporaside peracetate 1b, plus trace amounts of its homologues,
which were not further characterized.
Structure Elucidation. Because the molecular weight of the
peracetylated neurosporaside 1b was higher than 2000, the upper
m/z limit of our ESI mass spectrometer, only a doubly charged
[M þ 2Na]2þ ion at m/z 1025 was visible in its ESI mass
spectrum. Therefore, a small amount of 1b was deacetylated with
MeONa/MeOH to give compound 1a. The mass spectrum
of compound 1a showed a clear pseudomolecular [M þ Na]þ
ion peak at m/z 1354.8. A high-resolution measurement gave
m/z 1354.8411, corresponding to the molecular formula
C66H125NO25 (calcd 1354.8433).
Table 1. 1H and 13C NMR Data (CDCl3) for Neurosporaside
Peracetate (1b)
position
1
δH (J in Hz)b
δC, multc
a
3.88 (dd, 10.8, 3.6)
66.5 CH2
b
3.63 (dd, 10.8, 3.4)
4.28 (dddd, 9.0, 8.4, 3.6, 3.4)
6.74 (d, 9.0)
2
48.3 CH
2-NH
3
4
5
5.12 (dd, 3.1, 8.4)
4.87 (ddd, 10.0, 3.1, 3.1)
1.62 (m)
71.6 CH
73.2 CH
28.4 CH2
a
b
1.58 (m)
10
20
30
40
50
60
4.44 (d, 8.0)
100.8 CH
68.8 CH
71.0 CH
67.4 CH
72.1 CH
66.4 CH2
5.09 (dd, 10.4, 8.0)
4.99 (dd, 10.4, 3.4)
5.37 (dd, 3.4, 1.0)
3.86a
a
3.76 (dd, 10.5, 6.4)
3.69 (dd, 10.5, 5.7)
4.52 (d, 8.0)
b
100
200
300
400
500
600
100.6 CH
68.6 CH
70.9 CH
67.1 CH
71.7 CH
66.7 CH2
5.13 (dd, 10.4, 8.0)
5.04 (dd, 10.4, 3.4)
5.38a
3.92 (ddd, 7.1, 5.8, 1.1)
3.87a
a
The planar structure of neurosporaside was elucidated by
extensive 1D and 2D NMR analysis. In the 1H NMR spectrum of
the peracetyl derivative 1b were present (i) an intense aliphatic
chain signal at δ 1.25; (ii) several signals corresponding to
oxymethine and oxymethylene groups between δ 5.25 and
3.45; (iii) an amide NH doublet at δ 6.70; and (iv) 16 acetyl
methyl singlets between δ 2.22 and 1.96. These signals are
b
3.55 (dd, 9.5, 5.8)
4.42 (d, 7.7)
1000
2000
3000
4000
5000
6000
1IV
2IV
3IV
4IV
5IV
6IV
102.6 CH
72.8 CH
72.0 CH
67.0 CH
70.6 CH
61.2 CH2
95.4 CH
70.7 CH
69.5 CH
68.3 CH
67.6 CH
61.7 CH2
3.80 (dd, 10.3, 7.7)
4.93 (dd, 10.3, 3.4)
5.38a
3.86a
1
indicative of a GSL structure. The H NMR spectrum also
4.09 (d, 6.7)
showed a 6H triplet at δ 0.87 in the methyl region, indicating
that both alkyl chains in the ceramide unit are unbranched.
Analysis of the 2D-NMR COSY enabled assignment of all the
proton signals for the polar part of the sphinganine up to H2-6
(see Table 1). The R-hydroxy substitution of the fatty acid
residue was revealed by the absence in the 1H NMR spectrum of
1b of the characteristic fatty acid R-proton CH2 signal
and the presence of an acetoxymethine proton resonating at
δ 5.13 (H-2V), coupled with a methylene at δ 1.80 (H-3V),
which was, in turn, coupled with protons resonating in the
broad alkyl chain signal at δ 1.25. In addition, a ROESY
correlation of the signal at δ 5.13 with the amide NH doublet
enabled connection of the NH to this R-hydroxy fatty acid unit.
Therefore, the ceramide portion of the molecule is composed of
a trihydroxylated, saturated sphinganine and an R-hydroxy fatty
5.42 (d, 3.7)
4.81 (dd, 10.3, 3.7)
5.36 (dd, 10.2, 9.5)
5.08 (dd, 10.1, 9.5)
4.10 (ddd, 10.1, 4.2. 2.4)
4.26 (dd, 12.2, 4.3)
4.05 (dd, 12.2, 2.4)
a
b
1V
170.1 C
2V
5.14 (dd, 7.0, 5.0)
74.0 CH
3V
1.84 (m)
31.9 CH2
Ac’s
CH3
CO
2.219, 2.139, 2.136, 2.125, 2.093,
2.084, 2.070, 2.059, 2.053, 2.052,
2.050, 2.019, 2.009, 1.994, 1.970, 1.962
21.2ꢀ20.7 CH3
1
1
acid residue. The H and 13C NMR chemical shifts and the
171.3ꢀ169.5 C
a Overlapped by other signals. b Additional H signals: δ 1.24 (broad
band, alkyl chain protons), 0.87 (t, J = 7 Hz, n-chain Me groups).
c Additional 13C signals: δ 29.2 (CH2), 14.3 (CH3, ω).
1
H NMR multiplicities of the ceramide signals were all very close
to those reported for the most common stereoisomer, i.e., that
composed of a D-ribo-phytosphingosine and a 2R-hydroxy
acid.7 This configuration for the ceramide of neurosporaside
was confirmed by microscale chemical degradation analysis, as
reported below.
The sugar chain of neurosporaside, on the other hand, is
unprecedented not only among natural GSLs but also in any
known glycoconjugate, so its structure elucidation is described
in detail here. The presence of four sugar units was revealed
by four resonances in the characteristic anomeric region of the
13C NMR spectrum at δ 95.4, 100.6, 100.8, and 102.6, which
were associated through the HMQC spectrum to the relevant
anomeric protons, resonating as doublets at δ 5.42 (J = 3.7 Hz,
H-1IV), 4.52 (J = 8.3 Hz, H-1II), 4.44 (J = 7.9 Hz, H-1I), and 4.42
(J = 7.7, Hz, H-1III). These protons were used as starting points
in the analysis of the COSY and z-TOCSY8 spectrum (see
Figure 1) for the sequential assignment of the proton signals in
each monosaccharide unit, while the relevant carbon signals were
assigned using the HSQC spectrum.
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ARTICLE
1
Figure 2. High-resolution MS/MS spectrum of neusporaside 1a.
Figure 1. (a) Midfield region H NMR spectrum of neurosporaside
peracetate (1b); (bꢀf) Sections of the z-TOCSY spectrum of 1b,
showing the spin system of Gal-I (b), Gal-II (c), Gal-III (d), Glc-IV
(e), and the sphingosine (f).
Scheme 1. Microscale Chemical Degradation of Neuro-
sporaside (1b)
Unfortunately, the small coupling constant between H-4 and
H-5 of the three galactose residues prevented us from assigning
protons at positions 5 and 6 of this sugar using this method (this
assignment is essential to determine the way that the sugars are
linked to each other; see below). This problem can usually be
overcome by looking at the cross-peak present in the ROESY
spectrum between H-3 and H-5 of each sugar. This, indeed,
allowed us to assign the protons at positions 5 and 6 of Gal-II, but
not to distinguish those of Gal-I and Gal-III because the chemical
shifts of H-5I and H-5III were coincident. A strong correlation
peak between H2-6III and C-4III in the HMBC spectrum was then
the key to assigning the former protons and, as a consequence,
also H-5III and the protons at positions 5 and 6 of Gal-I.
Once the assignment of the sugar protons and carbons was
completed, determination of the structure of the sugar chain was
straightforward. The high-field chemical shift of H-5I (δ 3.86),
H-5II (δ 3.92), H-5III (δ 3.86), and H-5IV (δ 4.10) showed that
the relevant carbon atoms are involved in acetal (as opposed to
ester) groups and, therefore, that all the sugars are in the pyranose
form. Analysis of coupling constants revealed that sugars I, II, and II
are β-galactopyranoses, because in each sugar all protons are axial,
except for H-4. Likewise, sugar IV is an R-glucopyranose, because
H-1IV is the sole equatorial proton. Finally, the glycosylation sites
and the sugar sequence were established by the HMBC cross-peaks
of H-1I with C-1, H-1II with C-6I, H-1III with C-6II, and H-2III
with C-1IV, and confirmed by the ROESY cross-peak of H-1I with
H-1b, H-1II with H-6Ib, H-1III with H-6IIb, and H-1IV with H-2III.
The molecular formula deduced from the high-resolution mass
spectrum indicated that the ceramide part of neurosporaside is
composed of 42 carbon atoms, but did not provide information on
how they are split between the sphingosine and the fatty acid.
This information was provided by a high-resolution MS/MS experi-
ment performed using the pseudomolecular ion of compound 1a
(m/z 1354.8411, C66H125O25NNa) as the precursor ion. The spec-
trum (Figure 2) contained peaks at m/z 1192.7885 (C60H115O20-
NNa, [M þ Na ꢀ hexose]þ), 1030.7361 (C54H105O15NNa, [M þ
Na ꢀ 2 hexoses]þ), 868.6835 (C54H105O15NNa, [M þ Na ꢀ 3
hexoses]þ), 689.2101 (C24H42O21Na, [tetrahexose þ Na]þ),
527.1573 (C18H32O16Na, [trihexose þ Na]þ), and 365.1050
(C12H22O11Na, [dihexose þ Na]þ), which confirmed the pre-
sence of an unbranched tetrasaccharide chain. More interestingly,
the MS/MS spectrum also contained two peaks at m/z
988.4921 (C42H79O23NNa, [M þ Na ꢀ fatty acyl]þ) and
826.4396 (C36H69O18NNa, [M þ Na ꢀ hexose ꢀ fatty acyl]þ),
which were indicative a C24 fatty acid and, therefore, of a C18
phytosphingosine.
Microscale degradation analysis (see Scheme 1) was performed to
establish the absolute configuration of two sugars and of the stereo-
genic centers present in the ceramide. Compound 1b (450 μg) was
subjected to acidic methanolysis with 1 M HCl in 92% MeOH. The
resulting mixture, composed of fatty acid methyl esters, free sphinga-
nines, and methyl glycosides, was dried, benzoylated, and separated
on normal-phase HPLC. The benzoylated fatty acid methyl ester 2,
the perbenzoylated phytosphingosine 3, and the methyl β-glyco-
sides4 and 5 were obtained in pure form (the corresponding methyl
R-glycosides coeluted in the conditions used). The absolute con-
figuration of 2-benzoyloxytetracosanoate 2 was determined as R
from its CD spectrum, matching that reported.9 Sphingosine 3 was
identified as (2S,3S,4R)-1,3,4-tri-O-benzoyl-2-benzamido-1,3,4-
octadecatriol (D-ribo-phytosphingosine perbenzoate),9 methyl gly-
coside 4 as methyl tetra-O-benzoyl-β-D-glucopyranoside,10 and
methyl glycoside 5 as methyl tetra-O-benzoyl-β-D-galactopyrano-
side10 by comparison of the respective 1H NMR and CD spectra
with those reported. Therefore, neurosporaside (1a) is 1-O-[R-D-
glucopyranosyl-(1f2)-β-D-galactopyranosyl-(1f6)-β-D-galactopy-
ranosyl-(1f6)-β-D-galactopyranosyl]-(2S,3S,4R)2-[2R-hydroxy-
tetracosanoylamino]-1,3,4-octadecanetriol.
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ARTICLE
Neurosporaside is a tetraglycosylated sphingolipid with a sugar
chain unprecedented among natural glycoconjugates. Neurosporaside
is present in N. crassa in lesser, but significant amounts (about 25%)
compared to the only other GSL detected, β-glucosylceramide.
Fungi exhibit great variability in lipid composition. In addition to
the functional roles, lipid patterns have been considered a potential
and useful tool for taxonomic classifications. Until now, GSLs from
fungi have been described only to a limited extent. Results obtained
so far point to a genus-/species-specific relationship, and this
information can be used as a part of a more complex taxonomic
approach. Screening for the GSL composition has now been made
easier and much more accurate by the huge improvement of
instrument-based analytical techniques. In this work, a combina-
tion of NMR and MS studies made possible the isolation and
stereostructural identification of a novel tetraglycosylated sphin-
golipid from a small amount of biological material.
Previous studies on the GLS composition of N. crassa reported
only β-glucopyranosylceramide to be present, except for a paper
published in 1974,11 in which a glycolipid with a general (Gal)3-
GluCer structure was reported from N. crassa. The compound was
poorly characterized, and neither the sugar sequence nor the
glycosylation positions were determined, so that it is not possible
to define which of the many possible compounds with this general
structure was actually found. In addition, no details were given on
the absolute stereochemistry of the sugars nor on the stereochem-
istry of the glycosidic linkages. These are of course extremely
relevant issues, because the interaction of GSLs with the receptors
(or other cellular components) that may determine their biological
activity is strongly dependent on the overall shape of the carbo-
hydrate chain (as we have demonstrated in the past with other
GSLs),12 for which glycosylation positions and anomeric config-
urations play as important a role as the number and the nature of the
sugar monomers do.
using a Varian Prostar 210 apparatus equipped with a Varian 350 refractive
index detector or a Varian 325 UV detector.
Biological Material. Wild-type Neurospora crassa strain 74-OR23-1
(FGSC 987)18 was cultured in 250 mL flasks containing 100 mL of Vogel's
minimal medium with 1.5% (w/v) sucrose as previously described.19 The
biomass obtained from 5 flasks of 22 h old cultures (5 ꢁ 100 mL) was
harvested by filtration, frozen in liquid nitrogen, and lyophilized.
Extraction and Isolation. Lyophilized N. crassa (1.2 g) was
extracted with a mixture of MeOH and CHCl3 (1:1, 3 ꢁ 500 mL) and
then with CHCl3 (3 ꢁ 500 mL); the combined extracts were partitioned
between H2O and n-BuOH. The organic layer was concentrated in vacuo
and afforded 614 mg of an oil, which was chromatographed on a column
packed with RP-18 silica gel. A fraction (88 mg) eluted with MeOH/CHCl3
(1:1) was further chromatographed on a SiO2 column, giving a fraction [11
mg, eluent EtOAc/MeOH (7:3)] mainly composed of glycolipids. This
fraction was peracetylated by treatment with Ac2O in pyridine for 12 h.
The acetylated glycolipids were subjected to HPLC separation on an SiO2
column [Phenomenex Luna SiO2, 5 μ, 250 ꢁ 4.60 mm; eluent n-hexane/
i-PrOH (8:2); flow 1 mL minꢀ1], affording a fraction composed of
β-glucosylceramide peracetate (4.8 mg), identified by comparison of its
1H NMR spectrum with that of an authentic sample, and a fraction (1.2 mg)
containing neurosporaside and its minor homologues. Final separation on
an RP-18 column [Phenomenex Luna C18; 5 μ, 250 ꢁ 4.60 mm; eluent
n-hexane/i-PrOH (8:2); flow 1 mL minꢀ1] yielded pure neurosporaside
peracetate (1b, 0.9 mg). A second 500 mL culture of N. crassa was extracted
and separated using the same procedure; β-glucosylceramide peracetate and
neurosporaside peracetate were obtained in approximatively the same
amounts (4.2 and 1.0 mg, respectively).
Neurosporaside peracetate (1b): colorless oil, [R]25D þ6.8 (CHCl3,
c 0.05); 1H and 13C NMR, Table 1; ESIMS (positive ion mode, MeOH)
m/z 1025 ([M þ 2Na]2þ).
Deacetylation of 1b. Compound 1b (50 μg) was dissolved in
95 μL of MeOH, and 5 μL of a 0.4 M solution of MeONa in MeOH was
added. The reaction was allowed to proceed for 18 h at 25 °C in a sealed
vial; then the reaction mixture was dried under nitrogen, and the residue
partitioned between water and n-BuOH. After removal of the solvent,
the organic layer was analyzed without any further purification by
ESIMS (positive ion mode, MeOH), m/z 1354.8 ([MþNa]þ), and
high-resolution ESIMS (positive ion mode, MeOH), m/z 1354.8411
(C66H125NO25 gives 1354.8433).
Identification of the structural components of the fungal
membrane is a prerequisite for the eventual understanding of fungal
growth and development, especially in cases where structural
abnormalities linked to the plasma membrane and/or its adjacent
cell wall prevail.13ꢀ16 In addition, the biosynthesis of GSLs that are
present in fungi, but absent in humans, may offer unique targets for
antifungal drugs.17 The identification of neuroporaside is only the
first step of a more general study focused on the comprehension of
lipid structural biology in fungi.
Degradation Analysis of 1b. Neurosporaside 1b (450 μg) was
subjected to acidic methanolysis with 1 M HCl in 92% MeOH (500 μL)
for 16 h at 80 °C. The resulting mixture was dried under nitrogen and
then benzoylated for 16 h at 80 °C with benzoyl chloride (10 μL) in
pyridine (200 μL) at 25 °C for 16 h. The reaction was quenched with
MeOH and then dried under nitrogen. Methyl benzoate was mostly
removed by keeping the residue under vacuum with a freeze-drier for
60 h. The mixture was purified on normal-phase HPLC [column
Phenomenex Luna SiO2; 5 μ, analytical 250 ꢁ 4.60 mm; eluent
n-hexane/iPrOH (99:1); flow 1 mL minꢀ1]. Four peaks present in
the chromatogram were identified as (R)-2-benzoyloxytetracosanoate
(2, tR = 3.7 min), D-ribo-phytosphingosine perbenzoate (3, tR = 8.3 min),
methyl tetra-O-benzoyl-β-D-glucopyranoside (4, tR = 10.6 min), and
methyl tetra-O-benzoyl-β-D-galactopyranoside (5, tR = 11.6 min), by
comparison of their retention times, 1H NMR spectra, and CD spectra
with those reported.9,10
’ EXPERIMENTAL SECTION
General Experimental Procedures. High-resolution ESIMS and
ESIMS/MS spectra were acquired on a Thermo LTQ Orbitrap mass
spectrometer. ESIMS experiments were performed on an Applied Biosys-
tems API 2000 triple-quadrupole mass spectrometer. The spectra were
recorded by infusion into the ESI source using MeOH as the solvent.
Optical rotations were measured at 589 nm on a Jasco P-2000 polarimeter
1
using a 10 cm microcell. H and 13C NMR spectra were determined
on a Varian UnityInova spectrometer equipped with a cryogenic probe at
700 and 175 MHz, respectively; chemical shifts were referenced to the
residual solvent signal (CDCl3: δH 7.26, δC 77.0). For an accurate
1
measurement of the coupling constants, the one-dimensional H NMR
spectra were transformed at 64K points (digital resolution: 0.09 Hz).
Homonuclear 1H connectivities were determined by COSY and z-TOCSY8
experiments. Through-space H connectivities were evidenced using a
’ ASSOCIATED CONTENT
1
Supporting Information. 1H, COSY, ROESY, TOCSY,
S
ROESY experiment with a mixing time of 450 ms. The HSQC spectra
b
were optimized for 1JCH = 142 Hz, and the HMBC experiments for 2,3JCH
=
HSQC, and HMBC NMR spectra of compound 1b. This material is
8.3 Hz. High-performance liquid chromatography (HPLC) was performed
available free of charge via the Internet at http://pubs.acs.org.
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ARTICLE
’ AUTHOR INFORMATION
(15) Resheat-Eini, Z.; Zelter, A.; Gorovits, R.; Read, N. D.; Yarden,
O. Fungal Genet. Rep. 2008, 55, 32–36.
(16) Ziv, C.; Kra-Oz, G.; Gorovits, R.; Maerz, S.; Seiler, S.; Yarden,
O. Mol. Microbiol. 2009, 74, 974–989.
(17) Nagiec, M. M.; Nagiec, E. E.; Baltisberger, J. A.; Wells, G. B.;
Lester, R. L.; Dickson, R. C. J. Biol. Chem. 1997, 272, 9809–9817.
(18) McCluskey, K. In Advances in Applied Microbiology; Laskin, A. I.;
Bennett, J. W., Eds.; Elsevier: New York, 2003; Vol. 52, pp 246ꢀ262.
(19) Gorovits, R.; Yarden, O. Eukaryotic Cell 2003, 2, 699–707.
Corresponding Author
*Tel: þ39 081 678504. Fax: þ39 081 678552. E-mail:
valeria.costantino@unina.it.
’ ACKNOWLEDGMENT
The research leading to these results has received funding
from the European Union's Seventh Framework Programme
FP7/2007-2013 under grant agreement no. 229893 (NatPharma)
to V.C. and from the Israel Science Foundation to O.Y. Mass and
NMR spectra were recorded at the “Centro di Servizi Interdiparti-
mentale di Analisi Strumentale”, Universitꢀa di Napoli “Federico II”.
The assistance of the staff is gratefully acknowledged.
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