Eur J Biochem 271, 4939–4949 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04464.x 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 Kang1,*, William Low1,*, Thomas Norberg3, Jill Meisenhelder2, Karin Hansson4, Johan Stenflo4, Guo-Ping Zhou5,6, Julita Imperial7, Baldomero M Olivera7, Alan C Rigby5,6 and A Grey Craig1 The Clayton Foundation Laboratories for Peptide Biology and 2Laboratory for Molecular and Cell Biology, The Salk Institute, La Jolla, CA, USA; 3Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; 4Department of Clinical Chemistry, University of Lund, Malmo General Hospital, Malmo, Sweden; 5Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 6Marine Biological Laboratory, Woods Hole, MA, USA; 7Department of Biology, University of Utah, Salt Lake City, UT, USA The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-GlaAsp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* ¼ 6-bromotryptophan and Thr* ¼ Gal-GalNActhreonine), 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-transhydroxyproline and glycosylated threonine The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized We hypothesized that tx5a contained the T-antigen, b-D-Gal-(1fi3)-a-D-GalNAc, one of the most 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: thomas.norberg@kemi.slu.se or A C Rigby, Center for Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA Fax: + 617 975 5505, Tel.: + 617 667 0637, E-mail: arigby@bidmc.harvard.edu Abbreviations: DI, deionized; DIPEA, N,N¢-diisopropylethylamine; DMF, N,N-dimethylformamide; EDT, 1,2-ethanedithiol; Gal, galactose; GalNAc, N-acetyl galactosamine; NMP, N-methylpyrrolidone; Gla, c-carboxyglutamic acid, HBTU, 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 tetrafluoroborate; TCEP, tris-(2-carboxyethyl)-phosphine hydrochloride; TPPI, time-proportional phase incrementation *Note: These authors contributed equally to the work (Received August 2004, revised 20 October 2004, accepted 27 October 2004) common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis While the resulting synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-CysCys-Thr*-Ala-Ala-Hyp-OH, where Trp* ¼6-bromotryptophan 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 1H homonuclear and 13C-1H heteronuclear 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-DGalNAc Keywords: Conus textile; glycopeptide synthesis The diverse array of peptides isolated from the venom of cone snails are known collectively as conotoxins or conopeptides (if they lack a disulfide-bonded architecture) The growing interest in these peptides stems from their ability to bind receptors and ion channels with high selectivity and unparalleled specificity A distinct feature of most conotoxins is their relatively small size (10–35 amino acid residues) combined with the presence of a high proportion of cysteine residues that are involved in disulfide bridging [1] In addition, many of the amino acid residues present in conotoxins have undergone post-translational modification; among the diverse array of modifications characterized to date are glutamic acidfic-carboxyglutamic acid [2], prolinefi4-trans-hydroxyproline [3], tryptophanfi6-Lbromotryptophan [4] and threonine/serinefiO-linked glycosylated threonine/serine [5–7] Conotoxin tx5a (or e-TxIX), which was purified recently by two independent laboratories from the venom of the mollusc-hunting cone snail, Conus textile, the Ôcloth-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 posttranslational modifications and two disulfide bonds In Ó FEBS 2004 4940 J Kang et al (Eur J Biochem 271) 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 homogeneity, 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 Ca2+ channels or that it might act on these channels via other mechanisms, such as through G-protein-coupled presynaptic receptors [9] The composition of the O-glycan on the threonine residue of tx5a was previously investigated [9] It was shown that the peptide contained N-acetylgalactosamine (GalNAc) and galactose (Gal) in approximately equal molar amounts; however, the anomeric stereochemistry and the glycan linkages were not determined We have previously characterized 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/L6-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 venom To better understand the incongruence of these peptides we reinvestigated the glycan linkage configuration of the isolated and purified native tx5a venom, using both 1H-1H homonuclear and 13C-1H heteronuclear two-dimensional NMR spectroscopy The data clearly indicated that the tx5a glycan is in an a-D-Gal-(1fi3)-a-D-GalNAc configuration Taken together, these data demonstrate that two Conus glycopeptides identified to date possess the same monosaccharide constituents within their glycan, Gal and GalNAc, but their interresidue linkages are different (alpha vs beta) The results suggest that O-glycosylation in Conus peptides is likely to be more complex 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 The manual synthesis was carried out on an Fmoc-Hypro Wang resin (Chem-Impex, Wood Dale, IL, USA; 0.4 g, 0.7 mmolỈg)1) Each cycle consisted of Fmoc deprotection with 20% piperidine in N-methylpyrrolidone (NMP), followed by Fmoc amino acid coupling using 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/L6-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 peptide resin (0.47 g) was treated with 4.5 mL of trifluoracetic acid in the presence of 250 lL thioanisole, 125 lL 1,2-ethanedithiol (EDT), and 125 lL deionized (DI) 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 peptide was taken up in 0.1% aqueous trifluoroacetic acid and 60% acetonitrile (2· 10 mL) Automated chemical synthesis was performed 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) was performed manually and then loaded onto the automated synthesizer for the remainder of the sequence Three-fold excess of amino acid derivatives were used in the coupling reactions with the exception of per- Scheme Sequence of addition of amino acid derivatives during the solid-phase glycopeptide synthesis The amino acids are numbered starting from the amino terminal according to accepted nomenclature As solid-phase peptide synthesis starts from the carboxy terminal, the order of addition is from higher to lower number Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur J Biochem 271) 4941 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 the automated synthesis to the same level as the manual synthesis (280 lmol) we carried out nine separate automated syntheses 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 lL EDT and 25 lL DI water at room temperature for 1.5 h After precipitation and washing of the cleaved peptide with cold MTBE (2· mL), the nine precipitates were collected and the peptide was taken up in 0.1% aqueous trifluoracetic acid and 60% acetonitrile (2· mL) Purification of the manual synthesis [per-O-acetylb-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 gradient with 0.1% aqueous 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% acetonitrile Because of the low yields from each synthesis, the material was combined for the following treatments We estimated that prior to summation, the yield from automated and manual synthesis were approximately equivalent The crude extract was loaded onto a 45 · 320 mm column packed with Vydac C18 15–20 lm particles and eluted using a preparative HPLC (PrepLC/ System 6000, Waters Corporation, Millford, MA, USA) equipped with a gradient controller, a variable wavelength detector (Waters, model 486) and Waters 1000 PrepPack cartridge chamber in 0.1% aqueous trifluoracetic acid, using a gradient of 60% acetonitrile in 0.1% aqueous trifluoracetic acid Each component was injected on analytical HPLC under isocratic conditions to check for purity and quantity Approximately 240 nmol of the hydrophilic component (eight aliquots at 30 nmol) and 150 nmol of hydrophobic component (five aliquots at 30 nmol) were lyophilized for the sugar de-O-acetylation reaction Each dried aliquot was treated with 500 lL 150 mM NaOCH3 in methanol for 20 at 25 °C and then quenched with 200 lL DI water Purification of the [b-D-Gal-(1fi3)-a-D-GalNAc-Thr10, Cys(t-butyl thiol)2,8, Cys(Acm)3,9] hydrophilic and hydrophobic products on preparative HPLC identified hydrophilic and hydrophobic components with an observed mass of 2252.2 m/z (ESI-MS), which are consistent with the theoretical peptide mass (2252.6 Da) Disulfide bond formation In preparation for the Cys2-8 disulfide reaction, each component was injected on analytical HPLC under isocratic conditions to check for purity and quantity Approximately 160 nmol of the hydrophilic component (20 aliquots at nmol) and 120 nmol of hydrophobic component (20 aliquots at nmol) were lyophilized Each dried aliquot was treated with 0.17 M citric acid (750 lL, pH 6.5) and M tris-(2-carboxyethyl)-phosphine hydrochloride (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 complexes (i.e creating peak broadening or multiple peaks when analyzed on HPLC), 1% CaCl2 (100 lL) was added to each aliquot Purification of the [b-D-Gal(1fi3)-a-D-GalNAc-Thr10, Cys2-8, Cys(Acm)3-9] hydrophilic and hydrophobic products by analytical HPLC indicated hydrophilic and hydrophobic components whose observed mass (ESI-MS) were consistent with the calculated peptide mass To test for completion of the Cys2-8 disulfide bridge formation, 20 mM K3Fe(CN)6 (15 lL) was added to 30 lL (1 nmol) of the hydrophilic component (pH 7) at 25 °C for 20 min, and then the pH was readjusted to using 50% aqueous acetic acid Coinjection of the untreated and K3Fe(CN)6-treated hydrophilic components on analytical HPLC indicated a difference in retention time indicative of formation of the disulfide bridge which was confirmed with ESI-MS analysis Following the injection of both hydrophilic and hydrophobic components onto an analytical HPLC column under isocratic conditions to ensure the purity and quantity of each peptide,  120 nmol of the hydrophilic component (30 aliquots at nmol) and 80 nmol of hydrophobic component (20 aliquots at nmol) were lyophilized for a Cys39 disulfide reaction Each dried aliquot was dissolved with 0.1% aqueous trifluoroacetic acid (400 lL) and 40 lL 1% CaCl2 at °C, and then treated with lL of 0.1% iodine in methanol at 25 °C for 15 Finally, lL of 2.5% 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-DGalNAc-Thr10]-tx5a hydrophilic and hydrophobic products on analytical HPLC (10 mm · 250 mm Vydak C18 ˚ 300 A pore size) with 0.1% aqueous trifluoroacetic acid as buffer A and 60% acetonitrile in 0.1% aqueous trifluoroacetic acid as buffer B (gradient 1% per min) resulted in components whose observed masses (ESI-MS) were consistent with the expected peptide masses Each component was injected on an analytical HPLC under gradient conditions to check for purity and quantity The hydrophilic component was collected at  20% acetonitrile (174 lg, 90 nmol) The hydrophobic component was collected at approximately 23% acetonitrile (66 lg, 34 nmol) ESI and matrix assisted laser desorption mass spectrometry (MALDI-MS) measurement of both components resulted in intense species consistent with the correct product (see below) Both the hydrophilic and hydrophobic components were found to be 99% pure as assessed using an orthogonal ion pairing agent system (triethylammonium phosphate, pH 2.3 as buffer A, 60% acetonitrile as buffer B with a gradient from 10 to 50% B in 40 min) Enzyme hydrolysis Approximately nmol of native tx5a, tx5a hydrophilic and tx5a hydrophobic were incubated with 25 mU b-galactosidase from bovine testes (Glyko, Inc., Novato, CA, USA) in Ó FEBS 2004 4942 J Kang et al (Eur J Biochem 271) 100 lL of 100 mM sodium citrate/phosphate pH at 32 °C for 24 h As a positive control of enzyme activity, contulakin-G, a b-D-Gal-(1fi3)-a-D-GalNAc containing glycopeptide, and native tx5a were simultaneously incubated and reacted with the same vial of the enzyme b-galactosidase Native tx5a, tx5a hydrophilic, and tx5a hydrophobic (each nmol) were incubated with 2.5 mU of endo-O-glycosidase (endo-a-N-acetylgalactosaminidase) (Prozyme, Inc., San Leandro, CA, USA) in 50 lL of 50 mM NaHPO4 pH 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-glycosidase In each case, the enzyme reactions were stopped with addition of 10 lL 10% 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 hydrophobic) were incubated with 50 mM TCEP for 30 at 32 °C prior to injection on reverse-phase HPLC, collection and analysis with ESI-MS Coelution The native and synthetic tx5a (hydrophilic and hydrophobic) were coinjected onto a Vydac C18 RP-HPLC column (2.1 · 150 mm) and eluted with a 1% per gradient from 0% B to 45% 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 fractions were analyzed with both ESI-MS and MALDI-MS Samples for electrospray analysis were diluted : with 1% acetic acid in methanol and infused at lLỈmin)1 into an Esquire LC electrospray quadrupole ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA) Previously, we have demonstrated 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 mixed with a-cyano-4-hydroxycinnamic 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 tx5a (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 chromatographed 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 mLỈh)1 The column fractions were monitored using absorption (A) at 280 and 214 nm Column fractions were subjected to direct Gla analysis following alkaline hydrolysis [11,12] The material in the major Gla-containing peak was further purified on a reverse-phase column (HyCrom C18, 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% trifluoroacetic acid, acetonitrile) NMR spectroscopy Native tx5a NMR samples were dissolved initially in 99.8% D2O and heated to 50 °C at a neutral pH of 7.0 in the presence of Chelex 100 to ensure that all trace metal ions were removed This sample was then lyophilized and redissolved in 350 lL of 99.96% D2O (0.7 mM) (Cambridge Isotope Laboratories, Andover, MA, USA), to a noncorrected pH of 5.60 and transferred to a mm NMR tube All spectra were acquired on a Varian Unity INOVA spectrometer with a proton frequency of 499.695 MHz (Varian Inc., Palo Alto, CA, USA) The carrier frequency was set on the water resonance, which was suppressed using presaturation 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 1H homonuclear and 13C-1H 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) in t1, with a spectral width of 8000 Hz in the observed (F2) dimension 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 all 512 acquired points) using the Varian processing software, VNMR (Varian Inc., 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 were acquired in t2, with 384 time-proportional phase incrementation (TPPI or States-TPPI) increments in t1 A 35 ms mixing time was used in collecting 256 summed scans employing the MLEV17 spinlock sequence A DQF-COSY spectrum was recorded with 4096 real t2 points, 64 summed scans, and 712 TPPI increments to ensure increased resolution The spectrum was multiplied by sine bell window functions shifted by 30° in t2 and 30° in t1 and zero-filled to a 2048 by 1024 (real) matrix A two-dimensional 13C-1H heteronuclear 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 1H and 13C dimensions, respectively A total of 256 summed scans were collected with a relaxation delay of 1.3 s All 1H and 1H-13C correlation assignments were performed using FELIX 2000, which is part of the INSIGHT suite of programs (Accelrys, San Diego, CA, USA) Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur J Biochem 271) 4943 Results After synthesis and deprotection of tx5a from the resin, we obtained two components with the expected mass, herein referred to as hydrophilic and hydrophobic 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 hydrophobic fractions correspond to the L/D 6-bromotryptophan isomers of tx5a In order to further compare these hydrophilic and hydrophobic 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 arrangement) When measured in the negative ionization mode, the ESI mass spectra of all three samples were almost identical in appearance Figure shows (A) the hydrophilic component and (B) the reduced native tx5a (a similar result was observed for the hydrophobic component, data not shown) In Fig 1B, three major species observed at m/z 994.2, 972.1 and 950.5 (identified as MR¢, MR¢¢ and MR¢¢¢) were interpreted as corresponding to [MR+Fe-5H]2–, [MR+Fe-CO2-5H]2– and [MR+Fe-2CO2-5H]2– where MR corresponds to the expected 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 CO2 (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 chromatography and capillary zone electrophoresis results (data Relative Intensity A 100 MR''' Relative Intensity 1000 800 1845 1835 1855 Mass (m/z) MR'' 600 MR' 400 200 875 900 925 950 975 1000 1025 1050 1075 m/z MR''' 100 Relative Intensity 800 Fig Electrospray mass spectrum of (A) chemically reduced synthetic hydrophilic cyclo 2-8, 3-9[6-L/D-bromo-Trp7, b-D-Gal-(1fi3)-aD-GalNAc-Thr10]-tx5a compared with (B) chemically reduced native tx5a where MRÂ ẳ [MR+Fe-5H]2 species Insets show the corresponding MALDI resolved isotope distributions of the [MR-CO2-H]– species Relative Intensity B MR' 600 1835 1845 1855 Mass (m/z) MR'' 400 200 875 900 925 950 975 m/z 1000 1025 1050 1075 Ó FEBS 2004 4944 J Kang et al (Eur J Biochem 271) not shown) Other species present in Fig correspond to sodium cationization (i.e +Na-H) of the intact and fragment ions Insets in Fig are the MALDI-MS resolved isotope distribution of the chemically reduced hydrophilic and native samples (the species corresponds to [MR2CO2-H]–, observed monoisotopic m/z 1844.5 and 1844.7, respectively, compared with the calculated monoisotopic [MR-2CO2-H]– mass of 1844.46 Da) After selective folding of the disulfide bridges of the hydrophilic component of tx5a, we observed similar ESI negative mass spectra from the synthetic hydrophilic and native tx5a, as shown in Fig (a similar result was observed for the hydrophobic component, data not shown) In Fig 2B, the M¢, M¢¢ and M¢¢¢ species observed at m/z 992.1, 970.1 and 948.0 were interpreted as corresponding with [M+Fe-5H]2–, [M+Fe-CO2-5H]2– and [M+Fe-2CO2-5H]2– where M corresponds to the expected average mass of native tx5a (m ¼ 1931.76 Da) The insets in Fig show the MALDI-MS (negative ion mode), resolved isotope distribution measurements for the hydrophilic and native species (observed monoisotopic at m/z 1840.13 and 1840.32, respectively, compared with the calculated monoisotopic [M-2CO2-H]– mass of 1840.42 Da) The mass shift of  Da (MR ) 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 clearly distinguished when analyzed under either nonreducing or reducing conditions In particular, RP-HPLC chromatography of chemically reduced native tx5a and the reduced hydrophilic Relative Intensity A M''' Relative Intensity 1500 100 1835 1845 1855 Mass (m/z) M'' 1000 M' 500 875 900 925 950 975 1000 1025 1050 1075 B Relative Intensity m/z 100 M'' Relative Intensity 1200 1845 1835 M' M''' 1855 Mass (m/z) 600 875 900 925 950 975 m/z 1000 1025 1050 1075 Fig Electrospray mass spectrum of (A) synthetic hydrophilic [6-L/D-bromo-Trp7, b-D-Gal-(13)-a-D-GalNAc-Thr10]-tx5a compared with (B) native tx5a where MÂ ẳ [M+Fe-5H]2) species Insets show the corresponding MALDI resolved isotope distributions of the [M-CO2-H]– species Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur J Biochem 271) 4945 Table 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 Hydrophilic tx5a Hydrophobic tx5a Native tx5a Reducing b-Galactosidase O-Glycosidase 23.8 27.8 25.9 26.6 27.9 27.0 25.3 27.6 25.9 26.1 29.1 25.9 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 with b-galactosidase, 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 contaminating 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 residue 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-glycosidase (Table 1) and an observed mass change as a result of the elimination of the entire glycan In contrast, the retention time of native tx5a did not change when incubated under these conditions The presence of contulakin-G (positive control) was used to validate the activity of the endo-O-glycosidase enzyme, which was unable 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, twodimensional DQF-COSY, TOCSY, NOESY and HSQC spectra of the native tx5a glycopeptide were collected in 99.96% D2O at 12 °C, pD 5.6 These data, in combination UV Absorption (210 nm) Chemically reduced hydrophilic tx5a Chemically reduced Native tx5a with data collected previously in 90 : 10 H2O/D2O enabled assignment of the amino acid and sugar spin systems of the glycopeptide [9] Interestingly, O-glycosylation of Thr10 perturbed the b-carbon 13C 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 attributed to the glycan moiety of tx5a, localized within a spectral envelope between 3.4 p.p.m and 4.0 p.p.m., remained 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 field proton signals that were tentatively assigned H2 and H3 for the respective monosaccharides (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 13C-1H HSQC spectrum that enabled each carbon to be correlated with its directly bonded proton or protons (Tables and 3) Strong NOEs between the anomeric and H2 protons and J1,2 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 3J2,3 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 NOE to the Table 1H, 13C chemical shifts and scalar coupling constants for the glycan monosaccharides in tx5a in 99.96% D2O at 285.5 K (relative to sodium 2,2-dimethyl-2-silapentane-5-sulfonate) 3Jx,y, 3-bond coupling constant GalNAc Gal Proton (1H) 13 H1 H2 H3 H4 4.79 3.94 3.78 3.48 103.2 52.2 77.4 75.8 4.82 3.55 3.44 3.76 GalNAc Gal 4.25 7.92 4.6 4.25 7.84 4.4 Retention Time (min) Fig 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 Jx,y (Hz) J1,2 J2,3 J3,4 H C H 13 C 98.6 72.4 74.0 73.8 Ó FEBS 2004 4946 J Kang et al (Eur J Biochem 271) Table tx5a Gal-GalNAc NOE interactions and their corresponding proton (1H) chemical shifts in 99.96% D2O at 285.5 K (relative to sodium 2,2-dimethyl-2-silapentane-5-sulfonate) Table NOEs between the tx5a peptide resonances and the protons (1H) within the Gal-GalNAc disaccharide The nomenclature represents that used in Figs and Chemical shift (p.p.m.) Proton (1H) Chemical shift (p.p.m.) Disaccharide: Gal-GalNAc Proton (1H) GalNAc:H1 GalNAc:H3 GalNAc:H3 Gal:H1 Gal:H1 Gal:H2 GalNAc:CH3 GalNAc:CH3 GalNAc:CH3 4.79 3.77 3.77 4.82 4.82 3.53 1.79 1.79 1.79 GalNAc:H2 GalNAc:H2 Gal:H1 Gal:H2 Gal:H3 Gal:H4 Gal:H2 Gal:H3 Gal:H4 3.95 3.95 4.82 3.53 3.43 3.72 3.53 3.43 3.72 Proton (1H) Chemical shift (p.p.m.) Proton (1H) Chemical shift (p.p.m.) GalNAc:H1 GalNAc:CH3 GalNAc:H3 GalNAc:H1 Gal:H2 Gal:H2 4.79 1.79 3.77 4.79 3.53 3.53 10ThrcCH3 12AlaaH 10ThrcCH3 10ThrbH 11AlaßCH3 12AlaaH 0.98 4.07 0.98 4.04 1.09 4.07 anomeric proton of Gal, which identified 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 Taken together, these data identified the glycan as a-D-Gal-(1fi3)a-D-GalNAc There are several NOEs between the glycan and the glycopeptide side-chain atoms of tx5a, which suggests that the monosaccharides are conformationally less flexible, ˚ well ordered and within A of these glycopeptide sidechains at 12 °C (Table 4) Specifically, the side-chain protons of Thr10, Ala12 and Hyp13 interact with the A tx5a glycan protons Several NOEs are observed between the anomeric proton of GalNAc and the side-chain atoms of Thr10; Thr10b (strong NOE) and Thr10CH3 (medium NOE) (Fig 5) For Ala12 the Ala12a and Ala12bCH3 side-chain protons demonstrate medium and strong NOEs, respectively, with the N-acetyl CH3 of GalNAc at 1.79 p.p.m., which may alter the magnetic and chemical environment of this moiety and help us to understand this fairly unique chemical shift frequency (Fig 5) In addition, there are several weak NOEs between the N-acetyl CH3 and GalNAc H3, GalNAc H4 and Gal H3, which further supported a well ordered carbohydrate moiety at 12 °C (Table 3) B Fig Two-dimensional 1H spectra of 0.7 mM tx5a (e-TxIX) collected in 100% D2O or 90% : 10% H2O/D2O, respectively, at 500 MHz (A) TOCSY spectrum collected in 100% D2O illustrating the alpha region of the data, which includes the monosaccharide resonances and (B) NOESY spectrum collected in 90% : 10% H2O/D2O (H2O resonance at 4.65 p.p.m.) of this same region collected with 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 the tx5a peptide including Gly6, Thr10 and Pro13 (A) Illustrates the intraresidue carbohydrate assignments GalNAc (GN) and Gal (G), respectively In B, many of these same intraresidue assignments are labeled in addition to the interglycosidic linkage between GNH3 of GalNAc and GH1 of Gal, which is labeled in bold The amino acids Gly6, Thr10 and Pro13 are represented by 6G, 10T and 13P Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur J Biochem 271) 4947 Fig A region of the 500 MHz 2D NOESY (320 ms) collected in 90% : 10% H2O/D2O illustrating the NOEs observed between the amino acids side chains of residues Thr10 and Ala12 and the carbohydrate moieties of GalNAc (GN) and Gal (G) The individual amino acids Thr10, Ala12 are represented by 10T and 12A, respectively, while GNH1 and GNH3 represents the H1 and H2 protons from GalNAc, and GNCH3 represents the methyl group (CH3) that is within the GalNAc acetyl group 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 assumed that the glycan moiety would be the T-antigen, as was previously shown for contulakin-G, i.e b-D-Gal-(1fi3)-a-D-GalNAc O-linked to threonine [6] The synthetic strategy is briefly outlined in Scheme During the chemical synthesis we used a selective cysteine deprotection strategy to obtain the 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 yield obtained, 0.027% (based on our rough estimated 50 : 50 split between automated and manual synthesis, the 90 nmol of hydrophilic and 34 nmol of hydrophobic tx5a analogs) is in contrast with yields (30%) previously obtained for nondisulfide bridge-containing glycopeptides using either manual or automated strategies [6] We note, however, that even in the synthesis of nondisulfide bridge-containing glycopeptides the yield is dramatically affected by the scale of the reaction, the excess of amino acids used, and the level of purity desired Here, our reaction 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%) of some expensive 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 have partially contributed to the very low yields We note also that determination of the stereochemistry of the 6-bromotryptophan residue as either L or D, and utilization of the appropriate resolved precursor would result in a significantly 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 the synthetic peptide could be hydrolyzed by b-galactosidase, as well as by endo-O-glycosidase, as one would expect for the glycan in a T-antigen configuration These enzymes were previously shown to also hydrolyze the glycan moiety of contulakin-G [6] However, the native tx5a peptide was not amenable to hydrolysis by these two glycosidases The failure to hydrolyze the native peptide was not due to the presence of an inhibitor in the native preparation as demonstrated when native and synthetic peptides were mixed The synthetic peptide was cleaved by the glycosidases, while the native peptide was resistant These data support that the intrinsic carbohydrate properties of the glycan moieties linked to these peptides are distinct and more importantly that the tx5a glycan is comprised of interglycosidic linkages that are not recognized and thus not cleaved by these enzymes These data permit us to conclude that, contrary to our expectations and prior results with contulakin-G [6], the glycan present 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 not 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 with the inability of the aforementioned glycosidic enzymes to hydrolyze the tx5a glycan linked to Thr10 identifies that 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 configuration of this glycan moiety we used standard two-dimensional homonuclear and heteronuclear (natural abundance) NMR spectroscopy Using the information gleaned from our two-dimensional COSY, NOESY and 13C-HSQC experiments we assigned the 1H and 13C chemical shifts of all nuclei with the exception of those that remained spectrally degenerate The anomeric protons identified in the COSY and TOCSY spectra (collected at 12 °C) at 4.79 p.p.m and 4.82 p.p.m (GalNAc and Gal, respectively) 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 13CHSQC experiment and the assignments completed using NOESY spectra collected at several mixing times (Fig 4B) Ó FEBS 2004 4948 J Kang et al (Eur J Biochem 271) These 13C 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 glycosylation site (as we already believed) The anomeric configuration and interglycosidic linkage patterns were identified using several through-bond scalar measurements, the 3J1,2 coupling constant between the anomeric (H1) and H2 protons (1H’s) of each carbohydrate moiety and resonance assignments Specifically, the small 3J1,2 and larger 3J2,3 scalar coupling constants identified that both carbohydrate moieties were in an alpha configuration In addition, the low field chemical shift 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 alpha configuration These data were confirmed by the strong NOE between the 1H at position (H3) of GalNAc and the anomeric (H1) 1H of Gal This linkage pattern helps us to better understand the resistance to hydrolysis by the aforementioned glycosidic enzymes, while identifying a linkage pattern that is disparate from that previously identified for contulakin-G [6] Interestingly, several additional NOEs were identified between the glycan linked to Thr10 and other tx5a residues as illustrated in Fig These NOEs support that the carbohydrate moieties interact with the glycopeptide, suggesting that the carbohydrate is conformationally well structured This apparent reduction in conformational flexibility (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 function although this requires further investigation It was completely unexpected 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 enzymes necessary to catalyze O-glycosylation of threonine 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-Thr structure for this glycan, 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-glycosylation differences of these peptides Recent studies have demonstrated that for some Conus peptide post-translational 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 specific and distinct secretory pathways Thus, enzymes 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 environmental changes that are specific for each 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 for contulakin-G (Conus geographus) This surprising result establishes that the O-glycosylation of serine and threonine residues in Conus peptides are likely to be more complex than had originally been anticipated, involving more than one specialized post-translational modification enzyme Acknowledgements This work was supported by the National 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 like to thank Jean E Rivier and Josef Gulyas for stimulating conversations and helpful advice References Craig, A.G., Bandyopadhyay, P & Olivera, B.M (1999) Posttranslationally modified neuropeptides from Conus venoms Eur J Biochem 264, 271–275 McIntosh, J.M., Olivera, B.M., Cruz, L.J & Gray, W.R (1984) Gamma-carboxyglutamate in a neuroactive toxin J Biol Chem 259, 14343–14346 Stone, B.L & Gray, W.R (1982) Occurrence of hydroxyproline in a toxin from the marine snail Conus geographus Arch Biochem Biophys 216, 756–767 Craig, A.G., Jimenez, E.C., Dykert, J., Nielsen, D.B., Gulyas, J., Abogadie, F.C., Porter, J., Rivier, J.E., Cruz, L.J., Olivera, B.M & McIntosh, J.M (1997) A novel post-translational modification involving bromination of tryptophan Identification of the residue, L-6-bromotryptophan, in peptides from Conus imperialis and Conus radiatus venom J Biol Chem 272, 4689–4698 Craig, A.G., Zafaralla, G., Cruz, L.J., Santos, A.D., Hillyard, D.R., Dykert, J., Rivier, J.E., Gray, W.R., Imperial, J., DelaCruz, R.G., Sporning, A., Terlau, H., West, P.J., Yoshikami, D & Olivera, B.M (1998) An O-glycosylated neuroexcitatory Conus peptide Biochemistry 37, 16019–16025 Craig, A.G., Norberg, T., Griffin, D., Hoeger, C., Akhtar, M., Schmidt, K., Low, W., Dykert, J., Richelson, E., Navarro, V., Macella, J., Watkins, M., Hillyard, D., Imperial, J., Cruz, L.J & Olivera, B.M (1999) Contulakin-G, an O-glycosylated invertebrate neurotensin J Biol Chem 274, 13752–13759 Luning, B., Norberg, T & Tejbrant, J (1989) Synthesis of monoă and disaccharide amino acid derivatives for use in solid phase peptide synthesis Glycoconj J 6, 5–19 Walker, C., Steel, D., Jacobsen, R.B., Lirazan, M.B., Cruz, L.J., Hooper, D., Shetty, R., DelaCruz, R.C., Nielsen, J.S., Zhou, L., Bandyopadhyay, P., Craig, A & Olivera, B.M (1999) The T-superfamily of conotoxins J Biol Chem 274, 30664–30671 Ó FEBS 2004 Glycopeptide tx5a synthesis and NMR analysis (Eur J Biochem 271) 4949 Rigby, A.C., Lucas-Meunier, E., Kalume, D.E., Czerwiec, E., Hambe, B., Dahlqvist, I., Fossier, P., Baux, G., Roepstorff, P., Baleja, J.D., Furie, B.C., Furie, B & Stenflo, J (1999) A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx Proc Natl Acad Sci USA 96, 5758–5763 10 Kalume, D.E., Stenflo, J., Czerwiec, E., Hambe, B., Furie, B.C., Furie, B & Roepstorff, P (2000) Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods J Mass Spectrom 35, 145–156 11 Fernlund, P., Stenflo, J., Roepstroff, P & Thomsen, J (1975) Vitamin K and the biosynthesis of prothrombin V Gammacarboxyglutamic acids, the vitamin K-dependent structures in prothrombin J Biol Chem 250, 6125–6133 12 Ciarns, J.R., Williamson, M.K & Price, P.A (1991) Direct identification of gamma-carboxyglutamic acid in the sequencing of vitamin K-dependent proteins Anal Biochem 199, 93–97 13 Nakamura, T., Yu, Z.G., Fainzilber, M & Burlingame, A.L (1996) Mass spectrometric-based revision of the structure of a cysteine-rich peptide toxin with gamma-carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile Protein Sci 5, 524–530 14 Wishart, D.S & Sykes, B.D (1994) Chemical Shifts as a Tool for Structure Determination Methods Enzymol 239, 363–392 15 Bandyopadhyay, P.K., Colledge, C.J., Walker, C.S., Zhou, L.-M., Hillyard, D.R & Olivera, B.M (1998) Conantokin-G precursor and its role in gamma-carboxylation by a vitamin K-dependent carboxylase from a Conus snail J Biol Chem 273, 5447–5450  ... two-dimensional NMR spectroscopy The data clearly indicated that the tx 5a glycan is in an a- D-Gal-(1fi3) -a- D-GalNAc configuration Taken together, these data demonstrate that two Conus glycopeptides... glycosylated at position Taken together, these data identified the glycan as a- D-Gal-(1fi3 )a- D-GalNAc There are several NOEs between the glycan and the glycopeptide side-chain atoms of tx 5a, which... b-D-Gal-(1fi3) -a- D-GalNAc containing glycopeptide, and native tx 5a were simultaneously incubated and reacted with the same vial of the enzyme b-galactosidase Native tx 5a, tx 5a hydrophilic, and tx5a