2Department of Medical Chemistry, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands, 3Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA, and 4Research and Development Group, NV Organon, Oss, The Netherlands
Received on October 6, 2000; revised on December 12, 2000; accepted on January 3, 2001.
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Abstract |
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Key words: fucosyltransferase/disulfide bridges/mass spectrometry/homology modeling
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Introduction |
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We (De Vries et al., 1995, 1997; Nguyen et al., 1998
; Vo et al., 1998
) and others (Legault et al., 1995
; Dupuy et al., 1999
) have studied the acceptor substrate specificity of the FucTs in detail and have also identified specific amino acids that affect the acceptor substrate specificity of this family of enzymes. These studies have demonstrated that amino acids occurring at both the N-terminus and the C-terminus of the catalytic domain are involved in acceptor substrate recognition. Other amino acids of the catalytic domain also have been identified as being critical for catalytic activity. These observations have demonstrated that amino acids from various regions of the catalytic domain participate in the function of FucTs, but at present it is unknown how these amino acids are organized to form the active enzyme. In fact, there is currently very little information on how any glycosyltransferase folds into its native form. The only information available on how portions of the protein structure of glycosyltransferases are arranged is from studies on bovine ß4-galactosyltransferase (ß4-GalT). This enzyme has been proposed to contain one disulfide bond that brings together two segments of the protein, separated by 103 amino acid residues (Yadav and Brew, 1991
; Wang et al., 1994
). Recently, a 3D structure for this enzyme has been solved based on X-ray crystallographic studies. Interestingly, the crystal structure has an arrangement of disulfide bonds that differs from the one that was assigned from chemical and mutation studies (Gastinel et al., 1999
).
Currently, there is no information available on the existence of disulfide bonds in FucTs. Among FucTs (including human, mouse, and chicken) four cysteine residues are highly conserved (Figure 1). One pair of these Cys residues is located at the N-terminus and another at the C-terminus of the catalytic domain. The Cys residues found at the C-terminus are also conserved in FucTs from Caenorhabditis elegans. Besides the four conserved cysteines, FucT VII contains two additional cysteines in close proximity to each other, located in the middle of the catalytic domain. In the present study, we characterized the participation of the cysteine residues of FucT VII in disulfide linkages. FucT VII is involved in the biosynthesis of fucosylated glycoconjugates required for a normal cellular inflammatory response (Maly et al., 1996). Thus, inhibitors of FucT VII could operate as anti-inflammatory drugs. Determination of the 3D structure of FucT VII would enable the rational design of specific enzyme inhibitors, making this enzyme an interesting target for 3D structure determination and modeling studies. As a step toward this goal we have prepared and purified large quantities of a soluble form of FucT VII and determined the disulfide pattern. Our analyses, using mass spectrometry (MS), demonstrated that all six Cys residues of FucT VII are involved in disulfide bonds. The pairing of these Cys residues was determined by proteolytic cleavage of the nonreduced protein, followed by MS analysis of the resulting peptides. Finally, we have used these data, in addition to a method of fold recognition/threading and homology modeling, to propose a model structure of FucT VII.
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Results |
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Verification of amino acid sequence and location of sites of N-linked glycosylation by MS
The peptide sequence of soluble FucT VII was analyzed using an LC/MS/MS method that involves chromatographically resolving peptides by micro-LC, and detecting and selecting peptide ions produced by electrospray ionization mass spectrometry (ESI/MS). Subsequently, each selected peptide ion is fragmented by collisionally induced dissociation and analyzed by MS/MS. The peptide fragments are primarily generated by cleavage at the amide bond along the backbone of the peptide in which the charge is retained on the N-terminus (b-type ions) or the C-terminus (y-type ions). The amino acid sequence is identified by correlating the fragmentation pattern of the peptide observed in the MS/MS spectrum with a predicted fragmentation pattern in the protein database. Based on the mass of the peptide and its MS/MS fragmentation pattern, the sequence of the peptide can be conclusively verified.
With a combination of two proteolytic enzymes, trypsin and chymotrypsin, following treatment with PNGase F to remove N-linked carbohydrates, more than 98% of the amino acid sequence of FucT VII (amino acids 53342) was confirmed (results summarized in Figure 2). Only one tripeptide (AA 295297) was not detected in the tryptic/chymotryptic fragments. No ion signals were detected for any contaminating proteins.
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An example of the results obtained for one of the peptides is given in Figure 3, which shows the full scan mass spectrum of the tryptic peptide containing AA 282294 at m/z 720.7 obtained after PNGase F treatment. The analysis of the ESI mass spectrum can be difficult due to the distribution of mixed multiply charged ions. This difficulty, however, is resolved by using a high-resolution zoom scan mode to distinguish the charged state of ion. The inset zoom scan mass spectrum shows the doubly charged peptide (M+2H)2+ ion at m/z 720.3. The assignment of double charge is based on the difference between the mass (0.5 units, a difference that is easily measured based on the mass accuracy (± 0.2 units) of the instrument) of the monoisotopic peak at m/z 720.3 and the isotopic peak containing 13C at m/z 720.8. The singly charged peptide ion (M+H) + detected at m/z 1439.6 is 1 mass unit greater than the mass expected for the tryptic peptide containing Asn291, resulting from the conversion of Asn291 to Asp291.
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None of the peptide fragments was found to be modified by the biotin probe, however two peptides (AA 205223 and 317329), containing a total of four alkylated cysteine residues (Cys211, Cys214, Cys318, and Cys321), were detected. Tryptic peptides containing the other Cys residues (Cys68 and Cys76) were not detected. However, an additional tryptic peptide (AA 7682) containing alkylated Cys76 could be detected after treatment of the tryptic digest with PNGase F. A tryptic peptide containing alkylated Cys68 was not observed, but a chymotryptic peptide (AA 5371) containing this alkylated Cys residue was identified. These results demonstrate that all of the Cys residues in FucT VII are involved in disulfide bonds.
To identify which of the Cys residues are linked together in disulfide bonds, FucT VII was denatured with urea (without reduction/alkylation) and digested with trypsin or chymotrypsin. The resulting peptides were separated and detected by LC/ESI/MS. As shown in Figures 58, LC/MS/MS analysis confirmed the presence of two peptides that contained disulfide bonded Cys residues: a chymotryptic peptide containing Cys211Cys214 and a tryptic peptide containing Cys318Cys321. Figure 5 shows the full scan mass spectrum of the tryptic disulfide containing peptide (AA 317329) at m/z 795.1. The zoom scan spectrum (Figure 5 insert) shows the isotopic peak distribution of a doubly charged ion (M+2H)2+ with the monoisotopic peak at m/z 794.9. The measured (M+H)+ = 1588.8 Da of the disulfide-containing peptide matches the calculated (M+H)+ = 1588.8 Da and thus confirms the presence of a disulfide linkage between Cys318and Cys321 (M+H+ would be equal to m/z 1590.8 if there were no disulfide linkage). The corresponding MS/MS spectrum of m/z 795 (Figure 6) contains dominant b- and y-ions (bn, n = 812; yn, n = 26), confirming the peptide sequence with a disulfide bond (AA 317329).
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Discussion |
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In the current study, we determined the disulfide linkages in a soluble form of FucT VII. FucT VII contains six Cys residues, two at the N-terminal end (Cys68 and Cys76), two in the middle (Cys211 and Cys214), and two at the C-terminal end of the molecule (Cys318 and Cys321). Four of these residues (Cys68, Cys76, Cys318, and Cys321) are highly conserved among all mammalian fucosyltransferases. Based on the distribution of Cys residues in the FucT VII molecule, four general folds (Figure 13) are possible if all the Cys residues are disulfide-linked. A "circle" involves the entire catalytic domain held in a loop by disulfide bonding between conserved Cys residues at either end of the catalytic domain. A "lariat" is when the central Cys residues are bonded to the Cys residues at one end or the other of the catalytic domain to form a smaller loop. A "pretzel" is formed when either end is connected to the central Cys residues. Finally, a "triple loop" involves fixation of short, sharp bends in the protein. We anticipated that the circle structure might have the highest probability, because it links the highly conserved N- and C-terminal Cys residues. However, MS revealed that in fact the closely spaced Cys pairs form disulfide bridges (Cys68Cys76, Cys211Cys214, and Cys318Cys321). No ions were detected that would be consistent with any other disulfide arrangements. Thus, we have found that the overall structure of FucT VII resembles the triple loop model. The function of the disulfide bends or small loops in this structure remains to be resolved. However, two versions of a snake toxin have been found to differ by the presence or absence of a similar small disulfide loop. The presence of this small loop determines whether the toxin binds to a peripheral nicotinic acetylcholine receptor or a chimeric form of a neuronal 7 receptor (Servent et al., 1997
).
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The information on the distribution of the disulfide bridges in FucT VII was used in combination with fold-recognition/threading and homology modeling to compute a 3D fold of FucT VII. This fold, an (/ß)8-barrel based on the structure of 1dhp, was compared to a fold for FucT VII based on the structure of 2bgu, similar to the one for FucT IV proposed by Breton et al. (1996)
. The (
/ß)8-barrel fold is observed in a number of glycosyl hydrolases, amylases, and other carbohydrate processing or binding proteins (Davies and Henrissat, 1995
) and was also found in the fold-recognition searches performed by Breton et al. (1996)
.
As an indicator of how well the proposed folds allow for the formation of a hydrophobic core, the lipophilic fraction of the solvent exposed protein surface was calculated for the two models. For the 2bgu-based model (Figure 12, left side) 43% of the surface is hydrophobic (26% for 2bgu itself) and for the 1dhp-based model 35% (Figure 12, right side, compared to 31% for 1dhp). Because the majority of conserved amino acids are expected to reside in the hydrophobic interior of the protein, only a small fraction of conserved residues would be expected to be solvent exposed. Calculation of the average fraction of exposed surface for 87 conserved amino acids in the FucT family makes the 1dhp-based model even more likely: The average fractions are 26% for the 2bgu-based model and 0% for the 1dhp-based model. Finally, location in the models of the two conserved 3-motifs (Breton et al., 1998
) did not argue in favor of one of the two models. However, several conserved Asp and Glu acid residues, which may be relevant for catalysis, are located at the surface of the (
/ß)8-barrel model, close to where the active sites of other carbohydrate processing enzymes are located (Davies and Henrissat, 1995
). These are residues Glu62, Asp146, Asp200, Asp235, and Glu239 of FucT VII.
Our results indicate that an (/ß)8-barrel fold for FucT VII based on 1dhp provides a better scaffold to rationalize the observed Cys pairings and places more hydrophobic amino acids in the core of the protein than a fold based on 2bgu. Therefore, the (
/ß)8-barrel fold seems more likely than the
- and ß-type fold of ß-glucosyltransferase proposed by Breton et al. (1996)
. Although our model is of low resolution and likely to be incorrect in details, it strongly suggests a fold consisting of alternating
-helices and ß-sheets. More detailed biophysical studies are required and have been initiated to identify the exact nature of the 3D structure of FucT VII.
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Materials and methods |
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Preparation of FucT VII
Construction of a mammalian expression vector containing FucT VII encoding DNA and subsequent production of a CHO cell line (stably transfected with this construct) secreting a soluble form of FucT VII will be described elsewhere. Growth medium (serum-free) was collected from a large-scale 3.6-L fermentor culture and stored at 4°C until use. FucT VII was purified from the medium (1.7 U/L) in one step by affinity chromatography on GDP-hexanolamine-Sepharose. FucT VII was eluted from the column by washing with a buffer (25 mM sodium cacodylate, pH 6.8) containing 0.2 M NaCl, 10 mM GDP, 1 mM MnCl2, 15% glycerol, and 0.05% sodium azide, concentrated on Centricon cartridges (Amicon) and stored at 4°C until use. Enzyme activity assays were performed as described (De Vries et al., 1997).
Modification of Cys residues
Forty micrograms of FucT VII (4 mg/ml in 0.2 M TrisHCl, pH 8.3, 5 mM EDTA, 1 mM NaN3) was added to 40 µl 10 M urea. Iodo-LC-biotin, dissolved in DMSO (a molar excess of 1.2 per potential SH group) was added, and the mixture was incubated in the dark for 2 h at room temperature. Reduction of potential disulfide bonds was carried out with a 500-fold molar excess of dithiothreitol (based on a maximum of three disulfide bonds/molecule) incubated for 15 min at 65°C. Carbamidomethylation of Cys residues, following reduction with dithiothreitol, was carried out by adding a 2.2-fold molar excess of iodoacetamide and incubating the mixture for 15 min in the dark at room temperature.
Digestion with trypsin/chymotrypsin
The concentration of urea in the denatured FucT VII sample (either Cys modified as above or not) was reduced by diluting the mixture with water to 2 M (if using trypsin) or 1 M (if using chymotrypsin). A 1:20 ratio (w/w) of protease/protein was added, and the mixture was incubated overnight at 37°C.
PNGase F digestion
PNGase F was dissolved in 100 mM sodium phosphate/25 mM EDTA at pH 7.2 at a concentration of 200 unit/ml. The PNGase F digestion was done either on intact FucT VII or following tryptic or chymotryptic digestion. PNGase F was added at a final concentration of 20 unit/ml and the reaction was incubated overnight at 37°C.
Monosaccharide composition analysis
The monosaccharide content of 200 µg purified FucT VII was determined by analysis of alditol acetetates using GLC/MS as described (Savage et al., 1986). The monosaccharide composition was calculated by comparing the profile with that obtained with a mixture of monosaccharide standards.
Sequence analysis of peptides by HPLC/MS/MS
LC/MS/MS analysis was performed using a Finnigan LCQ ion trap mass spectrometer (San Jose, CA) with an electrospray ionization (ESI) source. A positive voltage of 3 kV was applied to the electrospray needle, and the temperature of the stainless steel heating capillary was maintained at 250°C. An N2 sheath flow (65 scale) was applied to stabilize the ESI signal. The voltage at the exit of the heating capillary and the tube lens was held at 13 V and 5 V, respectively, to minimize the source-induced dissociation and optimize the ESI signal of the analyte. Ion injection was controlled by automatic gain control to avoid space charge effects. The full scan mass spectrum was acquired from m/z 300 to m/z 2000. The MS/MS experiments were executed with a relative collision energy of 35%. The LC/MS analysis was conducted using a Hewlett-Packard 1050 HPLC system (Palo Alto, CA) coupled to the LCQ. The HPLC system was operated at a flow rate of 0.3 ml/min. The mobile phase was split before the injector by a Tee-connector. One end of the Tee was connected to a microbore (150 x 1 mm, Vydac, 5 µm particle size) or capillary C18 column (150 x 0.18 mm, Nucleosil, 5 µm particle size) and a flow rate of 30 or 1.5 µl/min was established, respectively. The enzymatically digested peptides were eluted from the column using 0.05% TFA in water (mobile phase A) and 0.1% TFA in acetonitrile (mobile phase B) with a three-step linear gradient of holding 5% B in the first 5 min, followed by 5% to 15% B in the next 15 min, and 15% to 40% B in the last 40 min. The LC/MS/MS analysis was accomplished using an automated data acquisition procedure in which a cyclic series of three different scan modes were performed as follows: The data acquisition was conducted using the full scan mode to obtain the most intense peak (signal > 1.5 x 105 counts) as the precursor ion, followed by a high-resolution zoom scan to determine the charge state of the precursor ion and an MS/MS scan to determine the fragment ions of the precursor ion.
Fold recognition and homology modeling
An alignment of the amino acid sequence of the catalytic domain of FucT VII with ß-glucosyltransferase from bacteriophage T4, an enzyme that catalyzes the transfer of glucose from UDP-glucose to DNA, was obtained by aligning FucT VII to the alignment of FucT IV with ß-glucosyltransferase (PDB entry 2bgu) as published by Breton et al. (1996). To obtain alignments of the sequence of the catalytic domain of FucT VII with other protein folds, the 123D threading service, available from the World Wide Web (http://www-lmmb.ncifcrf.gov/~nicka/123D.html), was used with default parameters (Alexandrov et al., 1996
). The (
/ß)8-barrel fold of E. coli dihydrodipicolinate synthase, an enzyme that catalyzes the first step in biosynthesis of lysine in plants and bacteria, represented by PDB entry 1dhp, was used as a template for homology modeling starting from the sequence alignment proposed by the 123D program. Alignments were adjusted manually to place deletions and insertions outside of secondary structure elements. All homology modeling was performed with the Modeler version 4.0 package, distributed with Quanta 97 (MSI, San Diego, CA) using default parameters and without further energy refinement of the structures. For each homology model, five models were generated, of which the one with the lowest error function was used for further analysis. Visualization, secondary structure prediction, and all computations on the homology models were performed with Quanta 97.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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