insect Carausius morosus contains two hypertrehalosae-mic decapeptides, Cam-HrTH-I and Cam-HrTH-II, which differ only in the modification of Trp8 by a hexose on peptide I.. Results Assign
Trang 1from the stick insect Carausius morosus
Claudia E Munte1, Gerd Ga¨de2, Barbara Domogalla1, Werner Kremer1, Roland Kellner3and
Hans R Kalbitzer1
1 Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany
2 Department of Zoology, University of Cape Town, Rondebosch, South Africa
3 Target Research and Biotechnology, Merck KGaA, Darmstadt, Germany
In insects, peptidergic regulation by neuropeptides is
the most important form of communication to
con-trol not only growth, development and reproduction,
but also metabolic homeostasis [1] Fuel
mobiliza-tion, especially to fulfil the exceptionally high energy
demand during contraction of flight muscles, is
under neuroendocrine control by peptides of the
so-called adipokinetic hormone (AKH) family, as is the
case in all investigated insect orders [2,3] These
short peptides of 8–10 amino acids in length
are produced in the retro-cerebral corpora cardiaca
from precursor polypeptides by proteolytic cleavage
The sequence of AKH peptides is characterized
by phenylalanine, tryptophan and glycine residues
at positions 4, 8 and 9, respectively (Fig 1) The
N-terminal glutamine of the precursor is transformed into pGlu in the final product and the C-terminus is amidated Besides the general post-translational modifications at both termini, some AKH peptides are known to contain additional modifications The AKH peptide from the protea beetle Trichostheta fascicularis can be phosphorylated at Thr6 [4] AKH peptides are responsible for the measurable increase
in haemolymph lipids, carbohydrates and proline levels and are denoted accordingly as adipokinetic, hypertrehalosaemic (trehalose instead of glucose is the sugar circulating in the haemolymph of insects) and hyperprolinaemic
More interestingly in the context of the present study, and as first demonstrated in 1992 [5], the stick
Keywords
Carausius morosus; hypertrehalosaemic
hormone; NMR; protein C-mannosylation;
a-mannosyltryptophan
Correspondence
H R Kalbitzer, Institute for Biophysics and
Physical Biochemistry, University of
Regensburg, D-93040 Regensburg,
Germany
Fax: +49 941 943 2479
Tel: +49 941 943 2595
E-mail: hans-robert.kalbitzer@biologie.
uni-regensburg.de
(Received 7 November 2007, revised 13
December 2007, accepted 8 January 2008)
doi:10.1111/j.1742-4658.2008.06277.x
The hypertrehalosaemic hormone from the stick insect Carausius morosus (Cam-HrTH) contains a hexose covalently bound to the ring of the trypto-phan, which is in the eighth position in the molecule We show by solution NMR spectroscopy that the tryptophan is modified at its Cd1(C2) by an a-mannopyranose It is the first insect hormone to exhibit C-glycosylation whose exact nature has been determined experimentally Chemical shift analysis reveals that the unmodified as well as the mannosylated Cam-HrTH are not completely random-coil in aqueous solution Most promi-nently, C-mannosylation strongly influences the average orientation of the tryptophan ring in solution and stabilizes it in a position clearly different from that found in the unmodified peptide NMR diffusion measurements indicate that mannosylation reduces the effective hydrodynamic radius It induces a change of the average peptide conformation that also diminishes the propensity for aggregation of the peptide
Abbreviations
AKH, adipokinetic hormone; Cam-HrTH, hypertrehalosaemic hormone from Carausius morosus; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; HSQC, heteronuclear single quantum coherence; IL, interleukin; TSR, thrombospondin type 1 repeats.
Trang 2insect Carausius morosus contains two
hypertrehalosae-mic decapeptides, Cam-HrTH-I and Cam-HrTH-II,
which differ only in the modification of Trp8 by a
hexose on peptide I However, the exact nature of this
tryptophan glycosylation was previously unknown
because, due to the small amounts of naturally
avail-able peptide, only MS was feasible, which did not
allow determination of the exact type of the hexose
moiety Two years later, the second example of a
tryp-tophan modification was reported in a mammalian
enzyme, human RNAse 2 Using MS and NMR
spec-troscopy, the hexose was shown to be connected to the
Cd1 of the tryptophan ring via a C-glycosidic linkage
[6], and could be unambiguously identified by
subse-quent studies as an a-d-mannopyranose [7] This
tryp-tophan C-mannosylation was later found in another
mammalian protein, human interleukin (IL)-12 [8], and
was shown to be catalysed by a microsome-associated
transferase that uses dolychyl-phosphate-mannose as
donor of the glycosyl group [9] The transferase
recog-nizes the motif WXXW (where X is any amino acid)
[9,10] and C-mannosylates the first tryptophan of this
sequence in RNAse 2 and IL-12 Subsequently,
addi-tional mammalian proteins with such a modification
have been identified, such as human terminal
comple-ment proteins C6, C7, C8a, C8b and C9 [11],
proper-din [12] and thrombosponproper-din-1 [13,14]; all of them
containing thrombospondin type 1 repeats (TSR
mod-ules) In the TSR modules, WXXWXXX motifs are
found Here, more than one tryptophan can be
man-nosylated Variations of this motif can be found in C6,
C7 and in properdin [12], leading to the more general
recognition sequence (W⁄ Y ⁄ F)XXWXX(W ⁄ C ⁄ V) in
the TSR modules Since the number of modified tryp-tophan residues varies in these sequences, it is assumed that either features outside the motif determine the degree of modification or more than one C-mannosyl-transferase might exist
It still remains unresolved as to whether the trypto-phan glycosylation found in 1992 in the stick insect hormone is identical to that found in mammalian enzymes, especially because both differ significantly in tryptophan-glycosylation motifs In the present study,
we describe a detailed NMR analysis of the tryptophan modification in the C morosus hypertrehalosaemic pep-tide Cam-HrTH-I, that was only possible with the high sensitivity of a high field spectrometer (800 MHz) equipped with a cryoprobe In addition, NMR is used
to characterize the structure of Cam-HrTH in aqueous solution at the atomic level, as well as to identify possi-ble structural changes induced by tryptophan modifica-tion that might play a role in receptor recognimodifica-tion
Results
Assignment of peptide chemical shifts
As the modified peptide Cam-HrTH-I and the unmodi-fied Cam-HrTH-II were obtained from natural sources
by isolating the proteins from the corpora cardiaca of approximately 2000 stick insects, a 13C and⁄ or 15N enrichment was not feasible The concentration of the modified protein with approximately 60 lm was rather low to conduct 2D NMR spectroscopy Only the high sensitivity of an 800 MHz-NMR spectrometer equipped with a cryoprobe permitted a successful
Fig 1 Sequence comparison of AKH pre-cursors Only part of the N-terminal part of the sequences is shown, corresponding to the mature AKH (grey background) and the cleavage site (K,R)R The preceding glycine residue provides the C-terminal NH2-group Conserved residues are shown in bold.
*, precursor not known.
Trang 3assignment of the NMR lines The assignment of the
1H resonance lines was obtained by classical
homo-nuclear methods.13C NMR assignments were obtained
by1H,13C-heteronuclear single quantum coherence
(HSQC) spectra with different mixing times To
achieve identical experimental conditions and to
avoid-ing any systematic chemical shift changes that could
arise from experimental differences such as buffer
con-ditions and temperature, the modified and unmodified
peptides were mixed in a 1 : 4 ratio Because the two
peptides were added in different, well defined amounts,
the intensities of the resonance lines allowed the direct
identification of the two peptides in the NMR spectra
of the mixture The assignments of the peptide
reso-nances are summarized in the supplementary Tables S1
and S2
It had been previously shown by MS and amino
acid analysis that Cam-HrTH-I and Cam-HrTH-II
share the amino acid sequence
pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 [5] The only difference
found between both peptides is a modification of the
Cam-HrTH-I tryptophan residue at position 8 by an
unidentified hexose We can expect that the
assign-ments of the two peptides mainly differ around this
residue In agreement with this expectation, the first
four amino acids do not show significant distinctions
in their chemical shifts
Identification of the sugar moiety and the
modification site in tryptophan
By theoretical considerations, it can be argued that the
glycosylation of the tryptophan ring system occurs via
an N–C or C–C bond; the formation of such a bond
would result in the disappearence of the corresponding
proton signal Positions available for the glycosylation
of the ring system are Ne1, Cd1, Cf2, Cg2, Ce3and Cf3;
all of them were assigned in the unmodified peptide
One likely attachment site is the indolic Ne1 atom of
tryptophan As shown in Fig 2, however, the
corre-sponding He1diagonal peaks at 10.51 p.p.m and
10.15 p.p.m are still present for both peptides, but the
correlation peak to the Hd1, although clearly present in
the unmodified peptide, is completely absent in the
modified peptide This indicates that, instead of Ne1,
most probably the Cd1atom is modified Furthermore,
all tryptophan ring carbons directly bound to a proton
for the unmodified peptide could be detected by
1H,13C-HSQC spectroscopy; in contrast, the Cd1peak
was missing in the modified peptide (Table S2) This
would be expected because the Hd1proton necessary
for the insensitive nuclei enhanced by polarization
transfer is removed by the modification
In addition to the peptides’ peaks, five well-defined sugar spin systems were found in the homonuclear 2D NMR spectra (Fig 3A) Diffusion experiments showed that only one of the sugars diffuses with the same diffusion constant as the peptide and, thus, cor-responds to the Trp bound hexose The other four spin systems diffuse freely in the sample (Fig 3B) and have been assigned from their chemical shifts to a- and b-glucose, fructose and sucrose
The 1H and 13C resonances of the bound hexose could be completely assigned The 1H and 13C chemi-cal shift values of the bound hexose and of the trypto-phan ring system are very close to those described for other peptides containing a glycosylated tryptophan (Table 1) For one of these peptides, corresponding to amino acids 5–10 of RNase 2 [7] and studied in aque-ous solution under comparable conditions (300 K,
2H2O) to those of the present study, it could be con-clusively shown by NMR spectroscopy that the trypto-phan is a-mannopyranosylated at Cd1 The proton and carbon chemical shifts obtained are almost identical to those found for the modified Cam-HrTH-I peptide; the maximum chemical shift deviations are 0.07 p.p.m and 0.6 p.p.m for H1¢ and C1¢, respectively When taking into account the average chemical shifts reported for the 2-(a-d-mannopyranosyl)-tryptophan residues (Table 1), the agreement is almost perfect This strongly indicates that the tryptophan of
Fig 2 Selected region of the 800 MHz TOCSY spectrum showing the tryptophan indol ring spin systems of both Carausius morosus neuropeptides The sample contained approximately 60 l M Cam-HrTH-I and 240 l M Cam-HrTH-II in 90%1H 2 O, 10%2H 2 O, 0.1 m M
DSS, pH 5.4 Temperature 300 K W, Trp8 of the native peptide Cam-HrTH-II; W*, Trp8 in the modified peptide Cam-HrTH-I The dashed line indicates the missing1H e1 –1H d1 contact in Cam-HrTH-I.
Trang 4Cam-HrTH-I is also a-mannopyranosylated The
struc-ture of the glycosylated tryptophan is depicted
sche-matically in Fig 4
The tryptophan–hexose bond is further confirmed
by NOEs between the mannose H2¢ proton and the
strongly shifted Trp8 He1 proton of the modified
pep-tide (Fig 4) In the carbohydrate moiety, strong NOEs
are observed between H1¢ and H6¢ and between H2¢
and H3¢; a weak NOE between H1¢ and H2¢; an
ambiguous NOE between H3¢ and H5¢; but no NOE
between H3¢ and H4¢ Thus, the NOE-pattern observed
in the hexose corresponds closely to those in RNase 2 peptides and in pure 2-(a-mannopyranosyl)-trypto-phan, which further corroborates the identity of the hexose moiety as a-mannopyranose
Aggregation state of Cam-HrTH-I and the unmodified Cam-HrTH-II
To investigate the aggregation state of the processed peptides, we performed NMR diffusion measurements
on the sample containing both peptides Figure 3B shows the dependence of the line intensities on the gra-dient strengths used in the stimulated echo sequence for important components of the sample Diffusion data are shown for Cam-HrTH-I and its mannose moi-ety, Cam-HrTH-II, sucrose, glucose and 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), all contained in the same sample In addition, before and after the mea-surements, the signal dependence of polyacrylamide was measured to check the stability of the gradient sys-tem; as required, no signal decay was observed for the immobilized macromolecule The glucose and the sucrose molecules show a relatively fast signal decay,
as expected for small molecules, and therefore are not bound to the peptide By contrast, the mannose reso-nances decay with the same rate as those of the pep-tide signal of Cam-HrTH-I This is to be expected for mannose bound to the peptide because the diffusion constants should be identical Using DSS as a mole-cular mass reference, the effective molemole-cular masses of 1.45 ± 0.12 kgÆmol)1 and 1.96 ± 0.10 kgÆmol)1 are obtained respectively for the modified Cam-HrTH-I and the unmodified Cam-HrTH-II peptides (Table 2) The effective molecular masses are larger than those obtained under the assumption of the same shape fac-tor and density for the test compound and the refer-ence For the glycosylated peptide, the molecular mass calculated from the chemical structure is still almost in the error range of the molecular mass calculated from the diffusion constant for a monomer For the unmod-ified peptide, the effective mass is significantly larger than the calculated value for a compactly folded monomer but smaller than expected for a dimer In line with this observation, the effective transversal relaxation rates increase in good approximation pro-portionally to the effective masses: the transversal relaxation rates calculated from the linewidths of the
He1 resonances of tryptophan increase by a factor of 1.38, from 16.65 ± 0.31 s)1to 22.93 ± 0.31 s)1, which
is within the limits of error of the ratio of 1.35 obtained for the diffusion constants The results indi-cate a monomeric state of Cam-HrTH-I and probably
A
B
Fig 3 NMR spectroscopy of carbohydrates in the solution of
Cam-HrTH-I and Cam-Cam-HrTH-II (A) Selected region of the TOCSY
spectrum showing three sugar spin systems (B) Plot of ln(I ⁄ I 0 ) as
function of G 2 where I is the peak integral at a given gradient
strength G and I 0 is the intensity at G = 0 The sample contained
approximately 60 l M Cam-HrTH-I and 240 l M Cam-HrTH-II in
99.8% 2 H2O, 0.1 m M DSS, pH 5.4 Temperature = 300 K PAA
(polyacrylamide in 90%1H 2 O, 10% 2H 2 O) was used to check the
stability of the gradient system before and after measurement.
Trang 5also for the Cam-HrTH-II with respect to the experi-mental conditions of the study Interestingly, the modi-fied peptide has a smaller hydrodynamic radius than the unmodified peptide, in spite of the known increase
of 162 gÆmol)1 by the mannosylation, indicating a
Table 1 Carbohydrate modifications of tryptophan residues in protein fragments All chemical shifts are referenced to 2,2-dimethyl-2-sila-pentane-5-sulfonate (DSS); when other standards where used, the values were adapted as best as possible.
d ⁄ p.p.m <d> ⁄ p.p.m b d⁄ p.p.m d ⁄ p.p.m d ⁄ p.p.m d ⁄ p.p.m d ⁄ p.p.m Trp
Hexose
a RNase 2, glycosylated hexapeptide from human RNase 2 (amino acids 5–10) [6,7]; IL-12, peptide from human IL-12 (amino acids 316–322) [8]; C9(T2-1) W27, 2-(a-mannopyranosyl)- L -tryptophan at position 27 in a pentadecapeptide derived from complement C9 [11]; C9(T2-2) W27 and C9(T2-2) W30, 2-(a-mannopyranosyl)- L -tryptophan at positions 27 and 30 in the two-fold modified pentadecapeptide derived from com-plement C9 [11] b Average chemical shifts observed for all peptides except of Cam-HrTH-I c Resonance not assigned d Shift not reported.
Fig 4 Structure of the glycosylated tryptophan residue The
exper-imentally found NOEs between the a-mannose moiety and the
Trp8 are depicted as grey lines, where line thickness indicates the
strength of the NOE Ambiguous NOEs are represented by dashed
lines The mannose is depicted in the 1 C4conformation.
Table 2 Molecular masses and relative hydrodynamic radii of the Carausius morosus neuropeptides The sample contained approxi-mately 60 l M Cam-HrTH-I and 240 l M Cam-HrTH-II in 99.8% 2 H2O, 0.1 m M DSS, pH 5.4 and was measured at 300 K.
Compound R h ⁄ R h,DSSa M expb⁄ kgÆmol)1 M calcc⁄ kgÆmol)1 Cam-HrTH-I 1.950 ± 0.056 1.45 ± 0.12 1.308 Cam-HrTH-II 2.156 ± 0.035 1.958 ± 0.097 1.146
a Ratio of the hydrodynamic radii from peptide and DSS, calculated with Eqn (3) b Molecular mass experimentally obtained from the diffusion experiments on the basis of Eqn (4).cMolecular mass cal-culated from the chemical formula.
Trang 6more compact structure of the neuropeptide induced
by mannosylation
Conformational restraints of the peptide
Figure 5 shows the deviations Dd of the Cam-HrTH-I
and Cam-HrTH-II 1H and 13C chemical shifts from
random-coil values The latter were calculated on the
basis of the random-coil values of completely
dena-tured model peptides [15], which were corrected for the
effects of neighbours in the sequence [16] The values
for the N-terminal pGlu were taken from the 21-amino
acid long glycopeptide Gp21 that is assumed to exist
as random-coil in water [17] It is evident that the
chemical shifts deviate significantly from zero,
suggest-ing the peptide is not a random-coil but has some
residual structure In general, negative Ha and Cb and
positive Ca shift differences Dd are thought to indicate
a propensity for a-helical conformations, whereas the
opposite behaviour is indicative for b-pleated
confor-mations The chemical shift differences of
Cam-HrTH-II do not follow one of these patterns, thus providing
no evidence for the dominance of a certain type of
secondary structure in water It is more likely that the
peptide rather exists as an ensemble of structures in
solution and contains a significant number of locally
ordered (transient) conformers The observation of
sequential HN(i)– HN(i+1) and Hb(i)– HN(I+1) NOEs
within residues Thr3-Phe4-Thr5 and within residues
Asn7-Trp8-Gly9 (Table 3) would indicate a
preferen-tial structuring of these regions of the peptide
For the modified and unmodified peptide, the
back-bone chemical shift changes depicted in Fig 5 do not
differ significantly for the N-terminal amino acids
pGlu-1 to Phe4 and the C-terminal Thr10 This is also
true for the side chain residues of these amino acids
Consequently, the average conformation of these parts
of the structure is not perturbed by the modification
Within the tryptophan residue, NOEs are observed
in the modified peptide between the He3 and the two
Hbatoms These are clearly absent in the unmodified
peptide; instead, NOEs between the Hd1 and the two
Hbatoms are present This clearly indicates that some
reorientation of the tryptophan ring around its
b–c-bond occurs as a consequence of the mannosylation
Discussion
Hexose modification of Trp8
Our data clearly indicate that the HrTH from C
moro-susis glycosylated at the Cd1(C2) of Trp8 Along with
the MS data, the coupling patterns, chemical shifts
and NOEs indicate that the hexose bound is an a-mannopyranose linked via C1¢ to the Cd1 of the tryptophan ring Such a C–C tryptophan modification
A
B
C
Fig 5 Deviations of the random-coil values for the a-protons, a-carbons and b-carbons Graphs show the difference between the chemical shifts Dd of Cam-HrTH-I and Cam-HrTH-II and the sequence corrected random coil shifts [15,16] The pGlu shifts were taken from Lu et al [17] (A) H a , (B) C a and (C) C b chemical shifts of the glycosylated (dark grey) and the unmodified peptide (grey) Glycine a-protons chemical shifts have been replaced by the average chemical shift.
Trang 7has been previously observed for mammalian peptides
and proteins, such as RNase 2 [6,7], IL-12 [8],
proper-din [12] and other proteins of the complement system
[11], and the MUC5AC and MUC5B Cys subdomains
[18] Since the classical biochemical pathways produce
exclusively d-mannose [19] in mammals and insects, it
is safe to assume that the modification of the
Cam-HrTH-I peptide is an a-d-mannosylation
The1H and 13C chemical shifts of the modified
tryp-tophan residue are very close to that observed in
pep-tides prepared from these proteins that were analysed
in detail The NOE data for Cam-HrTH-I suggest that
the mannose is in a similar conformation to that
previ-ously observed in an unstructured peptide derived from
RNAse 2 [7] and in
d1-(a-d-mannopyranosyl)-l-trypto-phan isolated from human urine [20] In the
manno-pyranosyl-tryptophan dissolved in acidic methanol, the
1C4 conformation clearly dominates [20] In the unfolded RNase in aqueous solution, a dynamic equi-librium most likely exists between different conforma-tions [7] but a strong NOE between the H4¢ and H6¢ typical for an axial arrangement of the C6¢ was also observed Such an NOE is also observed in the Cam-HrTH-I peptide The conformational equilibrium of the mannose moiety is clearly influenced by its environ-ment and is changed in the natively folded RNAse [21]
Structural implications
It is important to note that the Cam-HrTH-I peptide differs from other C-mannosylated proteins in the gly-cosylation recognition sequence because it lacks the recognition sequence WXXW In the Cam peptide, the fourth amino acid of this motive is missing because the mannosylated Trp is at position 8 and the peptide has only ten amino acids Although the sequence of the precursor of Cam-HrTH is unknown, sequences of precursors from other peptides of the AKH peptide family do not contain a tryptophan at position 11 but residues that are part of the typical cleavage site Gly-Arg⁄ Lys-Arg This pattern is also expected for the Carausiusprecursor (Fig 1)
The nonrandom chemical shifts as well as the NOE-patterns show that both the modified and the unmodi-fied hypertrehalosaemic hormone of the stick insect have some residual local structures in aqueous solution but do not have a well-defined, unique 3D structure Especially in the sequence ranging from Thr3 to Thr5 and from Asn7 to Gly9, larger deviations from typical random-coil properties can be observed NMR experi-ments on other AKH peptides from other insects were performed in organic solvents such as dimethylsulfox-ide Under these conditions, NMR data suggested a b-turn formation between Phe4 and Trp8 [22], which was experimentally supported by an NOE contact between the HN of Ser5 and the HN of Trp8 By con-trast, in Cam-HrTH-II in water, such an NOE could not be observed
The mannosylation of Trp8 does not influence the chemical shifts of the first four N-terminal amino acids and the C-terminal threonine Because chemical shifts are very sensitive to structural changes, the average ensemble structure is probably not changed in this part
of the structure By contrast, significant changes of chemical shifts are observed in the central part of the peptide (amino acids 5–9) In addition, some changes
in NOE intensities are observed (Table 3) Most important are the NOE contacts between the b-protons
of Trp8 and its ring protons After mannosylation, medium intensity NOE cross peaks are observed to the
Table 3 Inter-residual and important intraresidual NOEs in
Carau-sius morosus neuropeptides The sample contained approximately
60 l M Cam-HrTH-I and 240 l M Cam-HrTH-II in 90% 1 H2O, 10%
2 H2O, 0.1 m M DSS, pH 5.4 and was measured at 300 K The NOE
intensities for the modified peptide were corrected to take into
account the sample concentration ratio Nontrivial sequential NOEs
are shown in bold Important intraresidual NOEs are shaded.
pGlu1 Ha– Leu2 HN Mediuma
Leu2 H a – Thr3 H N Strong a
Thr3 H a – Phe4 H N Strong a
Thr5 H a – Pro6 H d Stronga
Trp8 Hb2⁄ b3– Trp8 He3 Medium
Trp8 H b3 ⁄ b2 – Trp8 H e3 Medium
Trp8 H b2 ⁄ b3
Trp8 H b3 ⁄ b2
Gly9 H a – Thr10 H N Weak a
a Contact that could not be distinguished between the two
pep-tides because of chemical shift degeneracy b Because of the
sam-ple concentration ratio, weak NOEs observed in Cam-HrTH-II
cannot be excluded in Cam-HrTH-I.
Trang 8H1¢ proton of the sugar and to the He3of the ring; the
latter NOE is not observed in the unmodified peptide
but, instead, a cross peak to the Hd1atom (the atom
to be modified in HrTH-I) This indicates that, on
average over time, a different v2angle of Trp8 is now
favoured in the modified peptide, which allows a closer
Hb–He3contact
A striking difference between the two peptides can be
found when the diffusion constants are considered The
relative diffusion constant and the relative
hydrody-namic radius of the mannosylated peptide correspond
closely to that expected for a monomeric peptide
How-ever, the experimentally determined relative
hydrody-namic radius of the unmodified peptide is significantly
larger than that of the modified peptide, although its
molecular mass is somewhat smaller Two general
expla-nations for this behaviour can be given: (a) the shape
factor, and thus the hydrodynamic radius, is different in
the two peptides, with the mannosylated peptide being
more compact and (b) in contrast to the mannosylated
peptide, the unmodified peptide is partially aggregated
Under the assumption that the shape factor is
identi-cal for both peptides and that the modified peptide is
completely monomeric, the refined effective molecular
mass of the unmodified peptide can be calculated as
1.776 kgÆmol)1 Assuming we have a monomer–dimer
equilibrium in HrTH-II, approximately 54% would be
in the dimeric state under our conditions Such a
pro-cess would also explain the increase of the observed
linewidths in HrTH-II
However, the shape factor (including the effect of
the hydration shell) can be very different for peptides
and proteins Qualitatively, an increase of the
hydro-dynamic radius Rh is expected when a peptide is less
compactly folded By contrast to our observations, the
linewidths in a completely unfolded peptide are almost
independent of the size because the internal motion in
the peptide dominates the relaxation Most of the
pre-dictions of Rh reported for larger biopolymers do not
accurately apply for small peptides One example
com-prises theoretical work showing the radius of gyration
Rg of a compactly folded homopolymer to scale with
the number of structural units as Nv; the exponent m
equals 1⁄ 3 and 3 ⁄ 5 when going from well defined to
random-coil structures [23,24] In a first
approxima-tion, Rg and Rh are proportional, allowing the
hydro-dynamic radius of a polymer to be predicted based on
the number of its units For proteins, an equivalent
empirical equation has been defined [25] (see
Experi-mental procedures, Eqn (6) that yields to a good
approximation to Rh both for folded and unfolded
proteins However, for small peptides, they would
predict an increase in Rh precisely when a peptide
becomes folded, probably meaning that the extrapola-tion to small molecular masses is not valid here
Conclusion
Although other examples of C-mannosylated trypto-phans have been reported, this is the first time that this type of modification could be demonstrated to occur
in an insect in which this type of modification was first speculated to be present To date, any advantage for the stick insect in having this modified peptide remains known Possible advantages could be a better binding
to the AKH receptor, or that the modified form may not as readily be attacked by peptidases Mannosyla-tion leads to a change of the average orientaMannosyla-tion of the tryptophan ring and may thus provide a more suitable conformation for receptor recognition In addition, mannosylation appears to reduce the propensity of the neuropeptide for aggregation, a feature which may again be favourable for receptor interactions
Experimental procedures
Insects The stick insect C morosus was reared in the Zoology Department, University of Cape Town, at 298 K under a
12 : 12 h light⁄ dark cycle Insects were fed fresh ivy leaves
ad libitum Young adults were separated from the rest of the colony, and corpora cardiaca were dissected from ani-mals more than 2 weeks of age
Purification of the peptides Dissection of glands, preparation of methanolic extracts and isolation of the hypertrehalosaemic peptides Cam-HrTH-I and Cam-Cam-HrTH-II on RP-HPLC were performed
as described previously [5,26] The combined material from approximately 2000 corpora cardiaca was further purified
by RP-HPLC (Zorbax C8, 21· 250 mm; Agilent Technolo-gies, Waldbrunn, Germany)
Preparation of NMR sample The two samples of purified hypertrehalosaemic peptides were freeze-dried and then dissolved in 450 lL distilled water and 50 lL 2H2O The pH was adjusted to 5.4 by addition of HCl DSS was added to a final concentration of 0.1 mm The final sample had a peptide concentration of approximately 60 lm HrTH-I and 240 lm Cam-HrTH-II After performing a set of NMR experiments in water, the sample was newly freeze-dried and re-dissolved
in 500 lL2H2O for a new set of NMR experiments
Trang 9NMR spectroscopy
All NMR experiments were performed on a Bruker Avance
800 spectrometer (Bruker Biospin, Karlsruhe, Germany)
operating at a proton frequency of 800 MHz, equipped with
a TCI CryoProbe Spectra were recorded at 300 K The
water signal was suppressed by selective presaturation
1D1H NMR spectra were recorded with 64 K complex data
points and 1024 scans 2D data sets were recorded with 512
experiments in the t1dimension and 8 K complex t2
-dimen-sion Typically, 64–256 free induction decays were averaged
Phase sensitive detection in the t1-direction was obtained
with time-proportional phase incrementation [27] NOESY
[28] spectra were recorded with a mixing time of 600 ms to
allow normally weak NOEs to become more apparent
RO-ESY [29] spectra were recorded with a RORO-ESY spin-lock
pulse of 300 ms TOCSY [30] spectra were recorded using a
‘clean’ MLEV-17 [31] TOCSY transfer step of 80 ms
Dou-ble quantum filtered-COSY spectra were obtained according
to Rance et al [32] The gradient-enhanced natural
abun-dance 1H,13C-HSQC [33] spectra were recorded using
het-eronuclear J coupling constants of 115, 145 and 165 Hz
Decoupling during acquisition was achieved by the GARP
sequence [34] Because of the low peptide concentration,
typically recording times of 24 h were required to obtain
2D spectra with sufficient signal-to-noise ratio
Diffusion measurements [35] were performed using a
stimulated echo pulse sequence with gradient sandwiches
(gradient length of 1 ms) in 2H2O In addition, spoiler
gradients of 1 and 2 ms in length were used during
trans-verse evolution One thousand and twenty-four scans
were accumulated for each gradient strength
Time-domain data were processed using topspin 2.0 (Bruker)
and evaluated with the program auremol (Bruker) [36]
Assignment of proton resonance lines was performed
according to the standard strategy for homonuclear
spec-troscopy [37] using double quantum filtered-COSY and
TOCSY spectra for the identification of the spin systems
and NOESY⁄ ROESY spectra for the sequence-specific
assignment Assignment of the carbon resonance lines
could be obtained from a set of 1H,13C-HSQC spectra,
assuming J= 145 Hz for peptide aliphatic atoms,
J= 165 Hz for aromatic atoms and J = 115 Hz for
sugar for the calculation of the insensitive nuclei
enhanced by polarization transfer mixing times 13C
chem-ical shifts were referenced based on the ratio
recom-mended by IUPAC [38]
The chemical shift data are deposited in the BioMagRes
database (entry numbers 15620 and 15621)
Evaluation of the NMR diffusion measurements
In a solvent with viscosity g at absolute temperature T, the
diffusion constant Di of a compound si with a
hydrody-namic radius Rh,iis given by the Stokes–Einstein relation
Di¼ kT 6pgRh;i
ð1Þ where k is the Boltzmann constant The hydrodynamic radius Rh,iis defined as the radius of a sphere with a vol-ume Vh,iresulting in the same diffusion constant Di For a compound sihaving an effective volume fiVi, where fi is a characteristic shape factor, Eqn (1) becomes:
Di¼ kT 3g ffiffiffiffiffiffiffi 6p2 3
p 1ffiffiffiffiffiffiffi
fiVi 3
Assuming the same form factor for two different com-pounds siand s1, the unknown hydrodynamic ratio Rh,iof the compound sican be calculated from the known hydro-dynamic ratio Rh,1of compound s1by:
Rh;i¼ Rh;1
D1
Di
ð3Þ Correspondingly, if the mass M1of the compound s1is known, and assuming equal density of both compounds, the mass Miof the compound sican be obtained by:
Mi¼ M1
D1
Di
3
ð4Þ
Diffusion coefficients Dican be experimentally obtained from diffusion NMR experiments [39], since the signal intensity I(G,si) in dependence on the gradient strength G
of a compound siis given by:
IðG; siÞ ¼ Ið0; siÞecDiG2 ð5Þ According to Wilkins et al [25], the empirical hydro-dynamic radius of proteins can be calculated from the number N of residues by:
Rh;i¼ ANa
with A = 4.75 and 2.21, and a = 0.29 and 0.57, respectively for a compactly folded and a completely denatured protein
Acknowledgements
This work was financially supported by the Fonds der Chemischen Industrie and the Deutsche Forschungs-gemeinschaft to HRK; and the National Research Foundation of RSA (gun no 2053806) and the University of Cape Town to GG
References
1 Ga¨de G (1997) The explosion of structural information
on insect neuropeptides In Progress in the Chemistry of Organic Natural Products, Vol 71 (Herz W, Kirby GW, Moore RE, Steglich W & Tamm Ch, eds), pp 1–128 Springer Verlag Wien, New York, NY
Trang 102 Ga¨de G (1996) The revolution in insect neuropeptides
illustrated by the adipokinetic hormone⁄ red
pigment-concentrating hormone family of peptides Z
Natur-forsch 51c, 607–617
3 Ga¨de G (2004) Regulation of intermediary metabolism
and water balance of insects by neuropeptides Ann Rev
Entomol 49, 93–113
4 Ga¨de G, Simek P, Clark KD & Auerswald L (2006)
Unique translational modification of an invertebrate
neuropeptide: a phosphorylated member of the
adipo-kinetic hormone peptide family Biochem J 393, 705–
713
5 Ga¨de G, Kellner R, Rinehart KL & Proefke ML
(1992) A tryptophan-substituted member of the
AKH⁄ RPCH family isolated from a stick insect
corpus cardiacum Biochem Biophys Res Commun 189,
1303–1309
6 Hofsteenge J, Mu¨ller DR, de Beer T, Lo¨ffler A,
Richter WJ & Vliegenthart JFG (1994) New-type of
linkage between a carbohydrate and
protein-C-glycosyl-ation of a specific tryptophan residue in human RNase
Biochemistry 33, 13524–13530
7 de Beer T, Vliegenthart JFG, Lo¨ffler A & Hofsteenge J
(1995) The hexopyranosyl residue that is
C-glycosidi-cally linked to the side chain of tryptophan-7 in human
RNase Us is a-mannopyranose Biochemistry 34,
11785–11789
8 Doucey M-A, Hess D, Blommers MJ & Hofsteenge J
(1999) Recombinant human interleukin-12 is the second
example of a C-mannosylated protein Glycobiology 9,
435–441
9 Doucey M-A, Hess D, Cacan R & Hofsteenge J (1998)
Protein C-mannosylation is enzyme-catalysed and uses
dolichyl-phosphate-mannose as a precursor Mol Biol
Cell 9, 291–300
10 Krieg J, Hartmann S, Vicentini A, Gla¨sner W, Hess D
& Hofsteenge J (1998) Recognition signal for
C-man-nosylation of Trp-7 in RNase 2 consists of sequence
Trp-x-x-Trp Mol Biol Cell 9, 301–309
11 Hofsteenge J, Blommers M, Hess D, Furmanek A &
Miroshnichenko O (1999) The four terminal
compo-nents of the complement system are C-mannosylated on
multiple tryptophan residues J Biol Chem 274, 32786–
32794
12 Hartmann S & Hofsteenge J (2000) Properdin, the
posi-tive regulator of complement, is highly C-mannosylated
J Biol Chem 275, 28569–28574
13 Hofsteenge J, Huwiler KG, Macek B, Hess D, Lawler J,
Mosher DF & Peter-Katalinic J (2001) C-mannosylation
and O-fucosylation of the thrombospondin type 1
module J Biol Chem 276, 6485–6498
14 Gonzalez de Peredo A, Klein D, Macek B, Hess D,
Peter-Katalinic J & Hofsteenge J (2002)
C-mannosyla-tion and O-fucosylaC-mannosyla-tion of thrombospondin type 1
repeats Mol Cell Proteomics 1, 11–18
15 Schwarzinger S, Kroon GJA, Foss TR, Wright PE & Dyson HJ (2000) Random coil chemical shifts in acidic
8 m urea: implementation of random coil shift data in NMRView J Biomol NMR 18, 43–48
16 Schwarzinger S, Kroon GJ, Foss TR, Chung J, Wright
PE & Dyson HJ (2001) Sequence-dependent correction
of random coil NMR chemical shifts J Am Chem Soc
123, 2970–2978
17 Jianyun L & Halbeek H van (1996) Complete1H and
13
C resonance assignments of a 21-amino acid glycopep-tide prepared from human serum transferring Carb Res
296, 1–21
18 Perez-Vilar J, Randell SH & Boucher RC (2004) C-Mannosylation of MUC5AC and MUC5B Cys subdomains Glycobiology 14, 325–337
19 Nelson DL & Cox MM (2005) Lehninger: Principles of Biochemistry WH Freeman, New York
20 Gutsche B, Grun C, Scheutzow D & Herderich M (1999) Tryptophan glycoconjugates in food and human urine Biochem J 343, 11–19
21 Lo¨ffler A, Doucey M-A, Jansson AM, Mu¨ller DR,
de Beer T, Hess D, Meldal M, Richter WJ, Vliegenthart JFG & Hofsteenge X (1996) Spectroscopic and protein chemical analyses demonstrate the presence of C-man-nosylated tryptophan in intact human RNase 2 and its isoforms J Biochemistry 35, 12005–12014
22 Nair MM, Jackson GE & Ga¨de G (2001) Conforma-tional study of insect adipokinetic hormones using NMR constrained molecular dynamics J Comput Aided Mol Des 15, 259–270
23 Flory PJ (1953) Principles of Polymer Chemistry Cornell University Press, Ithaca, NY
24 De Gennes PG (1979) Scaling Concepts in Polymer Physics Cornell University Press, Ithaca, NY
25 Wilkins DH, Grimshaw SB, Receveur V, Dobson CM, Jones JA & Smith LJ (1999) Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques Biochem 38, 16424– 16431
26 Ga¨de G & Rinehart KL (1987) Primary structure of the hypertrehalosaemic factor II from the corpus cardiacum
of the Indian stick insect, Carausius morosus, deter-mined by fast atom bombardment mass spectrometry Biol Chem Hoppe-Seyler 368, 67–75
27 Marion D & Wu¨thrich K (1983) Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of1H-1H spin-spin coupling constants in proteins Biochem Biophys Res Commun
113, 967–974
28 Jeener J, Meier BH, Bachmann P & Ernst RR (1979) Investigation of exchange processes by two-dimensional NMR spectroscopy J Chem Phys 71, 4546–4553
29 Bax A & Davis DG (1985) 2D ROESY with cw spinlock for mixing phase sensitive using States-TPPI method J Magn Reson 63, 207–213