Báo cáo khoa học: Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is a-D-Gal-(1fi3)-a-D-GalNAc pot

Total chemical synthesis and NMR characterization of the glycopeptide
tx5a, a heavily post-translationally modified conotoxin, reveals that
the glycan structure is a-
D
-Gal-(1fi3)-a-
D
-GalNAc
James Kang
1,
*, William Low
1,
*, Thomas Norberg
3
, Jill Meisenhelder
2
, Karin Hansson
4
, Johan Stenflo
4
,
Guo-Ping Zhou
5,6
, Julita Imperial
7
, Baldomero M. Olivera
7
, Alan C. Rigby
5,6
and A. Grey Craig
1
1

The Clayton Foundation Laboratories for Peptide Biology and
2
Laboratory for Molecular and Cell Biology, The Salk Institute,
La Jolla, CA, USA;
3
Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden;
4
Department of
Clinical Chemistry, University of Lund, Malmo General Hospital, Malmo, Sweden;
5
Center for Hemostasis and Thrombosis
Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA;
6
Marine Biological Laboratory,
Woods Hole, MA, USA;
7
Department of Biology, University of Utah, Salt Lake City, UT, USA
The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-
Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where
Trp* ¼ 6-bromotryptophan and Thr* ¼ Gal-GalNAc-
threonine), isolated from Conus textile, causes hyperactivity
and spasticity when injected intracerebral ventricularly into
mice. It contains nine post-translationally modified residues:
four cysteine residues, two c-carboxyglutamic acid residues,
and one residue each of 6-bromotryptophan, 4-trans-
hydroxyproline and glycosylated threonine. The chemical
nature of each of these has been determined with the
exception of the glycan linkage pattern o n threonine and the
stereochemistry of t he 6-bromotryptophan residue. P revious
investigations have demonstrated that tx5a contains a

disaccharide composed of N-acetylgalactosamine ( GalNAc)
and galactose (Gal), but the interresidue linkage was not
characterized. W e h ypothesized that t x5a contained the
T-antigen, b-
D
-Gal-(1fi3)-a-
D
-GalNAc, one of the most
common O-linked g lycan structures, identifi ed p reviously in
another Conus glycopeptide, contalukin-G. We therefore
utilized the peracetylated form of this glycan attached to
Fmoc-threonine in an attempted synthesis. W hile the r esult-
ing s ynthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-
Cys-Thr*-Ala-Ala-Hyp-O H, where Trp* ¼6-bromotrypto-
phan and Thr* ¼ b-
D
-Gal-(1fi3)-a-
D
-GalNAc-threonine)
and the native peptide had almost identical mass spectra, a
comparison of their RP-HPLC chromatograms suggested
that the two forms were not identical. Two-dimensional
1
H
homonuclear and
13
C-
1
H h eteronuclear NMR spectroscopy
of native tx5a isolated from Conus textile was then used to

determine that the glycan present on tx5a indeed is not the
aforementioned T-antigen, but rather a-
D
-Gal-(1fi3)-a-
D
-
GalNAc.
Keywords: Conus textile; glycopeptide synthesis.
The diverse array of peptides isolated from the venom of
cone snails are known collectively as conotoxins or cono-
peptides (if they lack a disulfide-bonded architecture). The
growing interest in these pe ptides stems from t heir ability to
bind receptors and io n channels with high selectivity and
unparalleled specificity. A distinct feature of most conotox-
ins is their relatively small size (10–35 amino acid residues)
combined with the presence of a high proportion o f c ysteine
residues that are involved in disulfide bridging [1]. In
addition, many of the amino acid residues present in
conotoxins h ave undergone post-translational modification;
among the diverse array of modifications characterized
to date are glutamic acidfic-carboxyglutamic acid [2],
prolinefi4-trans-hydroxyproline [3], tryptophanfi6-
L
-
bromotryptophan [4] and threonine/serinefiO-linked gly-
cosylated threonine/serine [5–7].
Conotoxin tx5a (or e-TxIX), which was purified recently
by two i ndependent laboratories from the venom of the
mollusc-hunting cone s nail, Conu s t extile,theÔcloth-of-goldÕ
cone, is comprised of an unusually large number of amino

acids that are post-translationally modified [8–10].
Uniquely, this 13-amino acid peptide contains four post-
translational modifications and two disulfide bonds. In
Correspondence to T. Norberg, Department of Chemistry, Swedish
University of Agricultural Sciences, SE -750 07 Uppsala, Sweden.
Fax: + 46 18 673476, Tel.: + 46 18 671578,
E-mail: or A. C. Rigby, Center for
Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, M A 02115, USA.
Fax: + 1 617 975 5505, Tel.: + 1 617 667 0637,
E-mail:
Abbreviations: DI, deionized; DIPEA, N,N¢-diisopropylethylamine;
DMF, N,N-dimethylformamide; EDT, 1,2-ethanedithiol; Gal, galac-
tose; GalNAc, N-acetyl galactosamine; N MP, N-methylpyrrolidone;
Gla, c-carboxyglutamic acid, H BTU, O-(benzotriazol-1-yl)-
N,N,N¢,N¢-tetramethyluronium hexafluorophosphate; Hypro,
4-trans-hydroxyproline; MTBE, methyl tert-butyl ether; TBTU,
O-(benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium tetrafluorobo-
rate; TCEP, tris-(2-carboxyethyl)-phosphine hydrochloride;
TPPI, time-proportional phase incrementation.
*Note: These authors contributed equally to the work.
(Received 9 August 2004, revised 20 October 2004,
accepted 27 October 2004)
Eur. J. Biochem. 271, 4939–4949 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04464.x
total, nine of the 13 residues in tx5a are modified, making it
one of the most highly modified gene products identified to
date. The native peptide was purified to apparent homo-
geneity, and was reported to cause tremors, hyperactivity
and spastic gait when injected intra-cerebral ventricularly
[8]. An underlying mechanism for the biological activity of

the tx5a peptide was proposed by Rigby et al.who
suggested that tx5a may target presynaptic Ca
2+
channels
or that it might act on these c hannels via other mecha-
nisms, such as through G-protein-coupled presynaptic
receptors [9].
The c omposition of t he O-glycan on the t hreonine residue
of tx5a was previously investigated [9]. It was shown t hat the
peptide contained N-acetylgalactosamine (GalNAc) and
galactose ( Gal) in approximately e qual m olar amounts;
however, the anomeric stereoche mistry and the glyc an
linkages were not determined. We have previously charac-
terized contulakin-G, a glycopeptide isolated from Conus
geographus venom, and determined that this glycopeptide
possessed the same monosaccharide constituents as tx5a.
We further demonstrated that these monosaccharides were
linked in the b-
D
-Gal-(1fi3)-a-
D
-GalNAc configuration of
the T-antigen [6].
Here we report the synthesis of a peptide identical
in composition to that of tx5a, using a racemic
D
/
L
-
6-bromotryptophan derivative and a Thr10 derivative

carrying a b-
D
-Gal-(1fi3)-a-
D
-GalNAc glycan substituent.
However, the peptide synthesized proved to be disparate
from native tx5a isolated from the Conus textile ven om. To
better understand the incongruence of these peptides we
reinvestigated the glycan linkage configuration of the
isolated and purified native tx5a venom, using both
1
H-
1
H
homonuclear and
13
C-
1
H heteronuclear two-dim ensional
NMR s pectroscopy. T he data clearly i ndicated that the t x5a
glycan is in an a-
D
-Gal-(1fi3)-a-
D
-GalNAc configuration.
Taken together, th ese data demonstrate that two Conus
glycopeptides identified to date possess the same monosac-
charide constituents within their glycan, Gal and GalNAc,
but their interresidue linkages are different (alpha vs. beta).
The r esults suggest that O-glycosylation in Conus peptides is

likely to be more c omplex than had originally been expected
and highlights another post-translational modification that
the Conus species employ to adapt to their ever changing
environment.
Experimental procedures
Peptide synthesis
We carried out both manual and automated syntheses as
described below, using an Fmoc solid-phase strategy with
the amino acid derivatives shown in Scheme 1. The manual
synthesis was carried out on an Fmoc-Hypro Wang resin
(Chem-Impex, Wood Dale, IL, USA; 0.4 g, 0.7 mmolÆg
)1
).
Each cycle consisted of Fmoc deprotection with 20%
piperidine in N-methylpyrrolidone (NMP), followed by
Fmoc amino acid coupling u sing O-(benzotriazol-1-yl)-
N,N,N¢,N¢-tetramethyluronium tetrafluoroborate (TBTU)
and N,N¢-diisopropylethylamine (DIPEA) in NMP. To
avoid diketopiperazine formation, Fmoc-Ala-Ala (Bachem,
Torrance, CA, USA) was coupled as the first Fmoc amino
acid. A two-fold excess of Fmoc amino acids was used in
the coupling reactions with the exception of Fmoc-
D
/
L
-
6-bromotryptophan and per-O-acetylated Fmoc [b-
D
-Gal-
(1fi3)-a-

D
-GalNAc]-Thr [7] where a 20% excess was used.
The efficiency of the coupling reactions was checked using
the Kaiser ninhydrin test. The dried p eptide resin (0.47 g)
was t reated with 4.5 mL o f trifluoracetic acid in the presence
of 250 lL t hioanisole, 125 lL 1,2-ethanedithiol (EDT), a nd
125 lL d eionized (D I) water at room temperature for 1.5 h.
After precipitation and washing of the cleaved peptide with
cold methyl tert-butyl ether (MTBE, 2· 20 mL), the pe ptide
was taken up in 0.1% aqueous trifluoroacetic acid and
60% acetonitrile (2· 10 mL).
Automated chemical s ynthesis was performe d on an ABI
432A peptide synthesizer (Applied Bioysystems, Foster
City, CA, USA) employing O-(benzotriazol-1-yl)-N,N,
N¢,N¢-tetramethyluronium hexafluorophosphate (HBTU)/
DIPEA/DMF for coupling and piperidine/DMF for Fmoc
deprotection. Coupling of Fmoc-Ala-Ala to Fmoc-Hypro
Wang resin (50 mg) w as performed m anually and t hen
loaded onto the automated synthesizer for the remainder of
the sequence. Three-fold excess of amino acid derivatives
were used in the c oup ling reactions with the exception of per-
Scheme 1. Sequence of addition of amino acid
derivatives during the solid-phase glycopeptide
synthesis. The amino acids are numbered
starting f rom the amino terminal according to
accepted nomenclature . As solid-phase pe p-
tide synthesis starts from the carboxy terminal,
the order of addition is from higher to lower
number.
4940 J. Kang et al.(Eur. J. Biochem. 271) Ó FEBS 2004

O-acetylated Fmoc [b-
D
-Gal-(1fi3)-a-
D
-GalNAc]-Thr
where a 10% excess was used. The peptide synthesizer used
conductance monitoring to check the efficiency of the
coupling reactions. In order to scale up t he automated
synthesis to the same level as the manual synthesis
(280 lmol) we carried out nine separate automated synthe-
ses. Each dried peptide-resin (65 mg · nine aliquots ¼ total
weight, 585 mg) was treated with 900 lL of trifluoracetic
acid in the presence of 50 lL thioanisole, 25 lLEDTand
25 lL DI water at room temperature for 1.5 h. After
precipitation and washing of the cleaved peptide with cold
MTBE (2· 5 mL), the nin e precipitates were collected and
the p eptide was t aken up in 0.1% aqueous trifluoracetic acid
and 60% acetonitrile (2 · 5mL).
Purification of the manual synthesis [per-O-acetyl-
b-
D
-Gal-(1fi3)-a-
D
-GalNAc-Thr10, Cys(t-butyl thiol)2-8,
Cys(Acm)3-9]-tx5a crude product on an analytical HPLC
(1% per min gradient with 0.1% a queous trifluoroacetic
acid as buffer A and 60% acetonitrile in 0.1% aqueous
trifluoroacetic acid as buffer B ) gave two major components
whose observed mass, using ESI-MS, was consistent with
the expected product. A similar result was obtained from the

automated synthesis. The first component (ÔhydrophilicÕ)
eluted at  46% acetonitrile, the second (ÔhydrophobicÕ)
eluted at  48% a cetonitrile. B ecause of t he low y ields from
each synthesis, the material was combined for the following
treatments. We e stimated that prior to summation, the y ield
from automated and manual synthesis were approximately
equivalent. T he crude extract w as loaded onto a
45 · 320 mm column packed w ith Vydac C
18
15–20 lm
particles and eluted using a preparative HPLC (PrepLC/
System 6000, Waters Corporation, Millford, MA, U SA)
equipped with a gradient controller, a variable wavelength
detector (Waters, model 486) and Waters 1000 PrepPack
cartridge c hamber in 0.1% aqueous trifluoracetic acid, using
a gradient of 60% acetonitrile in 0.1% aqueous trifluorace-
tic acid. Each component was injected on analytical HPLC
under isocratic conditions to check for purity and quantity.
Approximately 240 nmol of the h ydrophilic component
(eight aliqu ots at 30 nmol) and 150 nmol of hydrophobic
component (five aliquots a t 30 nmol) were lyophilized for
the sugar de-O-acetylation reaction. Each dried aliquot was
treatedwith500lL 150 m
M
NaOCH
3
in methanol for
20 min at 25 °C and then quenched with 200 lLDIwater.
Purification of the [b-
D

-Gal-(1fi3)-a-
D
-GalNAc-Thr10,
Cys(t-butyl thiol)2,8, Cys(Acm)3,9] hydrophilic and hydro-
phobic products on preparative HPLC identified hydrophi-
lic and hydrophobic components with an observed mass of
2252.2 m/z (ESI-MS), which are consistent with the theor-
etical peptide mass (2252.6 Da).
Disulfide bond formation
In preparation for the Cys2-8 disulfide r eaction, each
component was injected on analytical HPLC under
isocratic conditions to check for purity a nd quantity.
Approximately 160 nmol of the h ydrophilic component
(20 aliquots at 8 nmol) and 120 nmol of hydrophobic
component (20 aliquots at 6 nmol) were lyophilized. Each
dried aliquot was treated with 0.17
M
citric acid (750 lL,
pH 6.5) and 1
M
tris-(2-carboxyethyl)-phosphine hydro-
chloride (TCEP) (150 lL) at 37 °C for 180 min, and then
quenched with 0.1% aqueous trifluoracetic acid (500 lL).
In order to minimize complications that result from these
Gla-containing peptides forming divalent metal ion com-
plexes (i.e. c reating peak b roadening o r multiple p eaks
when analyzed on HPLC), 1% CaCl
2
(100 lL) was
added to each aliquot. Purification of the [b-

D
-Gal-
(1fi3)-a-
D
-GalNAc-Thr10, Cys2-8, Cys(Acm)3-9] hydro-
philic and hydrophobic products b y analytical HPLC
indicated hydrophilic and hydrophobic components
whose observed mass (ESI-MS) were consistent with the
calculated peptide m ass. To test for completion of the
Cys2-8 disulfide bridge formation, 20 m
M
K
3
Fe(CN)
6
(15 lL)wasaddedto30lL (1 nmol) of the hydrophilic
component (pH 7) at 25 °C for 20 min, and then the pH
was readjusted to 5 using 50% aqueous acetic acid.
Coinjection of the untreated and K
3
Fe(CN)
6
-treated
hydrophilic components on analytical HPLC indicated a
difference in retention time indicative of formation of the
disulfide bridge which was confirmed w ith ESI-MS
analysis.
Following the injection of both hydrophilic and hydro-
phobic c omponents onto an a nalytical HPLC column under
isocratic conditions to ensure the purity a nd quantity of

each peptide,  120 nmol of t he hyd rophilic component
(30 aliquots at 4 nmol) and 80 nmol of hydroph obic com-
ponent (20 aliquots at 4 nmol) were lyophilized for a Cys3-
9 disulfide reaction. Each dried aliquot was dissolved with
0.1% aqueous trifluoroacetic acid (400 lL) and 40 lL1%
CaCl
2
at 0 °C,andthentreatedwith2lL of 0.1% iodine
in methanol at 25 °C for 15 min. Finally, 2 lLof2.5%
ascorbic acid in DI water was added to quench the reaction
and eliminate excess iodine.
HPLC purification of cyclo tx5a
Purification of the cyclo 2-8, 3-9[b-
D
-Gal-(1fi3) -a-
D
-
GalNAc-Thr10]-tx5a h ydrophilic and hydrophobic prod-
ucts on analytical HPLC (10 mm · 250 mm Vydak C
18
300 A
˚
pore size) with 0.1% aqueous trifluoroacetic acid as
buffer A and 60% acetonitrile in 0.1% aqueous trifluoro-
acetic acid as buffer B (gradient 1% per min) resulted in
components whose observed masses (ESI-MS) were consis-
tent with the expected p eptide m asses. Each component was
injected on an analytical HPLC under gradient conditions
to check for purity and quantity. The hydrophilic compo-
nent was collected at  20% acetonitrile (174 lg, 90 nmol).

The hydrophobic component was collected at approxi-
mately 23% acetonitrile (66 lg, 34 nmol). ESI a nd matrix
assisted laser desorption mass spectrometry (MALDI-MS)
measurement o f both components r esulted i n intense species
consistent with the correct product (see below). Both the
hydrophilic and h ydrophobic co mponents were found to be
99% pure as a ssessed using an orthogonal i on pairing agent
system (triethylammonium phosphate, pH 2.3 as buffer A,
60% a cetonitrile as buffer B with a g radient from 10 t o 50%
Bin40min).
Enzyme hydrolysis
Approximately 1 nmol of native tx5a, tx5a hydrophilic and
tx5a hydrophobic were incubated with 25 mU b-galactosi-
dase from bovine testes (Glyko, Inc., Novato, CA, USA) in
Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur. J. Biochem. 271) 4941
100 lLof100m
M
sodium citrate/phosphate pH 4 at 32 °C
for 24 h. As a positive control of enzyme activity, contul-
akin-G, a b-
D
-Gal-(1fi3)-a-
D
-GalNAc c ontaining glyco-
peptide, and native t x5a were simultaneously incubated a nd
reacted with the same vial of the enzyme b-galactosidase.
Native tx5a, tx5a hydrophilic, and tx5a hydrophobic (each
1 nmol) were incubated w ith 2 .5 mU of endo- O-glycosidase
(endo-a-N-acetylgalactosaminidase) (Prozyme, Inc., San
Leandro, CA, USA) in 50 lLof50m

M
NaHPO
4
pH 5 at
32 °C for 24 h. As a positive control of enzyme activity,
contulakin-G and native tx5a were coincubated with the
enzyme endo-O-glyco sidase. In each case, t he enzyme
reactions were stopped with addition of 10 lL10%
aqueous trifluoroacetic acid and immediately injected onto
RP-HPLC and fractions collected were collected and
analyzed with ESI and MALDI-MS.
Chemical reduction
The native and synthetic tx5a (hydrophilic and hydropho-
bic) were incubated with 50 m
M
TCEP for 30 min at 32 °C
prior to injection on reverse-phase HPLC, collection and
analysis with ESI-MS.
Coelution
The native and synthetic tx5a (hydrophilic and hydropho-
bic) were coinjected onto a Vydac C
18
RP-HPLC column
(2.1 · 150 mm) and eluted with a 1% per min gradient
from 0% B t o 4 5% B ( where buffer A was 0.055% aqueous
trifluoroacetic acid and buffer B was 0.05% trifluoroacetic
acid in 90% aqueous acetonitrile).
Mass spectrometry
HPLC purified fraction s were analyzed with both ESI-MS
and MALDI-MS. Samples for electrospray analysis were

diluted 1 : 1 with 1% acetic acid in methanol and infused at
1 lLÆmin
)1
into an Esquire LC electrospray quadrupole ion
trap mass spectrometer (Bruker Daltonics, Billerica, MA,
USA). Previously, w e h ave dem onstrated the mass accuracy
for our electrospray instrument for nonresolved isotopic
clusters of metal chelate complexes to be ± 1.0 m/z when
compared with the calculated average mass. Samples for
MALDI-MS analysis were mixe d with a-cyano-4-hydroxy-
cinnamic acid and irradiated with 282 nm irradiation from
a nitrogen laser using a DE-Star (Perceptive, Framingham,
MA, USA) mass spectrometer. The mass accuracy of the
MALDI instrument for resolved isotopic clusters is ±
0.2 m/z when compared with the calculated monoisotopic
mass.
Purification of native tx5a
Native tx 5a (e-TxIX) was purified from Conus textile venom
as described previously [9]. Briefly, the venom from Conus
textile cone snails was expressed manually. The lyophilized
venom extract (200 mg) was dissolved in 0.2
M
ammonium
acetate and chrom atographed on a Sephadex G50 superfine
column (2.5 · 92 cm) equilibrated with 0 .2
M
ammonium
acetate buffer, pH 7.5, and eluted with a flow rate of
9.2 mLÆh
)1

. The column fractions were monitored using
absorption (A) at 280 and 214 nm. Column f ractions were
subjected to direct Gla a nalysis following alkaline hydrolysis
[11,12]. The material in the major Gla-containing peak was
further purified on a reverse-phase column (HyCrom C
18
,
5 l;10· 250 mm) in 0.1% trifluoroacetic acid and eluted
with a linear acetonitrile gradient 20–40% B (Buffer A:
0.1% trifluoroacetic acid, water; Buffer B: 0.1% trifluoro-
acetic acid, acetonitrile).
NMR spectroscopy
Native tx5a NMR samples were dissolved initially in
99.8% D
2
O and heated to 50 °C at a neutral pH o f 7.0 in
the presence of Chelex 100 to ensure that all trace metal
ions were removed. Th is sample was then lyophilized
and redissolved in 350 lL of 99.96% D
2
O(0.7m
M
)
(Cambridge Isotope Laboratories, Andover, MA, USA),
to a noncorrected pH of 5.60 and transferred to a 4 mm
NMR tube. All spectra were acquired on a Varian Unity
INOVA spectrometer w ith a proton frequency of
499.695 MHz ( Varian Inc., Palo A lto, CA, USA). The
carrier frequency was set on the water resonance, which
was suppressed using presaturati on or a wet pulse

sequence. Preliminary one-dimensional spectra were
acquired over a range of temperatures (5–35 °C) with
16 000 real data points, 256 summed scans and a spectral
width of 8000 Hz. The final two-dimensional
1
H homo-
nuclear and
13
C-
1
H heteronuclear correlation data sets
were collected at 12 °C.
Two-dimensional NOESY spectra were recorded with
mixing times of 150 and 320 ms. A total of 2048 (or 4096)
real data points were acquired in t2, 512 time-proportional
phase increments (or States-TPPI) i n t 1, with a spectral
width of 8000 Hz in the observed ( F2) d imension. A total of
128 summed scans were collected with a relaxation delay of
1.3 s between scans. Spectra were processed with a sine bell
window function shifted by 30° in t2 (applied over 1024
points) and a sine bell window function shifted by 30° in t1
(applied over a ll 512 acquired points) using the Varian
processing software,
VNMR
(Varian I nc., Palo Alto, CA,
USA). All data were zero-filled to a 4096 by 2048 matrix
using the
VNMR
processing program. TOCSY spectra were
recorded and processed as described for the NOESY with

the exception that 4096 real data points w ere acquired i n t2,
with 384 time-proportional phase incrementation (TPPI o r
States-TPPI) increments in t1. A 35 ms mixing time was
used in collectin g 256 summed scans employing the MLEV-
17 spinlock sequence. A D QF-COSY spectrum was recor-
ded with 4096 real t2 points, 64 summed scans, and 712
TPPI increments to ensure increased resolution. The
spectrum w as multiplied by s ine b ell w indow functions
shifted by 3 0° in t2 and 3 0° in t1 and zero-filled t o a 2048 by
1024 (real) matrix. A two-dimensional
13
C-
1
H h eteronuclear
single quantum coherence (HSQC) spectrum was recorded
with 2048 real data points in t2, with 192 time-proportional
phase instrumentation (TPPI or States-TPPI) increments in
t1 and spectral widths of 8000 Hz and 17 591 Hz in the
1
H
and
13
C dimensions, respectively. A total of 256 summed
scans were collected with a relaxation delay of 1.3 s. All
1
H
and
1
H-
13

C correlation assignments were performed using
FELIX
2000, which is part of the
INSIGHT
suite of programs
(Accelrys, San Diego, CA, USA).
4942 J. Kang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Results
After synthesis and deprotection of tx5a from the resin, we
obtained two components with the expected m ass, herein
referred to as h ydrophilic and h ydrophobic. Because a
racemic mixture of
L
/
D
6-bromotryptophan was used in the
synthesis to insure that we would synthesize a tx5a analog
corresponding to the native peptide (irrespective of which
6-bromotryptophan isomer was incorporated in the native
tx5a peptide) we propose that the hydrophilic and hydro-
phobic fractions correspond to the
L
/
D
6-bromotryptophan
isomers of tx5a. In order to further compare these
hydrophilic and h ydrophobic fractions the mass spectra of
the synthetic products were determined following reduction
of the disulfide bonds (to remove potential complexity of
data due to the disulfide a rrangement). When measured in

the negative ionization mode, the ESI mass spectra of all
three samples were almost identical in appearance. Figure 1
shows (A) the hydrophilic component and (B) the reduced
native tx5a (a similar result was observed for the hydro-
phobic component, data not shown). In Fig. 1B, three
major species observed at m/z 994.2, 972.1 and 950.5
(identified as M
R
¢,M
R
¢¢ and M
R
¢¢¢) were interpreted as
corresponding to [M
R
+F e - 5 H ]
2–
,[M
R
+F e - C O
2
-5H]
2–
and
[M
R
+F e - 2 C O
2
-5H]
2–

where M
R
corresponds to the expec-
ted average mass of chemically reduced native tx5a (m ¼
1935.81 Da). As previously proposed [13], the fragment ions
are formed in the mass spectrometer from the facile loss of
CO
2
(e.g. from either of the two c-carboxyglutamic acid
residues or other acidic groups) rather than from synthetic
by-products based on the RP-HPLC, ion exchange chro-
matography and capillary zone electrophoresis results (data
1000
800
600
400
200
0
875 900 925 950 975
1000 1025 1050 1075
M
R
'
M
R
''
M
R
'''
m/z

Relative Intensity
18551845
1835
100
Mass (m/z)
Relative Intensity
800
600
400
200
0
875 900 925 950 1000
1025 1050
1075
975
Relative Intensity
m/z
M
R
'
M
R
''
M
R
'''
18551845
1835
100
Mass (m/z)

Relative Intensity
A
B
Fig. 1. Electrospray mass spectrum of (A)
chemically reduced synthetic hydrophilic cyclo
2-8, 3-9[6-
L
/
D
-bromo-Trp7, b-
D
-Gal-(1fi3)-a-
D
-GalNAc-Thr10]-tx5a compared with (B)
chemically reduced native tx5a where M
R
¢ ¼
[M
R
+F e - 5 H ]
2–
species. Insets show the cor-
responding MALDI resolved isotope distri-
butions of the [M
R
-CO
2
-H]
–
species.

Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur. J. Biochem. 271) 4943
not shown). Other species present in Fig. 1 correspond to
sodium cationization (i.e. +Na-H) of the intact and
fragment ions. Insets in F ig. 1 are t he M ALDI-MS resolved
isotope distribution of the chemically reduced hydrophi-
lic and n ative samples (the species corresponds to [ M
R
-
2CO
2
-H]
–
, observed monoisotopic m/z 1844.5 and 1844.7,
respectively, compared with the calculated monoisotopic
[M
R
-2CO
2
-H]
–
mass of 1844.46 Da).
After s elective f olding of the disulfide bridges o f t he
hydrophilic component of tx5a, we observed similar ESI
negative mass spectra from the synthetic hydrophilic and
native t x5a, as shown in F ig. 2 (a simila r result w as observed
for the hydrophobic component, data not shown). In
Fig. 2B, the M ¢,M¢¢ and M¢¢¢ species observed a t m/z 992.1,
970.1 and 948.0 were interpreted as corresponding with
[M+Fe-5H]
2–

,[M+Fe-CO
2
-5H]
2–
and [M+Fe-2CO
2
-5H]
2–
where M corresponds to the e xpected average mass of
native tx5a (m ¼ 1931.76 Da). The insets in Fig. 2 s how the
MALDI-MS (negative ion mode), resolved isotope distri-
bution measurements for the hydrophilic and native species
(observed monoisotopic at m/z 1840.13 and 1840.32,
respectively, compared with the calculated monoisotopic
[M-2CO
2
-H]
–
mass of 1840.42 Da). The mass shift of
 4Da (M
R
) M) confirms the formation of the two
disulfide bridges.
However, comparison of the retention times of the
hydrophilic tx5a, hydrophobic tx5a and native tx5a
(Table 1) reveals that the three peptides have different
chromatographic properties and can be c learly distinguished
when analyzed under either nonreducing or reducing
conditions. In particular, RP-HPLC chromatography of
chemically reduced native tx5a and the reduced hydrophilic

1500
1000
500
0
875 900 925 950 975 1000 1025 1050
m/z
Relative Intensity
1075
M'
M''
M'''
18551845
1835
100
Mass (m/z)
Relative Intensity
1200
600
0
875 900
925
950 975 1000
1025
1050 1075
M'
M''
M'''
m/z
Relative Intensity
18551845

1835
100
Mass (m/z)
Relative Intensity
A
B
Fig. 2. Electrospray mass spectrum of (A)
synthetic hydrophilic [6-
L
/
D
-bromo-Trp7,
b-
D
-Gal-(1fi3)- a-
D
-GalNAc-Thr10]-tx5a
compared with (B) native tx5a where M¢ ¼
[M+Fe-5H]
2)
species. Insets show the cor-
responding MALDI resolved isotope distri-
butions of the [M-CO
2
-H]
–
species.
4944 J. Kang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
tx5a that were coinjected (Fig. 3) reveals a small but
significant difference in the chromatographic retention time

of these two glycopeptides.
Similarly, when the chemically synthesized hydrophilic
tx5a or hydrophobic tx5a was incubated w ith b-galactosi-
dase, the retention time of the product (Table 1) and the
observed mass were altered as a result of the elimination of
the galactose residue as determined by MALDI-MS (data
not shown). In contrast, the retention time of native tx5a
did not change when incubated under these conditions. In
order to exclude the possibility that the absence of enzyme
hydrolysis was due to a co ntaminating enzyme inhibitor
present in the native tx5a preparation, we added a control
glycopeptide (contulakin-G) to this incubation mixture.
We observed that the enzyme was able to hydrolyze the
galactose r esidue on the control glycopeptide (data not
shown). In addition, both the hydrophilic tx5a and
hydrophobic tx5a peptides demonstrated a shifted HPLC
retention time following incubation with endo-O-glycosi-
dase (Table 1) and an observed mass change as a result of
the elimination o f the entire glycan. In contrast, the
retention time of native tx5a did not change when
incubated under these conditions. The presence of contul-
akin-G (positive control) was used to validate the activity
of the endo-O-glycosidase enzyme, which was un able to
hydrolyze native tx5a. Together these data suggest that the
glycan configuration of native tx5a is distinct from the
synthetic tx5a peptides and contulakin-G. Therefeore, two-
dimensional DQF-COSY, TOCSY, NOESY a nd HSQC
spectra of the native t x5a glycopeptide w ere collected in
99.96% D
2

Oat12°C, pD 5.6. These data, in combination
with data collected previously in 90 : 10 H
2
O/D
2
O enabled
assignment of the amino acid and sugar spin systems of the
glycopeptide [9]. Interestingly, O-glycosylation of T hr10
perturbed the b-carbon
13
C chemical shift (81.8 p.p.m.),
which is downfield from the expected chemical shift (67.9–
68.3 p.p.m.) and in support of the glycan linkage at this
site [14]. Several resonances that were at tributed to the
glycan moiety of tx5a, localized within a spectral envelope
between 3.4 p.p.m. and 4.0 p.p.m., remaine d unassigned
following our initial assignment of the glycopeptide
backbone and side chain resonances [9]. The resonances
of the monosaccharides residues GalNAc and Gal were
primarily assigned from DQF-COSY and TOCSY spectra
commencing with the anomeric protons at 4.79 and
4.82 p.p.m., respectively (Table 2). Both of these proton
resonances demonstrated strong correlation cross-peaks to
two additional high fi eld proton signals that were tenta-
tively assigned H2 and H3 for the respective monosaccha-
rides (Fig. 4A). These assignments were confirmed using
the single interproton scalar connectivity measured by the
DQF-COSY spectrum. The remaining GalNAc and Gal
proton resonances were assigned using the aforementioned
spectra in combination with NOESY data and a natural

abundance
13
C-
1
H HSQC spectrum that enabled each
carbon to be correlated with its directly bonded proton or
protons (Tables 2 and 3).
Strong NOEs between th e anomeric and H2 p rotons and
3
J
1,2
coupling constants of 4.25 Hz for both the GalNAc
and Gal monosaccharides identify an a configuration for
both anomeric centers within the glycan (Table 2). The
3
J
2,3
coupling constants were 7.92 and 7.84 Hz, respectively, for
the GalNAc and Gal monosaccharides. Furthermore, the
H3 resonance of GalNAc showed a strong N OE to the
Table 1. Comparison of the reverse-phase HPLC retention times (in minutes) of native tx5a, synthetic hydrophilic, synthetic hydrophobic peptides
under nonreducing and reducing conditions, and after incubation with b-galactosidase and O-glycosidase.
Nonreducing Reducing b-Galactosidase O-Glycosidase
Hydrophilic tx5a 23.8 26.6 25.3 26.1
Hydrophobic tx5a 27.8 27.9 27.6 29.1
Native tx5a 25.9 27.0 25.9 25.9
Retention Time (min)
UV Absorption (210 nm)
Chemically reduced Native tx5a
Chemically reduced hydrophilic tx5a

Fig. 3. Reverse-phase HPLC chromatography of a coinjection of
chemically reduced native tx5a and synthetic reduced hydrophilic cyclo
2-8, 3-9[6-
L
/
D
-bromo-Trp7, b-
D
-Gal-(1fi3)-a-
D
-GalNAc-Thr10]-tx5a.
Table 2.
1
H,
13
C chemical shifts and scalar coupling constants for the
glycan monosaccharides in tx5a in 99.96% D
2
O at 285.5 K (relative to
sodium 2,2-dimethyl-2-silapentane-5-sulfonate).
3
J
x,y
, 3-bond coupling
constant.
Proton (
1
H)
GalNAc Gal
1

H
13
C
1
H
13
C
H1 4.79 103.2 4.82 98.6
H2 3.94 52.2 3.55 72.4
H3 3.78 77.4 3.44 74.0
H4 3.48 75.8 3.76 73.8
3
J
x,y
(Hz) GalNAc Gal
3
J
1,2
4.25 4.25
3
J
2,3
7.92 7.84
3
J
3,4
4.6 4.4
Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur. J. Biochem. 271) 4945
anomeric proton of Gal, which i dentified that the Gal
residue is linked to the GalNAc monosaccharide through a

H1–H3 linkage (Fig. 4B). Furthermore, the low field C3
(Table 3) carbon chemical shift (77.4 p.p.m.) of the GalNAc
residue supports it being glycosylated at position 3. Taken
together, these data identified the glycan as a-
D
-Gal-(1fi3)-
a-
D
-GalNAc.
There a re several NOEs between the glycan and the
glycopeptide side-chain atoms of tx5a, which suggests that
the monosaccharides are conformationally less flexible,
well ordered a nd within 5 A
˚
of these glycopeptide side-
chains at 12 °C ( Table 4). S pecifically, the side-chain
protons of Thr10, Ala12 and Hyp13 interact with the
glycan protons. Several NOEs are observed between the
anomeric proton of GalNAc and the side-chain atoms of
Thr10; Thr10
b
(strong NOE) and Thr10
CH3
(medium
NOE) (Fig. 5). For A la12 the Ala12
a
and Ala12
bCH3
side-chain protons demonstrate medium a nd strong
NOEs, r espectively, w ith the N-ace tyl C H

3
of GalNAc
at 1.79 p.p.m., which may a lter the magnetic and chem-
ical environment of this moiety a nd help us to understand
this fairly unique chemical shift frequency (Fig. 5). In
addition, there are several weak NOEs between the
N-acetyl CH
3
and GalNAc H
3
,GalNAcH
4
and Gal H
3
,
which further supported a well ordered carbohydrate
moiety at 12 °C(Table3).
AB
Fig. 4. Two-dimensional
1
H spectra of 0.7 m
M
tx5a (e-TxIX) collected in 100% D
2
O or 90% : 10% H
2
O/D
2
O, respectively, at 500 MHz. (A)
TOCSY spectrum c ollected in 100% D

2
O illustrating the alpha region of the data, which includes the monos accharide resonances and (B) NOESY
spectrum collected in 90% : 10% H
2
O/D
2
O(H
2
O resonan ce at 4 .65 p.p.m.) of th is same re gion co llected w ith a mixing time of 320 ms. All data
were collected at 12 °C. Specific carbohydrate resonances are assigned in addition to protons of amino acids residues from t he tx5a peptide
including Gly6, Thr10 a nd Pro13. (A) Illustrates the intraresidue carbohydrate a ssignments GalNAc ( GN) and Ga l (G), respectively. In B , many of
these same intraresidu e assignments are labeled in addition to the interglycosidic linkage betw een GNH
3
of GalNAc and GH
1
of Gal, which is
labeled in bold. The amino acids Gly6, Thr10 and Pro13 are represented by 6G, 10T and 13P.
Table 4. NOEs between the tx5a peptide resonances and the protons
(
1
H) within th e Gal-GalNAc disaccharide. The n om enclature represents
that used in Figs 4 and 5.
Disaccharide: Gal-GalNAc tx5a
Proton (
1
H)
Chemical
shift (p.p.m.) Proton (
1
H)

Chemical
shift (p.p.m.)
GalNAc:H1 4.79 10ThrcCH
3
0.98
GalNAc:CH3 1.79 12AlaaH 4.07
GalNAc:H3 3.77 10ThrcCH
3
0.98
GalNAc:H1 4.79 10ThrbH 4.04
Gal:H2 3.53 11AlaßCH
3
1.09
Gal:H2 3.53 12AlaaH 4.07
Table 3. tx5a Gal-GalNAc NOE interactions and their corresponding
proton (
1
H) chemical shifts in 99.96% D
2
O at 285.5 K (relative to
sodium 2,2-dimethyl-2-silapentane-5-sulfonate).
Proton (
1
H)
Chemical
shift (p.p.m.) Proton (
1
H)
Chemical
shift (p.p.m.)

GalNAc:H1 4.79 GalNAc:H2 3.95
GalNAc:H3 3.77 GalNAc:H2 3.95
GalNAc:H3 3.77 Gal:H1 4.82
Gal:H1 4.82 Gal:H2 3.53
Gal:H1 4.82 Gal:H3 3.43
Gal:H2 3.53 Gal:H4 3.72
GalNAc:CH3 1.79 Gal:H2 3.53
GalNAc:CH3 1.79 Gal:H3 3.43
GalNAc:CH3 1.79 Gal:H4 3.72
4946 J. Kang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
The tx5a peptide from Conus textile has the greatest
diversity of post-translational modifications found in any
conotoxin hitherto characterized. There are two disulfide
crosslinks, a hydroxylated proline residue, a brominated
tryptophan residue, and two c-carboxylated glutamic acid
residues. In addition, there is an O-glycosylated threonine
residue, where the glycan moiety contains equimolar
amounts of GalNAc and Gal. We synthesized the tx5a
peptide with the disulfide connectivity characteristic of
the previously characterized T-superfamily (Cys2-Cys8,
Cys3-Cys9) and a ssumed that the glycan moiety w ould
be the T-antigen, as was previously shown for contula-
kin-G, i.e. b-
D
-Gal-(1fi3)-a-
D
-GalNAc O-linked to threo-
nine [6].
The synthe tic strategy is briefly outlined in Scheme 1.

During the chemical synthesis we used a selective cysteine
deprotection strategy to obtain t he correct disulfide-bonding
pattern. In addition, we investigated the relative merits of
manual vs. automated Fmoc synthesis of this extremely
complicated target molecule. The very low y ield obtained,
0.027% (b ased on our rough estimated 50 : 50 split between
automated and manual synthesis, the 90 nmol of hydro-
philic and 3 4 nmol of hydrophobic t x5a analogs) is in
contrast with yields (30%) previously obtained for nondi-
sulfide bridge-containing glycopeptides using either manual
or automated strategies [6]. We note, however, that even in
the synthesis of nondisulfide bridge-containing glycopep-
tides the yield is dramatically affected by the scale of the
reaction, the excess of amino acids used, and the level of
purity desired. H ere, our r eaction scale was limited by the
costs of the reagents and our desire to obtain peptides that
were of the highest purity. Also, the use of only a slight
excess ( 10–20%) o f s ome e xpensive amino acids contributed
to the low yield of the desired product, and increased the
formation of truncated products. In summary, by using a
selective Cys deprotection strategy we successfully obtained
the desired disulfide connectivity, but this may h ave partially
contributed to the very low yields. We note also that
determination of the stereochemistry of the 6-bromotryp-
tophan r esidue as either
L
or
D
, a nd utilization of the
appropriate resolved precursor would result i n a signifi-

cantly improved yield.
Surprisingly, the chemically synthesized peptides did not
coelute with the native peptide as demonstrated by
RP-HPLC. The difference between native and synthetic
peptides is most probably associated with the configuration
of the glycan moiety attached to Thr10. We demonstrated
that the glycan of t he synthetic peptide cou ld be hydrolyzed
by b-galactosidase, as well a s by e ndo-O-glycosidase, as one
would expect for the glycan in a T-antigen configuration.
These enzymes w ere previously sho wn to a lso hydrolyze the
glycan moiety of contulakin-G [6]. However, the native t x5a
peptide was not amenable to hydrolysis by these two
glycosidases. T he failure to hydrolyze the native peptide w as
not due to the presence of an inhibitor in the native
preparation a s demonstrated when native and synthetic
peptides were mixed. The synthetic peptide was cleaved by
the g lycosidases, while the native p eptide was resistant.
These data support that the intrinsic carbohydrate proper-
ties of the g lycan moieties linked to these peptides are
distinct and more importantly that the tx5a glycan is
comprised of interglycosidic linkages t hat are not recognized
and t hus not cleaved by t hese enzymes. These data p ermit u s
to conclude that, contrary to our expectations and prior
results with contulakin-G [6], the glycan p resent on the tx5a
peptide is not the T-antigen.
Initial investigations by our laboratory identified that
GalNAc and Gal are present in equivalent concentrations,
but we did n ot further determine the configuration of this
carbohydrate. However, the different elution profiles of the
native tx5a and the synthetic peptides constructed with the

carbohydrate in the T-antigen configuration combined w ith
the inability of the aforemen tioned glycosidic enzymes to
hydrolyze the tx5a glycan link ed to Thr10 identifies t hat the
difference must be attributable to the interglycosidic linkage
of the native tx5a glycan, which is clearly not in a typical
T-antigen configuration. To better characterize the confi-
guration of this glycan moiety we used standard two-dimen-
sional homonuclear and heteronuclear (natural abundance)
NMR spectroscopy. Using the information gleaned from
our two-dimensional COSY, NOESY and
13
C-HSQC
experiments we assigned the
1
Hand
13
C chemical shifts of
all nuclei with the exception of those that remained
spectrally degenerate. The anomeric protons identified in
the C OSY a nd TOCSY spectra (collected at 12 °C) at
4.79 p.p.m. and 4 .82 p.p.m. (GalNAc and Gal, r espectively)
provided a good starting place for the through-bond scalar
assignment within each sugar moiety (Fig. 4A). Most
spectral degeneracy was resolved through the use of a
13
C-
HSQC experiment and the assignments completed using
NOESY spectra collected at several mixing times (Fig. 4B).
Fig. 5. A region of the 500 MHz 2D NOESY (320 ms) collected in
90% : 10% H

2
O/D
2
O illustrating the NOEs observed between the
amino acids side chains of residues Thr10 and Ala12 and the carbohy-
drate moieties of GalNAc (GN) and Gal (G). The individual amino
acids Thr10, A la12 are represented by 10T and 12A, respectively, whil e
GNH
1
and GNH
3
represents the H1 and H2 protons from GalNAc,
and GNCH
3
represents the methyl group (CH
3
) that is within the
GalNAc acetyl group.
Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur. J. Biochem. 271) 4947
These
13
C data also identified that the b-carbon of Thr10,
was shifted to lower field (81.8 p.p.m.), which supported
that Thr10 was the g lycosylation sit e ( as w e a lready
believed). The anomeric configuration and interglycosidic
linkage patterns were identified using several through-bond
scalar measurements, the
3
J
1,2

coupling constant between
the anomeric (H1) and H2 p rotons (
1
H’s) of each carbo-
hydrate moiety and resonance assignments. Specifically, the
small
3
J
1,2
and larger
3
J
2,3
scalar coupling constants
identified that both carbohydrate moieties were in an alpha
configuration. In addition, the low field chemical s hift of the
C3-carbon of the GalNAc (77.4) strongly supported this
carbon as the interglycosidic linkage carbon. Together these
data suggested that the interglycosidic linkage between
GalNAc and Gal was 1–3 in the a lpha configuration. These
data were confirmed by the strong NOE between the
1
Hat
position 3 (H3) of GalNAc and the anomeric (H1)
1
Hof
Gal. This linkage pattern helps us to better understand the
resistance to hydrolysis by the aforementioned glycosidic
enzymes, while identifying a linkage pattern that i s d isparate
from that previously identified for contulakin-G [6]. Inter-

estingly, several additional NOEs were identified between
the glycan linked to Thr10 and other tx5a residues a s
illustrated in Fig. 5. These NOEs support that t he carbo-
hydrate moieties interact with the glyco peptide, suggesting
that the carbohydrate is conformationally well structured.
This apparent reduction in conformational fl exibility (on
the NMR time scale) has been identified previously in other
glycosylated peptides and may further support a functional
role of the glycan in receptor-mediated func tion although
this requires further investigation. It was completely unex-
pected that the only two characterized Conus peptides
containing the same sugar moieties attached to the same
aglycone residue, Thr, would have different configurations.
This strongly suggests that the post-translational e nzymes
necessary to catalyze O-glycosylation of t hreonine residues
are different for Conus geographus (contulakin-G) and
Conus textile (tx5a) venoms. This conclusion raises a
number of additional questions that necessitate further
investigation.
Specifically, what is the actual structure of the glycan
moiety in the native tx5a peptide? Our NMR data indicates
an a-
D
-Gal-(1fi3)-a-
D
-GalNAc-Thrstructureforthisgly-
can, and a renewed total synthesis effort is currently under
way to confirm this finding. Apart from the question
pertaining to the glycan configuration there are more
general and intriguing questions related to the O-glycosy-

lation differences of these peptides. Recent studies have
demonstrated that for some Co nus peptide post-transla-
tional modifications (such as for the conantokin peptide
family which are all c-carboxylated), a recognition signal
sequence present in the precursor sequence serves as a
binding site to recruit the appropriate enzyme that is
necessary for a specific post-translational modification [15].
One possibility is that different recognition signals in the
tx5a and contulakin-G precursors recruit different glycosyl
transferases.
An alternative explanation is centered on the fact that
these peptides belong to different peptide superfamilies that
may process peptides through s pecific and d istinct secretory
pathways. T hus, e nzymes that carry out the glycosylation to
give the configuration of the T-antigen might be packaged
in the secretory pathway of the contulakin family, but a
different set of enzymes may be packaged into the secretory
pathway for the T-superfamily of peptides to which tx5a
belongs. It is also feasible that these two Conus species have
taken advantage of this post-translational modification in
unique ways that allows them to accommodate evolutionary
and e nvironmental changes that are specific for e ach species.
These data demonstrate the feasibility of chemically
synthesizing peptides, such as tx5a, that possess multiple
post-translational modifications. This synthesis in and of
itself is a significant achievement in lieu of the complexity
and number of post-translationally modified amino acids
included. However, our synthetic efforts and subsequent
enzymatic degradation and NMR spectroscopy studies,
have revealed that the glycan configuration is not the same

as that previously discovered and reported f or co ntulakin-G
(Conus geographus). This surprising result establishes that
the O-glycosylation of serine and threonine residues in
Conus pep tides are likely to b e more complex than had
originally been anticipated, involving more than one
specialized post-translational modification enzyme.
Acknowledgements
This work was s upported b y the N ational Institutes of Health
(GM48677) (B.M.O.), the National Science Foundation (A.C.R.) and
conducted in part by the Foundation for Medical Research (A.G.C.).
We would l ike to thank Jean E. Rivier and J osef Gulyas for s timulating
conversations and helpful advice.
References
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Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur. J. Biochem. 271) 4949