mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 329 (2004) 199–206 www.elsevier.com/locate/yabio

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins Jean-Pierre Zanetta,a,¤ Alexandre Pons,a Colette Richet,b Guillemette Huet,b Philippe Timmerman,a Yves Leroy,a Anne Bohin,a Jean-Pierre Bohin,a Pierre-André Trinel,c Daniel Poulain,c and Jan Hofsteenged a CNRS Unité Mixte de Recherche 8576, Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille Bâtiment C9, 59655, Villeneuve d’Ascq Cedex, France b INSERM U560, Place de Verdun, 59045 Lille Cedex, France c INSERM E360, 1 Place de Verdun, 59045 Lille Cedex, France d Friedrich Miescher-Institut, Maulbeerstrasse 66, PO Box 2543, CH-4002, Basel, Switzerland

Received 19 November 2003 Available online 10 May 2004

Abstract C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identiWcation of these mannosylated residues in proteins is rather tedious. In a previous paper [Biochemistry 42 (2003) 8342], it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three diVerent steps of gas chromatography/mass spectrometry of heptaXuorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptaXuorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C(2)-mannosylated Trp could be quantitatively recovered and identiWed during the step of amino acid analysis. Analyses of diVerent samples indicated that this type of glycosylation is absent in bacteria and yeasts.  2004 Elsevier Inc. All rights reserved. Keywords: Monosaccharide; Amino acids; Gas chromatography; Mass spectrometry; C-mannoside

C-mannosylation of Trp ((C-Man-)Trp)1 residues is one of the most recently discovered types of glycosylation of proteins [1–3]. The biosynthesis of C-mannosylation occurs through a dolichol-linked precursor [4], the exact mechanism giving rise to the formation of a C–C bond between the
-mannose residue and the C(2)

¤

Corresponding author. Fax: +33-3-20-43-65-55. E-mail address: [email protected] (J.-P. Zanetta). 1 Abbreviations used: (C-Man-)Trp, C-mannosylation of Trp; HFB, heptaXuorobutyrate; EI, electron impact; CI, chemical ionization; rMR, relative molar response; GC/MS, gas chromatography/mass spectrometry; GPI, glycosyl-phosphatidylinositol; HFBAA, heptaXuorobutyric anhydride; LB, Luria-Bertani; Nle, norleucine. 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.02.033

atom of the indole ring of Trp. The reaction is catalyzed by a microsome-associated transferase, whose activity can be detected in various organisms and most organs from mice [4]. Based on several studies, a consensus sequence for C-mannosylation, Trp–x–x–Trp, was deduced [5], the C-mannosylation occurring on the Wrst Trp residue of the sequence, with the possibility that the second Trp residue might be replaced by another aromatic amino acid. Subsequently, however, exceptions to this rule were found. For instance, human Wbrinogen B, tenascin, and N Cam L1 contain this motif, but are not C-mannosylated. In these proteins a large hydrophobic amino acid follows the Wrst Trp, a feature that inhibits C-mannosylation (T. Smilda and J. Hofsteenge,

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unpublished results). On the other hand, thrombospondin type 1 repeats are C-mannosylated on Trp residues without a Trp or Phe at position +3 [6–8]. Therefore, speciWc techniques had to be developed to determine the C-mannoside substitution. These methods generally involved the proteolytic cleavage of the protein and mass spectrometric analysis of peptides having an additional mass corresponding to a substituted (C-Man-) Trp residue [9]. This approach is eYcient on puriWed molecules, but the interpretation of the data becomes diYcult for complex proteins. Since the C–C bond is not cleaved by classical techniques used in monosaccharide analysis (i.e., acid-catalyzed methanolysis) and since Trp is in large part destroyed during acid hydrolysis used for amino acid determinations, the direct determination of (C-Man-)Trp residues in proteins or mixtures of proteins remained impossible. Recently it was reported that the complete analysis of all classical constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample using three diVerent steps of gas chromatography/mass spectrometry (GC/MS) as heptaXuorobutyrate derivatives [10]. This methodology involved Wrst a mild acid hydrolysis and second a GC/ MS analysis of the liberated sialic acids as HFB derivatives of their methyl esters. The remaining material was submitted to acid-catalyzed methanolysis under anhydrous conditions, followed by GC/MS analysis of the HFB derivatives of the liberated O-methyl-glycosides. The material remaining after this step was subjected to a classical hydrolysis for peptide bond cleavage and the liberated compounds were analyzed as HFB derivatives of their isoamyl esters in the same GC/MS system [11]. Using this approach, it was observed that, during the acid-catalyzed methanolysis, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively [10]. These derivatives were stable to acid hydrolysis. Since monosaccharide derivatives were also stabilized as per-heptaXuorobutyrate derivatives of O-methyl-glycosides, it was suggested that it should be possible to quantitatively recover C-mannosides on Trp residues. Based on the analyses of puriWed (C-Man-)Trp, of peptides containing this amino acid, and of RNase 2 from human urine, the Wne chemical formula of the perHFB derivative of the isoamyl ester was determined using electron impact (EI) and chemical ionization (CI) mass spectrometry. Using speciWc reporter ions in the EI mode of ionization, traces of this compound could easily be identiWed and quantiWed in the third step of analysis, i.e., amino acid analysis. After determination of the relative molar response of the (C-Man-)Trp derivative on standard samples, this methodology was applied to diVerent samples, including mammalian, bacterial, and yeast materials.

Materials and methods Chemicals Diazogen was from Acros. HeptaXuorobutyric anhydride (HFBAA; puriss. grade) was from Fluka, Merck, or Acros. Standard amino acids were from Beckman and Pierce. RNase 2 from human urine was isolated as previously described [3,5]. Peptides from human thrombospondin 1 were isolated as described by Hofsteenge et al. [7]. (C-Man-)Trp amino acid glycoside was puriWed from human urine, essentially as described by Gutsche et al. [12]. PuriWed MUC2 [13] was kindly provided by Dr. I. Carlstedt (Lund, Sweden). Bacteria and yeast cultures Five bacterial strains representative of three subdivisions of the proteobacteria were analyzed: Rodobacter sphaeroides strain WS8, Azospirillum brasiliense strain CdRif ATCC29710, Xantomonas campestris pv. Citri strain N1, Erwinia chrisanthemi strain 3937, and Escherichia coli K-12 strain DF214. These cells were grown at 30 °C in LB broth without NaCl as previously described [14–18]. The cells were washed three times in water, followed by delipidation using chloroform methanol mixtures. The insoluble pellet (1 mg protein) was submitted to the complete procedure of GC/MS analysis. Candida albicans A serotype cells were cultured in 1-L Erlenmeyer Xask containing 500 mL of Sabouraud’s broth at 37 °C on an orbital shaker until conXuence [19]. Cells were broken with a French press and centrifuged. The supernatant and the delipidized pellet (1 mg protein each) were analyzed separately. GC/MS analysis of sialic acids The glycoprotein-bound sialic acids were analyzed as previously described [4]. BrieXy, the samples (1–20 g protein) were hydrolyzed (105 min at 80 °C in 2 M acetic acid), methyl-esteriWed using diazomethane, and transformed into heptaXuorobutyrate derivatives [20]. The volatile derivatives were analyzed by GC/MS in the EI mode of ionization (see below). This step of liberation and analysis of sialic acid was not an absolute requirement for the determination of C-mannosylation, but it could provide important additional information on the monosaccharide composition of sialylated glycoproteins. GC/MS analysis of monosaccharides After analysis of sialic acids, the samples were dried under a stream of nitrogen. Alternatively, if this analysis was not needed, the dry samples were submitted to acidcatalyzed methanolysis (20 h at 80 °C in 0.5–1 mL anhydrous methanol containing 0.5 M gaseous HCl [10,21]).

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After drying under a stream of nitrogen, the samples were transformed into HFB derivatives and analyzed by GC/MS in the same system [10]. The acid-catalyzed addition of MeOH suppressed the delocalization of the cycles of His and Trp, converting nitrogen of the cycle into a secondary amino group, which subsequently became quantitatively derivatized by HFBAA [10].

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quantitation of the diVerent constituents was performed on the total ion count of the MS detector using the Xcalibur software (Thermoquest–Finnigan). For ascertaining the mass of the diVerent derivatives, the MS analyses were also performed in the CI mode in the presence of ammonia (ionization energy 150 eV; source temperature 100 °C) with detection of positive ions.

GC/MS analysis of amino acids Results and discussion After monosaccharide analysis, the samples were dried under a stream of nitrogen and, after the addition of 1 nmol of norleucine (Nle) as the internal standard, the samples were submitted to acid hydrolysis (6 N HCl, 20 h at 100 °C under a nitrogen atmosphere). After evaporation under a stream of nitrogen, the samples were submitted to a short-time methanolysis (0.2 mL of 0.5 M HCl in anhydrous methanol, 1.5 h at 80 °C) to form methyl esters and then trans-esteriWed with 0.2 mL of 1.5 M HCl in redistilled isoamyl alcohol overnight at 100 °C [11]. This two-step derivatization esteriWcation procedure was required due to the quasi-insolubility of some derivatives in the isoamyl reagent. After drying under a stream of nitrogen and mild heating with a hair dryer, the mixture was derivatized with HFBAA and analyzed by GC/MS in the same system. As reported recently [10], this method allowed qualitative and quantitative determination of all amino acids as sharp peaks of HFB derivatives of their isoamyl esters and hexosamines as the HFB derivatives of their isoamyl-glycosides. His and Trp derivatives modiWed in the Wrst step of methanolysis were quantitatively recovered. GC/MS analysis For GC/MS analysis, the GC separation was performed on a Carlo Erba GC 8000 gas chromatograph equipped with a 25-m £ 0.32-mm CP-Sil5 CB Low bleed/ MS capillary column and 0.25-m Wlm phase (Chrompack France, Les Ullis, France). The temperature of the Ross injector was 260 °C and the samples were analyzed using the following temperature program: 90 °C for 3 min, then 5 °C/min until 260 °C, followed by a plateau of 20 min at 260 °C (although the analyses could be shortened, we recommend this temperature program to clean the GC column from unrelated compounds, especially very-long-chain fatty acid methyl esters). For routine experiments, the column was coupled to a Finnigan Automass II mass spectrometer (mass detection limit 1000 amu); for studies of masses larger than 1000, it was coupled to a Nermag 10-10H mass spectrometer (mass detection limit 2000 amu). The analyses were performed routinely in the EI mode (ionization energy 70 eV; source temperature 150 °C). To preserve the Wlament of the ionization source, the GC/MS records were performed 5 min after the injection of the sample. The

As previously reported [10], during the step of acidcatalyzed methanolysis used for the liberation of O- and N-glycans from glycoconjugates, the Trp residues (as the His residues) were transformed by an acid-catalyzed addition of a CH3OH molecule on the indole (imidazol) cycle. The structures of these derivatives (denoted as His# and Trp# herein) were determined using their mass spectra in the EI and CI modes of ionization [10]. The suppression of one double bond inhibited the delocalization of the double bonds in the cycle(s). This resulted in stable compounds, quantitatively recovered in the amino acid analysis step as the di-HFB derivatives of the isoamyl esters of methoxy derivatives of both Trp (molecular ion at m/z D 698) and His (molecular ion at m/z D 649). The exact position of the CH3O and H addition on either the C(2) or the C(3) carbon atoms of the indole ring could not be determined. Nevertheless, the C(3) position seemed likely, because the delocalization of the N(1) nitrogen atom electron doublet would lead to the appearance of a negative charge on the C(2) carbon atom susceptible to protonation, whereas the electronattractive eVect of the methylene moiety would lead to the appearance of a positive charge on the C(3) carbon atom susceptible to nucleophilic attack by CH3O¡. This view was compatible with previous studies demonstrating that the major by-product formed from Trp during acid hydrolysis was -3-oxindolyl-alanine [22,23]. The question whether, in (C-Man-)Trp#, the C-mannoside cycle was resistant to the hydrolysis step arose. In fact, as a result of the methanolysis and HFBAA steps, the C-mannose was present not as the free compound but rather as its per-HFB derivatives, these compounds being more resistant than nonderivatized sugars to acid hydrolysis [10]. This was substantiated by the quantitative recovery of the isoamyl-glycoside of hexosamines after acid hydrolysis used for the cleavage of most peptide bonds and the high recovery of Man or Glc in the GC/MS analysis of amino acids of glycoproteins. Since C-mannose was considered to be more stable to acid hydrolysis than Man-O-glycosides, it was suggested that (C-Man-)Trp# could possibly be recovered in good yield. GC/MS analyses of very heterogeneous mixtures of glycoproteins from human origin (especially bronchial and colonic mucins), using the routine apparatus with a

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Fig. 1. GC/MS in the EI mode of ionization on a Finnigan Automass II mass spectometer (mass limit of 1000 amu) of a mixture of glycoproteins (essentially mucins from lung mucus). (A) Chromatogram reconstitution for the ion at m/z D 880 characteristic of the proposed (C-Man-)Trp# derivative. (B) Total ion counts. (C) Fragmentation mass spectrum of the derivative obtained with the routine GC/MS apparatus with a mass limit of 1000 amu. Note in (A) and (B) the good separation of the compound from amino acids and from other constituents remaining after acid hydrolysis. Note also in (A) the eYciency of detection of the (C-Man-)Trp# derivative. When analyzing samples containing puriWed (C-Man-)Trp or the same included in a protein or peptide sequence, a minor peak was observed (about 10% of the total), likely corresponding to the stereoisomer of the nucleophilic attack of CH3-O¡ on the C(3) carbon atom of the indole ring during acid-catalyzed methanolysis. Note also, in addition to amino acids, hexosamine derivatives (recovered as per-HFB derivative of its isoamyl-glycoside) were present as major compounds.

mass limit of 1000 amu, presented a peak with intense ions at m/z D 880 and 667, together with minor ions potentially compatible with the expected structure of (C-Man-)Trp# (M D 1644; Fig. 1). This suggested that (C-Man-)Trp# could be recovered using the procedure depicted above. Therefore, studies were performed on standard compounds to verify that the postulated (C-Man-)Trp# was actually this compound. Applying an arbitrary relative molar response to this derivative of 1 relative to Nle gave 3.01 mol of this compound for 1 molecule of MUC2 (containing more than 5000 amino acids). This was in good relation with the occurrence of the consensus sequence W–x–x–W (two sequences) and the putative new consensus sequence [9] W–x–x–F (one sequence).

IdentiWcation of the (C-Man-)Trp# derivative (C-Man-)Trp, peptides containing this amino acid, and RNase 2 from human urine were subjected to acidcatalyzed methanolysis under anhydrous conditions and formation of HFB derivatives, followed by acid hydrolysis and formation of HFB derivatives of the isoamyl esters. In all cases, peaks corresponding to the putative (C-Man-)Trp found in mucins were observed (data not

shown). Furthermore, the relative abundances of these peaks were in gross proportion to that expected from previous studies. To determine the structure of the (C-Man-)Trp derivative, these standard compounds were analyzed on a mass spectrometer with a mass limit of 2000 amu. The fragmentation EI mass spectrum obtained for (C-Man-) Trp# is shown in Fig. 2B. This analysis did not allow detection of the molecular ion at m/z D 1644. The highest mass ion so far detected was at m/z D 1559 (i.e., M D 85), corresponding to the elimination of an isoamyl (M D 70) and a methyl group (M D 15). Although it was of extremely low intensity, this ion was clearly distinguished from the background, and chromatogram reconstitution for the ion perfectly Wtted with all other ions arising from this compound. In fact, all ions in the EI spectrum (the most representative ones are shown in Fig. 2B and Table 1) could be explained by the proposed structure shown in Fig. 2A. Therefore, it was concluded that this compound corresponded to the (C-Man-)Trp# derivative. As discussed above, uncertainties in the position of the CH3O group and the conWguration of the C(3) carbon atom of the modiWed indole ring remained. The presence of the ion at m/z D 71 in the spectrum of lung mucins (Fig. 1C) indicated the presence of an isoamyl ester on the carboxyl group of the molecule, a deduction

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203

Fig. 2. Proposed structure (A) and EI fragmentation mass spectrum (B) of the derivative of (C-Man-)Trp# obtained from the treatment of free (C-Man-)Trp. The addition of the O-methyl group was evidenced by a very minor ion at m/z D M ¡ 32 (m/z D 1612). The major addition (see legend of Fig. 1) of CH3O on the C(3) carbon atom of the indole ring was suggested by the simultaneous elimination of an isoamyl and a methyl group (ion at m/z D 1559; i.e., M ¡ (70 C 15)), the formation of which can be explained only by the formation of a six-atom ring.

reinforced by the presence of ions corresponding to losses of mass of 70, 85, and 114/115 from the molecular ion (Fig. 2B). The loss of mass of 15 (simultaneous to the loss of 70 D isoamyl group) suggested the presence of an O-methyl group, a conclusion reinforced by losses of mass of 32 (CH3OH) for interpreting some ions (Fig. 2, legend). The presence of the C-hexoside group was evidenced by the losses of mass of 947. Nevertheless, because of the absence of standard compounds with hexoses other than mannose, this method will still not allow one to identify the nature of the C-glycoside bound to the Trp residue. This point could probably be resolved in the future, considering that speciWc ions from the hexoside groups allow the determination of the exact nature of the hexose. Indeed, the EI analysis of HFB derivatives of O-alkyl-glycosides allowed a very easy discrimination between the diVerent monosaccharides [10,20,21], suggesting that such discrimination could be also possible in the study of C-hexosides. Interestingly, all fragment ions obtained on the routine GC/MS apparatus with masses lower than 1000 amu showed intensities similar to those obtained using the

other spectrometer. Therefore, the very intense ions at m/ z D 880 and 667 were considered as major reporter ions of (C-Man-)Trp# (as illustrated in Fig. 1). The intense ion at m/z D 591 was not considered to be a useful reporter ion, because of its presence in several unrelated compounds, including common contaminants of biological samples. The structure of (C-Man-)Trp was also analyzed in the chemical ionization mode in the presence of ammonia. The GC/MS analysis (and the direct introduction method) gave a very weak pseudo-molecular ion at m/ z D 1662 ([M + NH4]+) and higher-intensity ions corresponding to fragmentations (data not shown). The fragmentation of esters (here isoamyl esters) in the CI mode of ionization is a common feature. This point was documented by the presence of an ion at m/z D 1573 corresponding to the loss of the isoamyl group. Therefore, both the EI and the CI studies indicated that the high-mass ions corresponded to those of the structure of (C-Man-)Trp# (M D 1644) proposed in Fig. 2A. In the chromatograms of all samples containing (C-Man-)Trp, a minor peak representing less than 10%

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Table 1 Relative intensities and schematic explanation of fragment ions relevant to the structure of the (C-Man-)Trp# derivative shown in Fig. 2A obtained in the EI mode of ionization Ion

Intensity

Ion

Intensity

1559 1538 1510 1490 1461 1297 1243 1241 1217 1204 1180 1166 1155 1154 1153 1138 1128 1115 1104

0.08% M-15-70 0.23% M-19-71-16 0.07% M-19-71-16-28 0.04% 1559-69 0.07% M-169-14 0.21% 1510-213 0.15% 1510-70-197 0.18% M-18-19-169-197 0.14% M-213-214 0.13% M-213-214 0.14% M-70-197-197 0.25% M-19-32-213-214 1.93% M-19-43-213-214 2.65% M-19-43-214-214 1.39% M-19-43-214-215 0.52% 1154-16 0.42% 1538-197-213 0.27% M-32-70-213-214 0.85% M-114-213-213

1100 1020 941 881 880 863 862 861 684 683 668 667 666 592 591 356 355 282 281

0.37% 1510-197-213 0.27% 1217-197 0.15% 1154-213 80.08% M-115-197-224-227 78.10% M-115-197-224-227 3.28% 881-18 10.90% 881-19 16.35% 880-19 7.02% 881-197 7.66% 880-197 84.56% 881-213 100.00% 881-214 40.99% 880-214 37.67% M-946* 47.59% M-947 4.71% Core + CH3O + 2O 10.44% Core + CH3O + 2O 23.67% Core 43.47% Core

87, CH3(CH3)CHCH2CH2O; 115, CH3(CH3)CHCH2CH2OCñO; 169, CF3CF2CF2; 197, CF3CF2CF2CO; 213, CF3CF2CF2COO or CF3CF2CF2 CONH2; 214, CF3CF2CF2COOH; 947, HFB-substituted (C-Man) residue. The values of the intensities of the ions are only indicative since they may vary with the apparatus used and with experimental parameters such as the Wne adjustment of the column end into the ion source. Nevertheless, it can be observed that the reporter ions deWned in the text are actually major fragment ions.

of the major one and with an identical fragmentation pattern was observed (see, e.g., Fig. 1A). We assume that it is the diastereoisomer resulting from the alternative attack of CH3O¡ on the C(3) atom. This is corroborated by the absence of such a component in the Trp derivative. Because this secondary peak was sometimes contaminated by phthalate derivatives, it was not considered in studies on the determination of the relative molar response of (C-Man-)Trp#.

Determination (C-Man-)Trp

of

the

relative molar response of

The determination of the relative molar response (rMR) of (C-Man-)Trp# involved a quantitative analysis of two glycopeptides from human thrombospondin 1, containing both modiWed and unmodiWed Trp residues, and RNase 2 from human urine. As shown in Table 2, the analysis of the diVerent compounds allowed us to determine the rMR of (C-Man-)Trp# to be very close to 1.00, relative to Nle, the classical internal standard used in amino acids analyses. Using this rMR, the compositions of the glycopeptides with respect to all other amino acids were found to be in close agreement with the theoretical values. The major diVerence was observed for Thr (known to be partially destroyed during acid hydrolysis of polypeptide chains) as also observed previously [10]. The presence of additional Gly was observed, likely originating from contamination. Nevertheless, for these standard glycopeptides, having both (C-Man-)Trp and

Trp, it was evident that the two types of compounds could be recovered and quantiWed using this derivatization procedure. When degradation of these compounds did occur, this was less than 10% for both Trp# and (C-Man-)Trp#, as indicated by the calculated rMR. As a consequence, it was concluded that Trp# and (C-Man-)Trp# could be quantitatively determined using an rMR factor of 1.000 relative to Nle. These quantitative determinations can routinely be performed with nanomolar amounts of initial compound, when 0.5% of the sample is injected into the GC/MS apparatus. In fact quantitation of (C-Man-)Trp# could reliably be performed at the picogram level of the initial compound in the sample.

Absence of (C-Man-)Trp in bacteria and yeast The presence of major, speciWc reporter ions for (C-Man-)Trp# at m/z D 667 and 880 and its retention time relative to that of Nle allowed its speciWc identiWcation. Furthermore, based on the sensitivity of detection, it was calculated that (C-Man-)Trp# could be reliably identiWed at a signal/background ratio of 1 residue per 5 £ 106 amino acid residues. This sensitivity oVered the possibility to examine the presence of (C-Man-)Trp in diVerent organisms, including bacteria and yeasts. Analysis of total homogenates of human and mammalian cells indicated that (C-Man-)Trp was present in all samples so far analyzed starting from 10 g of initial protein material (data not shown). Therefore, the

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205

Table 2 Composition of standard glycopeptides RNase2 Ala Gly Val Thr Ser Leu Ile Pro Met Phe Asp Lys Tyr Glu Arg His Trp Cysb CMan

6 2 9 12 6 5 7 12 4 5 21 4 4 14 8 5 1 8 1

Th-RNase2 6.05 2.19 8.90 10.25 5.85 4.98 6.85 12.00 3.42 5.11 22.55 4.01 4.02 14.00 7.62 4.87 0.96 6.21 1.02

1 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 1 0 1

1.10 0.21 0.85

1.03

1.00

0.98 1.01

TSP1-(414–426)a

TSP1-(469–483)a

0 2 0 0 2 0 0 1 0 0 1 0 0 1 0 1 1 0 2

1 3 0 0 1 0 1 3 0 0 2 0 0 0 0 0 2 0 1

2.26

1.98

1.02

1.03

1.00 0.98 0.97 1.98

1.05 3.12

0.99 0.99 3.00

2.11

1.95 1.02

a

The numbers in parentheses are residue numbers as deWned in [7]. Recovered as S-carboxy-methyl-cysteine. The experiments were performed starting from the following quantities: RNase 2, 21 g; peptide ThRNase 2 (5–10) FT(C-Man)-WAQW, 1.3 nmol; peptide TSP1-(414–426), 4 nmol; peptide TSP1-(469–483), 6.5 nmol. Cman, (C-Man-)Trp#. These data were obtained assigning the rMR of 1.000 for the major peak of this compound. b

method oVered the possibility to perform a simple screening for the presence of (C-Man-)Trp in diVerent organisms. Previous studies on RNase 2 expressed in bacteria [24] have shown that C-mannosylation of this protein did not occur in this bacterium, but the exact reasons for this and the occurrence of C-mannosylation in other bacteria have not been examined. Therefore, we investigated delipidized total homogenates of Wve diVerent bacteria for the presence of (C-Man-)Trp. R. sphaeroides is a member of the alpha subdivision, whose genetic analysis is highly developed because it is a remarkable model for the study of bacterial photosynthesis. R. sphaeroides shows a close relationship to organisms that interact with eucaryotic hosts but are themselves free-living organisms. A. brasilense, another member of the
subdivision, is a nitrogen-Wxing bacteria which grows in close association with the roots of grasses. X. campestris is a plant pathogen of the  subdivision. E. chrysanthemi is a pathogenic enterobacterium ( subdivision) responsible for the soft-rot disease of a wide range of plants. E. coli K-12 is the well-known model of enterobacteria. In all these bacterial strains (C-Man-)Trp# was undetectable. Given the speciWcity and sensitivity described above, (C-Man-)Trp should have been easily identiWed over the background signals if it had been present. When looking for the W–x–x–W motif in protein data bases, 542 occurrences were found in the complete E. coli 0157:47 genome (http://www.infobiogen.fr/services/ analyseq/cgi-bin/patternp_in.pl) and 24,538 occurrences in the human genome (http://alces.med.umn.edu/dbmotif. html). The lower occurrence of the W–x–x–W consensus

sequence (about 50 times lower in bacteria than in human) was not suYcient to explain the absence of detection of (C-Man-)Trp in the proteins of bacteria. Because in these speciWc experiments the quantity of material injected on the GC/MS apparatus was saturating with regard to amino acids, the limit of sensitivity of the method for detecting (C-Man-)Trp was at least Wve orders of magnitude higher than the quantity of (C-Man-)Trp expected from the putative occurrence of the consensus sequence proposed above. Therefore, it was concluded that the absence of (C-Man-)Trp in bacteria was due to the actual absence of this type of glycosylation in bacteria. These data reinforced the previous observations that RNase 2 expressed in bacteria was not C-mannosylated [24]. We next analyzed C-mannosylation on yeast material. The analyses were performed on C. albicans because it is considered an extremely sophisticated yeast strain. As for bacteria, the GC/MS analyses indicated the complete absence of C-mannosides (C-hexosides and C-pentosides) in this strain. The results on this microorganism (able to synthesize huge amounts of mannosylated compounds and having machinery able to synthesize the precursor for C-mannosylation, dolichol-P-Man) supported the view that, although the precursor of the synthesis of (C-Man-)Trp is abundant in these organisms, the enzymes involved in the biosynthetic pathway of C-mannosides were absent. This agrees with the previous results that suggested that the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe did not possess the enzyme involved in C-mannosylation ([24]; S. Hartmann and J. Hofsteenge, unpublished results).

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Conclusion The analytical approach described here allows the qualitative and quantitative determination of C-mannosylation in puriWed glycoproteins and glycoprotein mixtures after acid hydrolysis. In fact, as discussed elsewhere [10], it allows the complete analysis of glycoproteins (sialic acid diversity, monosaccharide (fatty acids [25]) composition, and amino acid composition) provided the use of an initial additional step of analysis of sialic acids on the same sample. Furthermore, this approach can provide also the complete analysis of GPIanchored glycoproteins and proteoglycans [10,26], at levels ranging from 1 to 100 pmol of initial puriWed glycoprotein material.

[12]

[13]

[14]

[15]

[16]

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