Báo cáo Y học: Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding pot

Eur J Biochem 269, 977–988 (2002) Ó FEBS 2002 Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding Christoph Bohme1, Manfred Nimtz2, Eckart Grabenhorst3, Harald S Conradt3, Annemarie Strathmann1 ă and Hermann Ragg1 Faculty of Technology, University of Bielefeld, Germany; 2Molecular Structure Research and 3Protein Glycosylation, Gesellschaft fuăr Biotechnologische Forschung mbH, Braunschweig, Germany The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of : with the galactose being > 90% sialylated with a2 fi linked N-acetylneuraminic acid About 50% of the triantennary structures contain one sLex motif Proximal a1 fi fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in > 90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively a2 fi 3-linked N-acetylneuraminic acid units Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein’s origin Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin–Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor’s interaction with glycosaminoglycans Heparin cofactor II (HCII) is a single-chain glycoprotein with a carbohydrate content of about 10% circulating in blood [1] The protein is a member of the serpin (serine protease inhibitor) superfamily, most members of which inhibit serine proteases by forming SDS stable complexes with their target enzymes HCII functions as an inhibitor of cathepsin G, chymotrypsin and thrombin After addition of heparin or dermatan sulfate, the rate of HCII/thrombin interaction is enhanced several orders of magnitude and the inhibition rate approaches the value observed for thrombin inhibition by antithrombin (AT) in the presence of heparin, indicating that HCII may be an interesting anticoagulant and antithrombotic agent Increased plasma levels of HCII have been found under a variety of pathophysiological conditions, suggesting that the protein may be involved in the acute phase response [2,3] HCII has been biochemically characterized in some detail As deduced from the cDNA sequence, the mature human protein consists of 480 amino acids and contains three potential N-glycosylation sites at positions Asn30, Asn169 and Asn368 [4,5] In the mouse, two HCII variants that appear to differ in number and/or structure of their glycans circulate in blood [6] In addition, two tyrosine sulfation signals with the characteristic accumulation of acidic amino acids are present close to the N-terminus of all HCII sequences known [7,8] Sulfation of these tyrosine residues has been demonstrated for HCII secreted from a human hepatoma cell line [9] Composition and stoichiometry of the monosaccharide units of the inhibitor molecule have been analyzed [1,10] with mannose, galactose, N-acetylglucosamine, and sialic acid being the main elements of HCII glycans, the structure of the carbohydrate chains, however, is unknown Glycosylation may have profound effects on a variety of biological features of proteins [11] This is also evident for human AT, which exists in two isoforms (AT-a and AT-b) These variants differ substantially in their affinity for heparin (elution from heparin–Sepharose at about 0.8 and 1.2 M NaCl, respectively) due to differential N-glycosylation [12,13] As a consequence, AT-b is predominantly associated with endothelial and subendothelial cells [14,15], providing the vessel wall with a strongly antithrombotic surface HCII isolated from human plasma displays a low affinity for heparin as indicated by the low NaCl concentration required for its dissociation from heparin–Sepharose (about 0.25 M NaCl) Various experiments, however, have shown that HCII has a considerably higher intrinsic propensity for Correspondence to H Ragg, Faculty of Technology, University of Bielefeld, Universitatsstr 25, D-33501 Bielefeld, Germany ă Fax: + 49 521106 6328, Tel.: + 49 521106 6321, E-mail: hr@zellkult.techfak.uni-bielefeld.de Abbreviations: AT, antithrombin; CHO, Chinese hamster ovary; dhfr, dihydrofolate reductase; DMEM, Dulbecco’s modified Eagle’s medium; GAG, glycosaminoglycan; HCII, heparin cofactor II; HPAEC-PAD, high pH anion-exchange chromatography with pulsed amperometric detection; MEM, minimal essential medium; MTX, methotrexate; NeuAc, N-acetylneuraminic acid; PEG, poly(ethylene glycol); PNGase F, polypeptide:N-glycosidase F; sLex, sialyl Lewis X (Received 26 September 2001, revised December 2001, accepted 11 December 2001) Keywords: heparin cofactor II; glycosylation; tyrosine sulfation; heparin binding; serpins 978 C Bohme et al (Eur J Biochem 269) ă heparin binding, approaching that of AT-a HCII mutants devoid of an acidic region (positions 53–75), which resembles the C-terminal tail of hirudin, require > 0.7 M NaCl for elution from heparin–Sepharose [16–18], suggesting that this polyanionic domain may affect the heparin-binding properties of HCII Here, we report on the structure of posttranslational modifications of plasma-derived and recombinant human HCII from CHO cells and their effects on heparin binding MATERIALS AND METHODS Outdated citrated human plasma was a gift of Centeon AG Bovine insulin, MTX, PEG, polybrene, sodium chlorate, sulfate-deficient DMEM/Ham’s F12 : mixture, and tunicamycin were obtained from Sigma Culture media and fetal bovine serum were purchased from Life Technologies Heparin-HiTrap (5 mL), Mono Q HR5/5 and Superdex 200 HR 10/30 were from Pharmacia Standard grade BSA was supplied from Serva A Sartocon Micro 30-kDa MWCO cross flow module was from Sartorius Human transferrin was purchased from Bayer AG PeptideN4-(N-acetyl-4-D-glucosaminyl)asparagine amidase F (from Flavobacterium meningosepticum) from recombinant Escherichia coli was bought from Roche Molecular Biochemicals Vibrio cholerae sialidase was from Calbiochem Stable expression and amplification of HCII in CHO cells Plasmid pWTBI2 was constructed by ligating a 1.6-kb HindIII/EcoRI human HCII cDNA fragment into a HindIII/EcoRI-cleaved expression vector of the pSV2 plasmid series [19] The HCII cDNA spanned the protein coding sequence (including the signal peptide) down to the EcoRI site at position 1559 in the 3¢ untranslated region (numbering as described previously [4]) The expression of the thrombin inhibitor cDNA in this construct is regulated by the SV40 early promoter and the polyadenylation signal for SV40 early mRNAs Plasmid pWTBI2 and plasmid pSV2dhfr, which encodes a mouse dihydrofolate reductase cDNA under the control of the SV40 early promoter, were mixed at a ratio of : (w/w) and transfected into DHFR-deficient CHO cells (routinely cultured in Ham’s F12 medium) by the polybrene method, following established procedures [20] After 2–3 weeks of selection in nucleoside-free a-MEM containing 10% dialyzed serum, mM L-glutamine and 0.3 mM L-proline, individual colonies were picked and expanded Cell lines expressing increased amounts of HCII were isolated by augmenting the MTX concentration in increments (0.05, 0.25, 2.5, and 100 lM, respectively) The amount of recombinant HCII secreted from MTXselected cell clones was determined by a sandwich ELISA employing a monoclonal mouse anti-HCII capturing Ig and a horseradish peroxidase-coupled monoclonal mouse antiHCII detection antibody Assays were performed in 96-well microtiter plates (Nunc) with HCII from human plasma as standard The enzyme-catalyzed oxidation of 3,3¢,5,5¢ tetramethylbenzidine was monitored at 405 nm with a microtiter plate autoreader (BioTek Instruments) Cells were counted using the trypan blue exclusion assay and the specific HCII productivity (lg per 106 cells per day) was calculated Ó FEBS 2002 Treatment of HCII expressing CHO cells with inhibitors of N-glycosylation and tyrosine sulfation Recombinant CHO cells producing human HCII were grown close to confluency in DMEM/Ham’s F12 medium After washing twice with serum-free medium, cells were incubated in serum-free medium in the presence of tunicamycin at the concentrations indicated in the figure legends After h, fresh medium containing the same concentration of the glycosylation inhibitor was added Three days later, the conditioned medium was harvested and inspected by Western blotting for the presence of HCII as described previously [21] Treatment of cells with sodium chlorate (20 mM) followed the same procedure, except that cells were cultivated in sulfate-deficient DMEM/Ham’s F12 : mixture during the presence of the sulfation inhibitor Purification of HCII from plasma and from recombinant CHO cells HCII from outdated citrated human plasma of a single blood donor was isolated essentially as described previously, including precipitation with barium chloride and poly(ethylene glycol) [22], chromatography on heparin–Sepharose and on Mono Q [6] Highly purified HCII suitable for glycoanalysis was obtained through inclusion of a final gel filtration step on Superdex 200 HR 10/30 For mass production of recombinant HCII, cells from the CHO clone Bi2/100/6/13/3/19 were trypsinized and cultured in a Superspinner [23], consisting of a Duran flask (capacity L) equipped with a magnetic membrane stirrer in order to improve the oxygen supply by bubble-free aeration The device was placed on a stirring plate in a CO2 incubator together with a small membrane pump, which supplied the membrane stirrer with the incubator gas The starting volume (550 mL) was inoculated with 2.5 · 105 cellsỈmL)1 from five confluent T175-flasks, which had been propagated in DMEM/Ham’s F12 medium with 2% fetal bovine serum To remove bovine HCII, the serum content was successively reduced further in a combined repeated/fed batch process To this end, the cell suspension culture was diluted to a cell density of 2.5 · 105 cellsỈmL)1, filled up to the maximum working volume (1 L) with serum-free DMEM/Ham’s F12 medium supplemented with gỈL)1 BSA, mM glutamine, 10 lgỈmL)1 bovine insulin, 10 lgỈmL)1 human transferrin and antibiotics, and grown to the early stationary phase After two further dilution steps, the cells were finally cultivated for days in serumfree medium supplemented with 0.5 gỈL)1 BSA, amino acids, and glucose For isolation of recombinant HCII, the cells were removed by centrifugation, and after addition of phenylmethanesulfonyl fluoride (10 lM), the supernatant was concentrated about threefold in a cross-flow ultrafiltration module and dialyzed against 10 mM Tris/HCl, mM EDTA, 10 lM phenylmethanesulfonyl fluoride, mM NaCl, pH 7.4 The following isolation procedures included fractionation on heparin–Sepharose and chromatography on Mono Q essentially as described above, except that the Tris/EDTA buffer system was used for heparin–Sepharose chromatography For glyco-analysis, HCII was rechromatographed on heparin–Sepharose with a linear 0–1 M NaCl gradient (40 mL) and on Superdex-200 HR 10/30 Purity Ó FEBS 2002 Heparin cofactor II: N-glycans and sulfation (Eur J Biochem 269) 979 was assessed by reducing SDS/PAGE on 10% gels run in a Tris/glycine buffer system, according to the manufacturer’s protocol (Novex), and subsequent visualization of proteins by Coomassie blue staining The homogeneity of HCII from plasma and from recombinant CHO cells was checked by N-terminal sequence analysis using an Applied Biosystems ProciseTM instrument Reduction, carboxamidomethylation and tryptic digestion of HCII from plasma and from recombinant CHO cells One to two nanomoles of the purified protein were reduced, carboxamidomethylated and digested with trypsin; RP-HPLC separation of resulting peptides was performed as described previously [24] Separation of N-glycans by Mono Q anion-exchange chromatography Oligosaccharides were liberated quantitatively by treatment of the glycoprotein preparations (2 mgỈmL)1) with 15 U of recombinant PNGase F in 50 mM sodium phosphate buffer (pH 7.2) for h at 37 °C In order to separate native N-glycans according to charge, desalted oligosaccharides were dried using a SpeedVac and redissolved in 0.5 mL of MilliQ water The N-glycans were applied to a Mono Q HR 5/5 column at room temperature and eluted at mLỈmin)1 with a mixture of water (solvent A) and 0.5 M NaCl (solvent B) Chromatographic conditions were: a 5-min isocratic run with 100% A, followed by a linear gradient to 10% B for 15 min, an isocratic run using 10% B for 10 and a final linear gradient to 100% B over Oligosaccharides were detected by their ultraviolet absorption at 206 nm Fractions of 0.5 mL were collected Enzymatic and chemical removal of sialic acid Vibrio cholera neuraminidase (2.5 lL; mL)1) was added to the samples containing 0.5 nmol of total oligosaccharides in 25 lL of sodium acetate, pH 5.0, mM CaCl2 and 0.02% NaN3 (w/v) The reaction mixture was incubated for h at 37 °C For the chemical removal of NeuAc, N-glycans were incubated in 100 lL of 0.2% trifluoroacetic acid for h at 82 °C High-pH anion-exchange chromatography with pulsed amperometric detection Purified native and desialylated oligosaccharides were analyzed by high-pH anion-exchange (HPAE) chromatography using a Dionex BioLC system (Dionex, Sunnyvale, CA, USA) equipped with a CarboPac PA1 column (0.4 · 25 cm) in combination with a pulsed amperometric detector (PAD) [25,26] Detector potentials (E) and pulse durations (T) were: E1: + 50 mV, T1: 480 ms; E2: + 500 mV, T2: 120 ms; E3: ) 500 mV, T3: 60 ms, and the output range was 500–1500 nA The oligosaccharides were then injected into the CarboPac PA1 column that was equilibrated with 100% solvent C Elution (flow rate of mLỈmin)1) was performed by applying a linear gradient (0–20%) of solvent D over a period of 40 followed by a linear increase from 20 to 100% solvent B over Solvent C was 0.1 M NaOH in bidistilled H2O, solvent D consisted of 0.6 M NaOAc in solvent C Reduction and permethylation of oligosaccharides The enzymatically liberated N-glycans were reduced and permethylated as described previously [27] MALDI-TOF MS The reduced and carboxamidomethylated tryptic peptides of the HCII preparations were subjected to positive ion matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, using a Bruker REFLEX time-of-flight (TOF) instrument equipped with delayed-extraction and reflectron systems and a N2 laser (337 nm) operating with 3-ns pulse width and 107)108 WỈcm)2 irradiance at the surface of 0.2 mm2 spots In addition to the positive mode standard procedure for the detection of sulfated peptides using the reflectron for enhanced resolution, the peptide mixture was also analyzed in the positive and negative linear ion mode One-microliter samples containing equal volumes of peptide solution (10 pmolỈlL)1) and the ultraviolet-absorbing matrix [19 mg a-cyano-4-hydroxycinnamic acid in 400 lL acetonitrile and 600 lL 0.1% (v/v) trifluoroacetic acid in H2O] were spotted onto the stainless steel target and dried at room temperature Determination of the molecular masses of reduced and permethylated oligosaccharides was carried out similarly in the positive ion mode using the reflectron One-microliter samples containing equal volumes of reduced and permethylated oligosaccharide solution ( 10 pmolỈlL)1) and a-cyano4-hydroxycinnamic acid matrix were mixed and spotted on the target Sodium chloride was added to the matrix to a final concentration of lM in order to guarantee the exclusive generation of sodium adducts of the carbohydrate molecular ions Tandem electrospray ionization (ESI) mass spectrometry The peptide samples were dissolved in a : mixture of MeOH/H2O (the reduced and permethylated oligosaccharide samples in : MeOH/H2O) to a concentration of  pmolỈlL)1, and gold-coated nanospray glass capillaries (Protana, Odense, Denmark) were filled with lL of this solution The tip of the capillary was placed orthogonally in front of the entrance hole of a QTOF II mass spectrometer (Micromass, Manchester, England) equipped with a nanospray ion source, and a voltage of  800 V was applied For collision-induced dissociation experiments, parent ions were selectively transmitted from the quadrupol mass analyzer into the collision cell Argon was used as the collision gas and the kinetic energy was set from )15 to )60 eV for optimal fragmentation The resulting daughter ions then were separated by an orthogonal TOF mass analyzer RESULTS Expression of HCII from CHO cells Human HCII cDNA was expressed under the control of the SV40 early promoter in DHFR-deficient CHO DUKX-B1 980 C Bohme et al (Eur J Biochem 269) ă cells, which had been cotransfected with plasmid pSV2dhfr More than 20 individual clones growing in selection medium were examined, but initially no cell lines producing sufficient HCII detectable by the ELISA technique were identified Therefore, individual clones were isolated at random, exposed to 0.05 lM MTX, expanded, and checked for the presence of human HCII mRNA by RT-PCR Positive cell lines were picked and after several rounds of selection with increasing MTX concentrations, cell lines expressing up to 17 lg HCII per 106 cells per day were isolated Production, purification and characterization of recombinant and plasma HCII The high producer clone Bi2/100/6/13/3/19, selected in medium containing 100 lM MTX was cultivated in suspension in a 1-L superspinner by successively reducing the serum concentration During the production phase, the serum-free cultivated cells accumulated HCII to a concentration of about 11 lgỈmL)1 within a 5-day period The recombinant inhibitor and HCII from plasma were purified to apparent homogeneity as assessed by SDS/PAGE (not shown) and subjected to sequence analysis The N-terminus of the inhibitor consisted of a single sequence (GSKGPLDQLEKGGE), irrespective of whether HCII had been isolated from plasma or from recombinant CHO cells Thus, these results are in agreement with the accurate and efficient cleavage of the 19-amino-acid signal peptide in CHO cells [5,28] Characterization of the tryptic peptides of serum-derived and recombinant HCII Natural and recombinant HCII were characterized further by peptide mapping After reduction and carboxamidomethylation, the proteins were digested with trypsin and the resulting peptide mixtures were subjected to RP-HPLC mapping The elution profiles of both protein preparations were almost identical (data not shown) This result was further corroborated by MALDI-TOF-MS analysis of the tryptic peptide mixtures (compare Fig and Table 1) Most signals could be assigned, resulting in a sequence coverage of almost 70% The pattern of peptides obtained from both proteins was very similar, indicating an identical polypeptide backbone of the natural and the recombinant HCII Position 218, for which an amino-acid polymorphism has been reported (Lys or Arg [4,5]), is populated with a lysine residue in the plasma derived protein We note that higher amounts of peptides containing oxidized methionine were detected in the recombinant polypeptide preparation (see legend to Table 1) Identification of sulfated tyrosine peptides and glycosylation site occupancy in trypsin digests of serum-derived and recombinant HCII Human HCII from HepG2 cells has been found to be sulfated at two tyrosine residues [9] In our experiments, however, solely the unmodified forms of the corresponding tryptic peptides T43–65 and T66–101 were detected when the cleavage products from both protein preparations were analyzed by the standard MALDI-TOF-MS techniques Ó FEBS 2002 Even after desialylation by mild acid hydrolysis, no MALDI signals corresponding to N-glycopeptides were identified in the pertinent spectra (Fig 1) The tryptic peptides of both protein preparations were therefore additionally subjected to ESI-MS analysis Using this ionization technique, the tyrosine containing peptides T43–65 and T66–101 were found predominantly in their post-translationally modified forms (Fig 2) These findings can be explained by the pronounced instability of tyrosine sulfate under the conditions routinely applied for positive ion MALDI-TOF-MS of peptides A complete series of sequence-specific fragment ions was only found with the smaller sulfopeptide upon ESI-MS/MS, whereas the larger one yielded an intense signal generated by elimination of SO3 and a relatively weak fragment due to peptide cleavage at the proline residue The daughter ion spectra of the sulfated and desulfated peptide ENTVTNDWIPEGEEDDDYLDLEK(43–65) obtained after increasing the collision energy were found to be almost identical (data not shown), confirming the amino-acid sequence of the corresponding peptide from the recombinant and the serum-derived HCII As no modified fragment ions were detected after collisioninduced dissociation from the sulfated peptide, the position of sulfation could not be deduced from this spectrum Such behaviour can be explained by the spontaneous elimination of SO3 from any possible peptide fragment generated Therefore, the determination of the sequence position of sulfated tyrosine residues appears to be impossible by classical mass spectrometric peptide sequencing techniques However, in the case of the peptides under consideration here, the position of sulfation is unambiguous, as both peptides contain only a single tyrosine residue, and sulfation at Ser/Thr residues is improbable, in view of the characteristic consensus sequence for tyrosine sulfation [29,30] present in both peptides In addition to the identification of tyrosine sulfate residues detected with the ESI technique, we were also able to identify the dominant glycoforms of all three potential glycopeptides [NLSMPLLPADFHK(30–42), DFVNASSK(166–173), SMTNR(T) (365–370)] after desialylation by mild acid hydrolysis (Table 1) As the corresponding unmodified peptides were neither detectable by MALDI nor by ESI-MS, we deduce from these results that all three potential HCII N-glycosylation sites are completely glycosylated This conclusion is also supported by the SDS/ PAGE pattern of HCII from CHO cells treated with limited concentrations of tunicamycin (Fig 3) In contrast to the serum-derived protein, which predominantly contained a diantennary carbohydrate structure attached to each glycosylation site, the monofucosylated (plus 146 Da) derivative was observed for the glycopeptides of the recombinant protein The expected linkage position of this fucose unit to the proximal GlcNAc residue could be confirmed by MS/MS of the respective molecular ions by the identification of a fragment ion generated by the cleavage of the chitobiose bond of the N-glycan derived from serum HCII Generally, intense carbohydrate specific fragments were detected (fragment ions generated by elimination of monosaccharide residues from the molecular ion, as well as intense pure carbohydrate fragments), but only very weak peptide sequence specific fragment ions (data not shown) Ó FEBS 2002 Heparin cofactor II: N-glycans and sulfation (Eur J Biochem 269) 981 Fig MALDI-TOF-MS tryptic peptide fingerprints of natural (A) and recombinant (B) HCII Amino-acid sequence, calculated, and experimentally detected masses of the peptide fragments are summarized in Table Both protein preparations yielded a very similar peptide pattern, suggesting an identical polypeptide backbone The oxidation rate of the methionine residues, however, was markedly higher for the recombinant protein, as can be clearly seen, e.g for the peptides T311-343 [m/z 3672.1) or T420-449 [m/z 3206.2], each containing a single methionine residue We failed to detect any of the N-glycopeptides using the MALDI technique, even after desialylation The dominant glycoforms of all three glycopeptides, however, were readily observed employing the ESI technique Peptides T43-65 and T66-101, which had been reported to be sulfated [9], were observed only in the desulfated form due to elimination of SO3 By ESI-MS (see Fig 2) it could be unequivocally demonstrated that serum HCII as well as the recombinant species are predominantly sulfated *, incompletely cleaved peptide Detailed characterization of the N-glycans from serum and CHO cell-derived HCII For a more detailed investigation of the N-glycan structures, we performed additional mass spectrometric analyses of the oligosaccharides liberated by PNGase F After reduction and permethylation of the total glycan mixtures, the MALDI-TOF spectra depicted in Fig revealed the presence of a mainly diantennary disialylated structure and approximately 15% of triantennary trisialylated chains in both proteins (compare legend to Fig 4), confirming the results obtained from the ESI-MS analysis of the tryptic peptides described above The CHO cell-derived N-glycans were almost completely fucosylated at the proximal GlcNAc residue, whereas only about 10% of the serum protein showed fucosylation of the diantennary and triantennary N-glycans Approximately 60% of the triantennary structures isolated from the serum HCII contained a fucose residue In order to determine the linkage position of the fucose to the triantennary structure, ESI-MS/MS analysis was performed on the triply charged molecular ion [M + 3Na]3+ after isolation of the trisialylated oligosaccharide fraction by anion exchange chromatography on a Mono Q column [31] From the resulting daughter ion spectrum (Fig 5), we conclude that the fucose unit was not linked to the proximal GlcNAc residue, which is characteristic for the CHO cell-derived material, but was rather attached to a peripheral GlcNAc residue as is indicated by the weak fragment at m/z 1021.6 [NeuNAc-Hex(dHex)HexNAc + Na] and its more intense secondary fragment at m/z 646.3 [HO-Hex-(dHex)HexNAc + Na] (see fragmentation scheme and legend of Fig 5) This Ó FEBS 2002 982 C Bohme et al (Eur J Biochem 269) ă Table Amino-acid sequence and tryptic peptides of human HCII The peptides indicated below were detected by MALDI-TOF- and/or ESI-MS (see Figs and 2, respectively) in tryptic digests of human HCII isolated from serum or recombinant CHO cells, respectively, indicating their identical polypeptide backbones In the serum-derived protein  10–20% of the methionine residues were oxidized, whereas in the recombinant polypeptide about 50% of these residues occurred in the oxidized form Peptides smaller than 650 Da were not detected (ND) With the inclusion of the glycopeptides detected only by ESI-MS, a sequence coverage of more than 80% was achieved N-glycosylation consensus sequences are underlined, sulfated tyrosine residues are typed in bold w, m, s ¼ weak, middle or strong signal, respectively GP, glycopeptide glyc, glycan Amino-acid sequence Residue nos [M + H]+ Calculated GSK GPLDQLEK GGETAQSADPQWEQLNNK NLSMPLLPADFHK ENTVTNDWIPEGEEDDDYLDLEK IFSEDDDYIDIVDSLSVSPTDSDVSAGNILQLFHGK SR IQR LNILNAK FAFNLYR VLK DQVNTFDNIFIAPVGISTAMGMISLGLK GETHEQVHSILHFK DFVNASSK YEITTIHNLFR K LTHR LFR K NFGYTLR SVNDLYIQK QFPILLDFK TK VR EYYFAEAQIADFSDPAFISK TNNHIMK LTK GLIK DALENIDPATQMMILNCIYFK GSWVNKFPVEMTHNHNFR LNER EVVK VSMMQTK GNFLAANDQELDCDILQLEYVGGISMLIVVPHK MSGMK TLEAQLTPR VVER WQK SMTNR(T) TR EVLLPK FK TR NYNLVESLK LMGIR MLFDK NGNMAGISDQR IAIDLFK HQGTITVNEEGTQATTVTTVGFMPLSTQVR FTVDRPFLFLIYEHRTSCLLFMGR VANPSR S 1–3 4–11 12–29 30–42 43–65 66–101 102–103 104–106 107–113 114–120 121–123 124–151 152–165 166–173 174–184 185 186–189 190–192 193 194–200 201–209 210–218 219–220 221–222 223–242 243–249 250–252 253–256 257–277 278–295 296–299 300–303 304–310 311–343 344–348 349–357 358–361 362–364 365–370 370–371 372–377 378–379 380–382 383–391 392–396 397–401 402–412 413–419 420–449 450–473 474–479 480 ND 899.5w 1973.1m GP1a 2739.4mb 3912.1mb ND ND 785.5w 930.6s ND 2952.7w 1660.9m GP2a 1405.9m ND ND ND ND 870.5s 1079.8wd 1120.8w ND ND 2312.3w 857.5m ND ND 2500.4wc 2201.3me ND ND 823.5w 3672.1m ND 1028.7m ND ND GP3a ND ND ND ND 1079.8wd ND ND 1162.7w 819.6 m 3206.2s 3017.8mc,e ND ND 291.2 899.5 1972.9 1482.8 + glyc 2739.2 3911.9 262.1 414.3 785.5 930.5 58.3 2952.5 1660.8 867.4 + glyc 1406.7 147.1 526.3 435.3 175.1 870.4 1079.6 1120.6 248.2 274.2 2312.1 857.4 361.2 430.3 2500.2 2201.1 530.3 474.3 823.4 3671.8 552.2 1028.6 502.3 461.2 608.3 + glyc 276.2 698.4 294.2 389.2 1079.6 588.3 653.3 1162.5 819.5 3206.5 3017.5 643.3 105.0 a Glycopeptide; using ESI-MS, the most abundant glycoforms could be detected (diantennary complex type with two NeuAc residues (serum protein) or diantennary complex type with proximal fucose and two NeuAc units (recombinant protein) b Peptide containing a sulfated tyrosine Due to the easy elimination of SO3, MALDI-MS allowed the detection of only the desulfated molecular ion With ESI-MS, however, the predominant presence of a sulfopeptide was unequivocally detected (see text and Fig 2) c Peptide bearing a carboxamidomethylated cysteine residue d The presence of two peptides with identical molecular masses but different amino-acid sequences could be unequivocally shown by ESI-MS/MS e Peptide with one missed cleavage site Ó FEBS 2002 Heparin cofactor II: N-glycans and sulfation (Eur J Biochem 269) 983 interpretation was further corroborated by the detection of the intense fragment at m/z 316.2 [GlcNAc-ol + Na] instead of the fragment characteristic for proximally fucosylated N-glycans at m/z 490 [dHex-HexNAc-ol + Na] Even the linkage position of the fucose residue at the N-acetyllactosamine antennae could be assigned from the daughter ion spectra, due to the well-known preferential elimination of the 3-linked substituent of the GlcNAc residue of the N-glycan antenna [32] A weak, but reproducible signal at m/z 440.3 was detected, which is generated by the elimination of fucose from the fragment ion at m/z 646.3 Therefore, fucose must be linked to O-3 of a GlcNAc residue, indicating the presence of a sLex unit rather than a sLea motif in the N-linked oligosaccharides from serumderived HCII HPAEC-PAD mapping of the desialylated oligosaccharides enabled the quantitation of the basic oligosaccharide chains present in both glycoprotein preparations and demonstrated again the very similar sialylation degree of both proteins Table summarizes the glycosylation characteristics of recombinant and serum HCII based on mass spectrometry results and HPAEC-PAD mapping using oligosaccharides of known structure as standard Heparin binding properties of HCII treated with inhibitors of post-translational modifications In order to investigate the influence of post-translational modifications on heparin binding, recombinant CHO cells were treated with inhibitors of tyrosine sulfation and N-glycosylation under conditions that allowed partial inhibition of these modifications After dialysis, the conditioned medium was fractionated on heparin–Sepharose with a linear NaCl gradient (Fig 6) HCII produced in the presence of 20 mM sodium chlorate dissociated in a bimodal manner from the affinity matrix The first peak is observed at  280 mM NaCl, a concentration characteristic for HCII synthesized in the absence of the sulfation inhibitor A second peak is present at  430 mM NaCl, and a considerable amount of HCII eluted at still higher ionic strength, a property not associated with HCII from cells Fig Mass region of the triply charged molecular ions of the two tyrosine sulfated tryptic peptides recorded by ESI-MS of (A) serum-derived HCII and (B) the recombinant protein from CHO cells The detected molecular ions of these peptides (43-ENTVTNDWIPEGEEDDDYLDLEK-65 and 66-IFSEDDDYIDIVDSLSVSPTDSDVSAGNILQLFHGK-101) are compatible with the presence of one tyrosine O4-sulfate ester in each peptide (calculated m/z of monoisotopic masses for the triply charged monosulfated molecular ions [M + 3H]3+: 940.4 and 1331.3, corresponding to a molecular mass of 2818.1 and 3990.8 Da, respectively) Arrows indicate the expected positions for the molecular ions of the triply charged unsulfated peptide species Approximately 10% of the smaller peptide were present in its unmodified form in both protein preparations In contrast, the larger peptide from the natural protein was completely sulfated, while about 20–30% of this fragment from the recombinant protein did not contain this modification 984 C Bohme et al (Eur J Biochem 269) ă Fig Electrophoretic resolution of recombinant HCII from CHO cells incubated with various concentrations of tunicamycin After concentration ( fivefold), the medium was fractionated by SDS/PAGE and examined by Western blotting for the presence of HCII Lanes 1–5, HCII from cells treated with the indicated concentrations of tunicamycin; lane 6, recombinant HCII from an independent experiment For comparison, PNGase F-treated HCII from CHO cells (lane 7) and purified HCII from recombinant CHO cells (lane 8) were included grown in normal medium A shift towards elution at higher salt concentrations, albeit less pronounced, was also observed for the totally unglycosylated  64 kDa HCII form, secreted from CHO cells grown in the presence of lgỈmL)1 tunicamycin Similar results were obtained with HCII from HepG2 cells cultivated in the presence of tunicamycin (not shown) DISCUSSION In this investigation we have analyzed the structure of posttranslational modifications of human HCII from circulating blood and genetically modified CHO cells and their effects on heparin binding All three potential N-glycosylation sites were found to be populated with complex type carbohydrate chains Interestingly, sLex motifs were detected in triantennary oligosaccharides of plasma HCII, whereas only trace amounts of this structural motif were present in the diantennary glycan fraction It remains to be determined which structural features discriminate N-glycans for addition of this modification, and which types of a1,3/4-fucosyltransferases [33] are able to decode the involved signals The presence of sLex structures in HCII is an intriguing finding in the light of reports which indicate that several proteins associated with the acute phase response in humans contain altered glycostructures [34,35] and that HCII levels in plasma are increased under inflammatory conditions [2,3] Therefore, it could be possible that the N-glycan structures of HCII are also changed under certain (patho)-physiological situations We have detected triantennary oligosaccharides containing the Lex motif also in a1-microglobulin, hemopexin, and in a1-antitrypsin, whereas human serum AT contains this Ó FEBS 2002 structural motif almost exclusively in 5% of the diantennary oligosaccharides (H S Conradt & M Nimtz, unpublished observations) It remains to be established whether the amounts of sLex containing oligosaccharides synthesized by the liver are accomplished by a concomitant increase in the levels of the a2 fi sialyltransferase and a1 fi fucosyltransferase VI sLex units linked to cell-bound molecules on the surface of leukocytes have been found to interact with selectins exposed on endothelial cells [36] It may be envisaged that sLex bearing HCII molecules could interfere with these processes resulting in decreased plasma levels of the inhibitor On the other hand, interaction of HCII molecules with receptors recognizing sLex-carrying structures on vessel-lining cells could lower the risk of thrombotic events Blood from a single donor was used in this work for the analysis of post-translational modifications As glycan structures between individuals may differ, further investigations may determine whether qualitative or quantitative changes in the carbohydrate pattern of HCII correlate with specific (patho)-physiological situations There were several features specific for human HCII expressed in CHO cells; compared to plasma HCII, only a1 fi fucosylation of the recombinant HCII was observed and sLex structures were not detected (the trace amounts of difucosylated oligosaccharides contain an additional fucose residue a1 fi linked to galactose, thus constituting the Lewis H motif (for review see [37]) The NeuAc units are linked in a CHO cell-characteristic manner by a2 fi bonds, consistent with the lack of a2 fi sialyltransferase activity in CHO cells [38,39] Tyrosine sulfate has been implicated in several biological roles like leukocyte adhesion and haemostasis [40] HCII contains two adjacent sequences (positions 53–62 and 69–75, respectively) with similarity to the consensus signals characteristic for tyrosine sulfation The presence of tyrosine O4-sulfate esters in this domain, which resembles the acidic C-terminal tail of hirudin, has previously been reported for HCII from HepG2 cells [9] We were not able to detect this modification when routine positive ion MALDI-TOF conditions were used This issue has recently been addressed [41]; tyrosine sulfated peptides readily eliminate SO3 and therefore solely the desulfated peptide form was detected Even when applying negative ion MALDI in the linear mode, as has been recommended [41], we could detect only very small amounts of the sulfated molecular ion signal compared to the nonsulfated peptide signal Therefore, the detection and quantitation of polypeptide modification by sulfate provides a major challenge to mass spectrometric analysis In contrast to the situation with peptide phosphorylation, which can be detected in the positive as well as in the negative ion mode, it is difficult or even impossible to detect tyrosine sulfation by MALDI-TOF-MS With the electrospray technique applying very soft nozzle/skimmer conditions, degradation of the peptide sulfates was minimized or almost avoided During MS/MS experiments on both sulfated peptides, we observed a very facile elimination of SO3 even under very mild conditions where peptide bonds remained completely intact The ESI-MS results presented here clearly show that serum HCII as well as its recombinant counterpart Ó FEBS 2002 Heparin cofactor II: N-glycans and sulfation (Eur J Biochem 269) 985 Fig MALDI-TOF mass spectra of the reduced and permethylated total N-glycans enzymatically liberated from human HCII isolated (A) from human serum or (B) produced by genetically engineered CHO cells The following complex type carbohydrate structures were assigned to the detected molecular ions [M + Na]+: 2622, diantennary monosialylated with one fucose residue; 2796, diantennary monosialylated with two fucose residues; 2809, diantennary disialylated; 2983, diantennary disialylated with one fucose residue; 3432, triantennary disialylated with one fucose residue; 3619, triantennary trisialylated; 3793, triantennary trisialylated with one fucose residue; 4242, tetraantennary trisialylated with one fucose residue (monoisotopic masses) The major difference between the oligosaccharides from the natural protein compared to its recombinant counterpart is the almost complete proximal fucosylation and a slightly lower degree of sialylation of the recombinant material The triantennnary trisialylated N-glycan with one fucose residue (marked by an arrow) is the only major molecular ion observed in both glycoproteins ESI-MS/MS (compare Fig 5), however, showed that the fucose residue in the triantennary structure from the serum protein is not linked to the proximal GlcNAc, but peripherally to an N-acetyllactosamine antenna, thus constituting a Lex motif F ¼ fragment; * ¼ artefacts due to the insertion of CH2O or CO2 expressed by CHO cells are almost quantitatively sulfated at the two tyrosine residues at positions 60 and 73, respectively The nearly complete tyrosine sulfation of the recombinant HCII molecules was unexpected, as several reports showed incomplete sulfate ester modification of recombinant proteins expressed in CHO cell lines [42,43] Such individual differences indicate that the efficiency of tyrosine sulfation may depend on additional signals [44] and/or accessability to the modifying enzyme HCII from CHO cells incubated with inhibitors of post-translational modifications eluted at higher ionic strength from heparin–Sepharose than inhibitor molecules isolated from untreated cells In the case of tunicamycin, this may be the consequence of reduced sterical hindrance of heparin binding due to the missing N-glycans, similar to the situation observed with AT [45,46] This may especially apply to the carbohydrate chain linked to Asn169, a position in proximity to a site involved in heparin binding by HCII [47,48] Inhibition of tyrosine sulfation had an even more profound effect on the interaction between HCII and heparin The observation that higher NaCl concentrations are required for the dissociation of the unsulfated inhibitor from the heparin affinity matrix indicates that the N-terminal acidic domain may affect the GAG binding domain, although it can not be excluded that this effect is due to the reduction in the protein’s overall negative charge In summary, we present evidence for a very strong similarity of HCII from human serum and its recombinant counterpart from CHO cells with respect to tyrosine sulfation and N-glycosylation The remarkable identity of oligosaccharide antennarity and the extent of sialylation observed in both preparations represents an example of the importance of polypeptide structure governing protein N-glycosylation, as it is known that CHO cells have a high capacity to synthesize N-glycans of high tetraantennarity with considerable N-acetyllactosamine repeats, which however, were not detected in our recombinant glycoprotein We demonstrate that tyrosine sulfation and N-glycans individually affect heparin binding of the inhibitor These findings may be exploited to generate HCII variants with increased heparin affinity Ó FEBS 2002 986 C Bohme et al (Eur J Biochem 269) ă Fig ESI daughter ion spectrum of the triply charged parent ion [M + 3Na]3+ of a reduced and permethylated triantennary trisialylated N-glycan with one fucose residue isolated from serum HCII The detected fragment ions, in particular the signal at m/z 646.3 [HO-Hex-(dHex-HexNAc) + Na], as well as the accompanying signal at m/z 440.3, which is generated by the elimination of the fucose residue from the former ion, suggest a linkage to O-3 of the GlcNAc residue and therefore a Lex structure The signal at m/z 1021.6 [NeuAc-Hex-(dHex-HexNAc) + Na] again clearly indicates the presence of a peripheral fucose as shown in the fragmentation scheme This is confirmed by the detection of a signal at m/z 316.2, which is typical for an unfucosylated proximal GlcNAc-ol residue The presence of very small amounts of an isomeric carbohydrate structure including a proximal fucose is indicated by the weak signal at m/z 490.3 characteristic for this structural feature It should be noted that CHO cells predominantly produce proximally fucosylated structures and very small amounts of structures with an additional fucose linked a1 fi to the galactose residue of an acetyllactosamine antenna (LeH-motif) Table N-glycan structures of serum-derived and recombinant HCII After enzymatic liberation of the N-glycans from both protein preparations, the carbohydrates were subjected to HPAEC-PAD after enzymatic desialylation The major oligosaccharide from serum HCII was identified as a diantennary type II N-acetyllactosamine oligosaccharide (Gal2GlcNAc2Man3GlcNAc2), whereas the major glycan of the CHO cell-derived HCII contained the same structure with an additional proximally a1 fi linked fucose ND, not detected % Oligosaccharides in Structure Serum HCII CHO cell HCII Diantennary Diantennary + prox fucose Diantennary + Lewis fucose Triantennary Triantennary + prox fucose Triantennary + periph fucose (Lex) a2 fi or a2 fi sialylation of terminal Galb1–4GlcNAc 84 ND 7.5 ND 8.7 82 ND 1.5 7.6 ND 96 90 HCII may provide a valuable tool to study the fidelity of post-translational modifications in recombinant cell lines engineered with new transferases, e.g fucosyltransferases or sulfotransferases for the production of polypeptides which are modified in novel ways and which may be used for the study of the functional significance of post-translational protein modifications Ó FEBS 2002 Heparin cofactor II: N-glycans and sulfation (Eur J Biochem 269) 987 ACKNOWLEDGEMENTS We thank Dr H Karges, Centeon AG, Marburg, for human plasma samples We are grateful to Ulrike Beutling, Sabrina Herrmann, 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Church, F.C (1991) Role of lysine 173 in heparin binding to heparin cofactor II J Biol Chem 266, 8129–8135 48 Pratt, C.W., Whinna, H.C & Church, F.C (1992) A comparison of three heparin-binding serine proteinase inhibitors J Biol Chem 267, 8795–8801  ... modifications on heparin binding, recombinant CHO cells were treated with inhibitors of tyrosine sulfation and N-glycosylation under conditions that allowed partial inhibition of these modifications... In summary, we present evidence for a very strong similarity of HCII from human serum and its recombinant counterpart from CHO cells with respect to tyrosine sulfation and N-glycosylation The remarkable... of posttranslational modifications of human HCII from circulating blood and genetically modified CHO cells and their effects on heparin binding All three potential N-glycosylation sites were found