Báo cáo khoa học: C-mannosylation in the hypertrehalosaemic hormone from the stick insect Carausius morosus doc

C-mannosylation in the hypertrehalosaemic hormone from the stick insect Carausius morosus Claudia E Munte1, Gerd Gade2, Barbara Domogalla1, Werner Kremer1, Roland Kellner3 and ă Hans R Kalbitzer1 Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany Department of Zoology, University of Cape Town, Rondebosch, South Africa Target Research and Biotechnology, Merck KGaA, Darmstadt, Germany 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 November 2007, revised 13 December 2007, accepted January 2008) The hypertrehalosaemic hormone from the stick insect Carausius morosus (Cam-HrTH) contains a hexose covalently bound to the ring of the tryptophan, 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 CamHrTH are not completely random-coil in aqueous solution Most prominently, 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 doi:10.1111/j.1742-4658.2008.06277.x In insects, peptidergic regulation by neuropeptides is the most important form of communication to control not only growth, development and reproduction, but also metabolic homeostasis [1] Fuel mobilization, especially to fulfil the exceptionally high energy demand during contraction of flight muscles, is under neuroendocrine control by peptides of the socalled 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, 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 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 repeats FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS 1163 C-mannosylation in HrTH from a stick insect C E Munte et al Fig Sequence comparison of AKH precursors 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 insect Carausius morosus contains two hypertrehalosaemic 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 available 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 tryptophan modification was reported in a mammalian enzyme, human RNAse Using MS and NMR spectroscopy, 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 subsequent studies as an a-d-mannopyranose [7] This tryptophan 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 recognizes the motif WXXW (where X is any amino acid) [9,10] and C-mannosylates the first tryptophan of this sequence in RNAse and IL-12 Subsequently, additional mammalian proteins with such a modification have been identified, such as human terminal complement proteins C6, C7, C8a, C8b and C9 [11], properdin [12] and thrombospondin-1 [13,14]; all of them containing thrombospondin type repeats (TSR modules) In the TSR modules, WXXWXXX motifs are found Here, more than one tryptophan can be mannosylated 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 1164 the TSR modules Since the number of modified tryptophan residues varies in these sequences, it is assumed that either features outside the motif determine the degree of modification or more than one C-mannosyltransferase might exist It still remains unresolved as to whether the tryptophan 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 peptide 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 possible structural changes induced by tryptophan modification that might play a role in receptor recognition Results Assignment of peptide chemical shifts As the modified peptide Cam-HrTH-I and the unmodified 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 FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS C E Munte et al assignment of the NMR lines The assignment of the H resonance lines was obtained by classical homonuclear methods 13C NMR assignments were obtained by 1H,13C-heteronuclear single quantum coherence (HSQC) spectra with different mixing times To achieve identical experimental conditions and to avoiding any systematic chemical shift changes that could arise from experimental differences such as buffer conditions and temperature, the modified and unmodified peptides were mixed in a : 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 resonances 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-ThrPro-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 by an unidentified hexose We can expect that the assignments of the two peptides mainly differ around this residue In agreement with this expectation, the first four amino acids 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, Ce3 and 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 corresponding He1 diagonal 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 Cd1 atom is modified Furthermore, all tryptophan ring carbons directly bound to a proton for the unmodified peptide could be detected by H,13C-HSQC spectroscopy; in contrast, the Cd1 peak was missing in the modified peptide (Table S2) This would be expected because the Hd1 proton necessary for the insensitive nuclei enhanced by polarization transfer is removed by the modification C-mannosylation in HrTH from a stick insect Fig 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 lM CamHrTH-I and 240 lM Cam-HrTH-II in 90% 1H2O, 10% 2H2O, 0.1 mM 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 missing 1He1 – 1Hd1 contact in Cam-HrTH-I 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, corresponds 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 chemical shift values of the bound hexose and of the tryptophan 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 [7] and studied in aqueous solution under comparable conditions (300 K, H2O) to those of the present study, it could be conclusively shown by NMR spectroscopy that the tryptophan 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 FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS 1165 C-mannosylation in HrTH from a stick insect C E Munte et al between H3¢ and H4¢ Thus, the NOE-pattern observed in the hexose corresponds closely to those in RNase peptides and in pure 2-(a-mannopyranosyl)-tryptophan, which further corroborates the identity of the hexose moiety as a-mannopyranose A Aggregation state of Cam-HrTH-I and the unmodified Cam-HrTH-II B Fig NMR spectroscopy of carbohydrates in the solution of CamHrTH-I and Cam-HrTH-II (A) Selected region of the TOCSY spectrum showing three sugar spin systems (B) Plot of ln(I ⁄ I0) as function of G2 where I is the peak integral at a given gradient strength G and I0 is the intensity at G = The sample contained approximately 60 lM Cam-HrTH-I and 240 lM Cam-HrTH-II in 99.8% 2H2O, 0.1 mM DSS, pH 5.4 Temperature = 300 K PAA (polyacrylamide in 90% 1H2O, 10% 2H2O) was used to check the stability of the gradient system before and after measurement Cam-HrTH-I is also a-mannopyranosylated The structure of the glycosylated tryptophan is depicted schematically in Fig The tryptophan–hexose bond is further confirmed by NOEs between the mannose H2¢ proton and the strongly shifted Trp8 He1 proton of the modified peptide (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 1166 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 gradient strengths used in the stimulated echo sequence for important components of the sample Diffusion data are shown for Cam-HrTH-I and its mannose moiety, Cam-HrTH-II, sucrose, glucose and 2,2-dimethyl2-silapentane-5-sulfonate (DSS), all contained in the same sample In addition, before and after the measurements, the signal dependence of polyacrylamide was measured to check the stability of the gradient system; 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 resonances decay with the same rate as those of the peptide 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 molecular mass reference, the effective molecular masses of 1.45 ± 0.12 kgỈmol)1 and 1.96 ± 0.10 kgỈmol)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 factor and density for the test compound and the reference 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 unmodified 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 proportionally 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)1 to 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 indicate a monomeric state of Cam-HrTH-I and probably FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS C E Munte et al C-mannosylation in HrTH from a stick insect Table Carbohydrate modifications of tryptophan residues in protein fragments All chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS); when other standards where used, the values were adapted as best as possible RNase 2a Cam-HrTH-I d ⁄ p.p.m Trp He1 He3 Hf3 Hg2 Hf2 Cd1 Ce3 Cf3 Cg2 Cf2 Hexose H1¢ H2¢ H3¢ H4¢ H5¢ H6¢ H6¢¢ C1¢ C2¢ C3¢ C4¢ C5¢ C6¢ ⁄ p.p.mb 10.51 7.66 7.17 7.27 7.49 –c 121.2 122.2 125.3 114.4 10.47 7.59 7.12 7.21 7.43 – 121.1 121.9 125.2 114.2 5.15 4.36 4.08 3.96 3.88 4.22 3.73 69.3 70.6 73.0 71.4 –c 61.8 5.19 4.42 4.07 3.94 3.83 4.18 3.77 69.9 70.7 73.0 71.3 81.4 62.1 IL-12a C9(T2-1) W27a C9(T2-2) W27a C9(T2-2) W30a d ⁄ p.p.m d ⁄ p.p.m d ⁄ p.p.m d ⁄ p.p.m d ⁄ p.p.m –d 7.65 7.14 7.20 7.42 –d –d –d –d –d –d 7.53 7.11 7.20 7.42 –d –d –d –d –d –d 7.54 7.08 7.21 7.43 –d –d –d –d –d –d 7.56 7.14 7.24 7.46 –d –d –d –d –d 5.18 4.44 4.07 3.93 3.83 4.18 –d –d –d –d –d –d –d 5.18 4.44 4.07 3.94 3.82 4.18 –d –d –d –d –d –d –d 5.18 4.39 4.05 3.93 3.81 4.16 –d –d –d –d –d –d –d 5.20 4.43 4.06 3.93 3.81 4.16 –d –d –d –d –d –d –d 10.47 7.67 7.14 7.20 7.41 –d 121.1 121.9 125.2 114.2 5.22 4.42 4.09 3.96 3.87 4.21 3.77 69.9 70.7 73.0 71.3 81.4 62.1 a RNase 2, glycosylated hexapeptide from human RNase (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 complement C9 [11] b Average chemical shifts observed for all peptides except of Cam-HrTH-I c Resonance not assigned d Shift not reported Table Molecular masses and relative hydrodynamic radii of the Carausius morosus neuropeptides The sample contained approximately 60 lM Cam-HrTH-I and 240 lM Cam-HrTH-II in 99.8% 2H2O, 0.1 mM DSS, pH 5.4 and was measured at 300 K Compound Rh ⁄ Rh,DSSa Mexpb ⁄ kgỈmol)1 Mcalcc ⁄ kgỈmol)1 Cam-HrTH-I Cam-HrTH-II 1.950 ± 0.056 2.156 ± 0.035 1.45 ± 0.12 1.958 ± 0.097 1.308 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) c Molecular mass calculated from the chemical formula Fig Structure of the glycosylated tryptophan residue The experimentally 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 1C4 conformation also for the Cam-HrTH-II with respect to the experimental conditions of the study Interestingly, the modified peptide has a smaller hydrodynamic radius than the unmodified peptide, in spite of the known increase of 162 gỈmol)1 by the mannosylation, indicating a FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS 1167 C-mannosylation in HrTH from a stick insect C E Munte et al more compact structure of the neuropeptide induced by mannosylation A Conformational restraints of the peptide Figure 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 denatured 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, suggesting 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 conformations The chemical shift differences of Cam-HrTHII 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 preferential structuring of these regions of the peptide For the modified and unmodified peptide, the backbone chemical shift changes depicted in Fig 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 Hb atoms These are clearly absent in the unmodified peptide; instead, NOEs between the Hd1 and the two Hb atoms are present This clearly indicates that some reorientation of the tryptophan ring around its b–cbond occurs as a consequence of the mannosylation Discussion Hexose modification of Trp8 Our data clearly indicate that the HrTH from C morosus is glycosylated at the Cd1 (C2) of Trp8 Along with the MS data, the coupling patterns, chemical shifts 1168 B C Fig 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) Ha, (B) Ca and (C) Cb 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 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 FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS C E Munte et al C-mannosylation in HrTH from a stick insect Table Inter-residual and important intraresidual NOEs in Carausius morosus neuropeptides The sample contained approximately 60 lM Cam-HrTH-I and 240 lM Cam-HrTH-II in 90% 1H2O, 10% H2O, 0.1 mM 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 Contact Cam-HrTH-I a N pGlu1 H – Leu2 H Leu2 Ha – Thr3 HN Thr3 Ha – Phe4 HN Thr3 Hb – Phe4 HN Thr3 HN – Phe4 HN Phe4 Ha – Thr5 HN Phe4 Hb2 ⁄ b3 – Thr5 HN Phe4 Hb3 ⁄ b2 – Thr5 HN Phe4 HN – Thr5 HN Thr5 Ha – Pro6 Hd Thr5 Hb – Pro6 Hd Pro6 Ha – Asn7 HN Asn7 Ha – Trp8 HN Asn7 Hb2 ⁄ b3 – Trp8 HN Asn7 Hb3 ⁄ b2 – Trp8 HN Asn7 HN – Trp8 HN Trp8 Hb2 ⁄ b3 – Trp8 He3 Trp8 Hb3 ⁄ b2 – Trp8 He3 Trp8 Hb2 ⁄ b3 – Trp8 Hd1 Trp8 Hb3 ⁄ b2 – Trp8 Hd1 Trp8 Ha – Gly9 HN Trp8 Hb2 ⁄ b3 – Gly9 HN Trp8 Hb3 ⁄ b2 – Gly9 HN Trp8 HN – Gly9 HN Gly9 Ha – Thr10 HN Medium Stronga Stronga Weaka Weaka Strong –b –b –b Stronga Weaka Strong Strong –b –b –b Medium Medium Cam-HrTH-II a Ambiguous –b –b –b Weaka C4 conformation clearly dominates [20] In the unfolded RNase in aqueous solution, a dynamic equilibrium most likely exists between different conformations [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 CamHrTH-I peptide The conformational equilibrium of the mannose moiety is clearly influenced by its environment and is changed in the natively folded RNAse [21] Structural implications Strong Weak Weak Weak Strong Strong Weak Weak Weak Medium Medium Strong Weak Weak Weak a Contact that could not be distinguished between the two peptides because of chemical shift degeneracy b Because of the sample concentration ratio, weak NOEs observed in Cam-HrTH-II cannot be excluded in Cam-HrTH-I has been previously observed for mammalian peptides and proteins, such as RNase [6,7], IL-12 [8], properdin [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 CamHrTH-I peptide is an a-d-mannosylation The 1H and 13C chemical shifts of the modified tryptophan residue are very close to that observed in peptides 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 previously observed in an unstructured peptide derived from RNAse [7] and in d1-(a-d-mannopyranosyl)-l-tryptophan isolated from human urine [20] In the mannopyranosyl-tryptophan dissolved in acidic methanol, the It is important to note that the Cam-HrTH-I peptide differs from other C-mannosylated proteins in the glycosylation 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 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 not contain a tryptophan at position 11 but residues that are part of the typical cleavage site GlyArg ⁄ Lys-Arg This pattern is also expected for the Carausius precursor (Fig 1) The nonrandom chemical shifts as well as the NOEpatterns show that both the modified and the unmodified hypertrehalosaemic hormone of the stick insect have some residual local structures in aqueous solution but 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 experiments on other AKH peptides from other insects were performed in organic solvents such as dimethylsulfoxide 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 contrast, 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 FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS 1169 C-mannosylation in HrTH from a stick insect C E Munte et al H1¢ proton of the sugar and to the He3 of the ring; the latter NOE is not observed in the unmodified peptide but, instead, a cross peak to the Hd1 atom (the atom to be modified in HrTH-I) This indicates that, on average over time, a different v2 angle of Trp8 is now favoured in the modified peptide, which allows a closer Hb–He3 contact A striking difference between the two peptides can be found when the diffusion constants are considered The relative diffusion constant and the relative hydrodynamic radius of the mannosylated peptide correspond closely to that expected for a monomeric peptide However, the experimentally determined relative hydrodynamic radius of the unmodified peptide is significantly larger than that of the modified peptide, although its molecular mass is somewhat smaller Two general explanations 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 identical 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 kgỈmol)1 Assuming we have a monomer–dimer equilibrium in HrTH-II, approximately 54% would be in the dimeric state under our conditions Such a process 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 hydrodynamic 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 predictions of Rh reported for larger biopolymers not accurately apply for small peptides One example comprises 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 ⁄ and ⁄ when going from well defined to random-coil structures [23,24] In a first approximation, Rg and Rh are proportional, allowing the hydrodynamic 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 Experimental 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 1170 becomes folded, probably meaning that the extrapolation to small molecular masses is not valid here Conclusion Although other examples of C-mannosylated tryptophans 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 Mannosylation leads to a change of the average orientation 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 animals more than weeks of age Purification of the peptides Dissection of glands, preparation of methanolic extracts and isolation of the hypertrehalosaemic peptides CamHrTH-I and 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 Technologies, 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 Cam-HrTH-I and 240 lm CamHrTH-II After performing a set of NMR experiments in water, the sample was newly freeze-dried and re-dissolved in 500 lL 2H2O for a new set of NMR experiments FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS C E Munte et al C-mannosylation in HrTH from a stick insect NMR 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 1D 1H NMR spectra were recorded with 64 K complex data points and 1024 scans 2D data sets were recorded with 512 experiments in the t1 dimension and K complex t2-dimension 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 ROESY [29] spectra were recorded with a ROESY spin-lock pulse of 300 ms TOCSY [30] spectra were recorded using a ‘clean’ MLEV-17 [31] TOCSY transfer step of 80 ms Double quantum filtered-COSY spectra were obtained according to Rance et al [32] The gradient-enhanced natural abundance 1H,13C-HSQC [33] spectra were recorded using heteronuclear 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 ms) in 2H2O In addition, spoiler gradients of and ms in length were used during transverse evolution One thousand and twenty-four scans were accumulated for each gradient strength Timedomain 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 spectroscopy [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 chemical shifts were referenced based on the ratio recommended 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 hydrodynamic radius Rh,i is given by the Stokes–Einstein relation Di ¼ kT 6pgRh;i ð1Þ where k is the Boltzmann constant The hydrodynamic radius Rh,i is defined as the radius of a sphere with a volume Vh,i resulting in the same diffusion constant Di For a compound si having an effective volume fiVi, where fi is a characteristic shape factor, Eqn (1) becomes: Di ¼ kT pffiffiffiffiffiffiffi pffiffiffiffiffiffiffi : 3g 6p2 fi Vi ð2Þ Assuming the same form factor for two different compounds si and s1, the unknown hydrodynamic ratio Rh,i of the compound si can be calculated from the known hydrodynamic ratio Rh,1 of compound s1 by: Rh;i ¼ Rh;1 D1 Di ð3Þ Correspondingly, if the mass M1 of the compound s1 is known, and assuming equal density of both compounds, the mass Mi of the compound si can be obtained by:  3 D1 4ị Mi ẳ M1 Di Diffusion coefficients Di can 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 si is given by: IG; si ị ẳ I0; si ịecDi G ð5Þ According to Wilkins et al [25], the empirical hydrodynamic radius of proteins can be calculated from the number N of residues by: Rh;i ẳ ANia 6ị 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 Forschungsgemeinschaft to HRK; and the National Research Foundation of RSA (gun no 2053806) and the University of Cape Town to GG References Gade G (1997) The explosion of structural information ă 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 FEBS Journal 275 (2008) 1163–1173 ª 2008 The Authors Journal compilation ª 2008 FEBS 1171 C-mannosylation in HrTH from a stick insect C E Munte et al Gade G (1996) The revolution in insect neuropeptides ă illustrated by the adipokinetic hormone ⁄ red pigmentconcentrating hormone family of peptides Z Naturforsch 51c, 607–617 Gade G (2004) Regulation of intermediary metabolism ă and water balance of insects by neuropeptides Ann Rev Entomol 49, 93–113 Gade G, Simek P, Clark KD & Auerswald L (2006) ă Unique translational modication of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic 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C-mannosylation in HrTH from a stick insect C E Munte et al H1¢ proton of the sugar and to the He3 of the ring; the latter NOE is not observed in the unmodified peptide but, instead, a cross peak to the Hd1... 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 insect Carausius morosus contains two hypertrehalosaemic. .. E Munte et al C-mannosylation in HrTH from a stick insect Table Inter-residual and important intraresidual NOEs in Carausius morosus neuropeptides The sample contained approximately 60 lM Cam-HrTH-I