Neighboring cysteine residues in human fucosyltransferase VII are engaged in disulfide bridges, forming small loop structures

Theodora de Vries1,2, Ten-Yang Yen3, Rajesh K. Joshi3, Janet Storm2, Dirk H. van den Eijnden2, Ronald M. A. Knegtel4, Hans Bunschoten4, David H. Joziasse2 and Bruce A. Macher3

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Among {alpha}3-fucosyltransferases ({alpha}3-FucTs) from most species, four cysteine residues appear to be highly conserved. Two of these cysteines are located at the N-terminus and two at the C-terminus of the catalytic domain. FucT VII possesses two additional cysteines in close proximity to each other located in the middle of the catalytic domain. We identified the disulfide bridges in a recombinant, soluble form of human FucT VII. Potential free cysteines were modified with a biotinylated alkylating reagent, disulfide bonds were reduced and alkylated with iodoacetamide, and the protein was digested with either trypsin or chymotrypsin, before characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry. More than 98% of the amino acid sequence for the truncated enzyme (beginning at amino acid 53) was verified. Mass spectrometry analysis also demonstrated that both potential N-linked sites are occupied. All six cysteines in the FucT VII sequence were shown to be disulfide-linked. The pairing of the cysteines was determined by proteolytic cleavage of nonreduced protein and subsequent analysis by mass spectrometry. The results demonstrated that Cys68–Cys76, Cys211–Cys214, and Cys318–Cys321 are disulfide-linked. We have used this information, together with a method of fold recognition and homology modeling, using the ({alpha}/ß)8-barrel fold of Escherichia coli dihydrodipicolinate synthase as a template to propose a model for FucT VII.

Key words: fucosyltransferase/disulfide bridges/mass spectrometry/homology modeling


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Fucosyltransferases (FucTs) comprise a family of highly homologous enzymes differing in substrate requirements, tissue distribution, and developmental expression (reviewed by Oriol et al., 1999Go). In humans six {alpha}3-FucTs have been cloned (Kukowska-Latallo et al., 1990Go; Goelz et al., 1990Go; Lowe et al., 1991Go; Kumar et al., 1991Go; Weston et al., 1992aGo,b; Koszdin and Bowen, 1992Go; Sasaki et al., 1994Go; Natsuka et al., 1994Go; Kaneko et al., 1999Go): FucTs III (Kukowska-Latallo et al., 1990Go), V (Weston et al., 1992aGo), and VI (Koszdin and Bowen, 1992Go; Weston et al., 1992bGo), the genes of which are all located closely together on chromosome 19 (Reguigne-Arnould et al., 1995Go), are mostly expressed in endocrine tissues and share more than 85% amino acid homology; FucT IV, which is predominantly expressed in myeloid type cells and brain (Goelz et al., 1990Go; Lowe et al., 1991Go; Kumar et al., 1991Go); and FucT VII, of which the expression is limited to leukocytes and high endothelial cells of the venule (Sasaki et al., 1994Go; Natsuka et al., 1994Go). The recently cloned FucT IX is expressed in brain and stomach and is most likely involved in neuronal development and compaction of the embryo (Kaneko et al., 1999Go). In addition, related enzymes have been identified in and cloned from other species, ranging from apes to bacteria (see references in Oriol et al., 1999Go).

We (De Vries et al., 1995Go, 1997; Nguyen et al., 1998Go; Vo et al., 1998Go) and others (Legault et al., 1995Go; Dupuy et al., 1999Go) 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, 1991Go; Wang et al., 1994Go). 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., 1999Go).

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., 1996Go). 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.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1. Alignment (Clustal method) of conserved Cys residues (labeled C) in human {alpha}3-fucosyltransferases.

 

    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Preparation of FucT VII
A soluble form of FucT VII was prepared by replacing the cytoplasmic tail and transmembrane domain by a suitable leader sequence, which directs the protein into the culture medium. Stable CHO transfectants, cultured in serum-free medium, produced FucT VII in good yield (1 mg/L). The protein was purified to homogeneity in one step by affinity chromatography on GDP-hexanolamine-Sepharose in almost quantitative yield. After an initial characterization (including an analysis of acceptor specificity) the protein was used in subsequent reactions and analyses.

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 53–342) was confirmed (results summarized in Figure 2). Only one tripeptide (AA 295–297) was not detected in the tryptic/chymotryptic fragments. No ion signals were detected for any contaminating proteins.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2. Schematic representation of the results obtained by LC/MS/MS analysis of tryptic (shown by line below sequences) and chymotryptic (shown by line above sequences) fragments from FucT VII. Peptides involved in N-glycosylation (Asn80 and Asn291) were also detected, as shown in bold. The sequence shown represents AA 39–342 of FucT VII.

 
FucT VII contains two potential N-linked glycosylation sites (Asn81-Arg-Ser and Asn291-Glu-Ser), and these were demonstrated to be glycosylated using a combination of PNGase F treatment and MS analysis. If FucT VII was digested with trypsin or chymotrypsin without prior PNGase F treatment, peptides containing Asn81 or Asn291 were not detected. However, after PNGase F treatment tryptic peptides (AA 76–82 and AA 282–294), containing Asn81-Arg-Ser and Asn291-Glu-Ser were identified at m/z 858.4 and 1439.6, respectively. These masses are one mass unit larger than expected for each peptide (m/z 857.4 and 1438.7) assuming they each contain an Asn residue but correspond to the correct masses for the peptides after the release of N-linked glycans by PNGase F converts the Asn residues to Asp residues.

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 282–294 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.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. The full scan mass spectrum of the N-linked Asn291 residue containing tryptic peptide (AA 282–294) at m/z 1439.6 for (M+H)+. The inset of zoom scan from m/z 718 to m/z 724 demonstrates that m/z 720.3 is the doubly charged monoisotopic peak for the N-linked containing peptide. The measured (M+H)+ = 1439.6 Da supports the conclusion that Asn was converted to Asp by PNGase F treatment. The ion at m/z 1137.4 is a doubly charge ion for a peptide derived from trypsin.

 
The corresponding MS/MS spectrum of the doubly charged ion at m/z 720.3 is shown in Figure 4A and illustrates the dominant fragment ions observed when fragmentation occurs from the N- (b2–12) and C-terminus (y2–11). Figure 4B presents an expanded view of the spectrum from m/z 930 to 940 and m/z 1044 to 1054 and shows that the N-terminal fragments, b9 and b10, are detected at m/z 934.3 and 1049.2, respectively. The mass difference (m/z 114.9) between the fragments is consistent with peptides of -Met (b9) and -Met-Asp (b10) and provides unambiguous confirmation that the sequence contains -Asp- in place of -Asn-. There was no evidence for any other posttranslational modifications. However, it is possible that a portion of some of the peptide segments in the protein is posttranslationally modified in other ways.




View larger version (42K):
[in this window]
[in a new window]
 
Fig. 4. (A) The corresponding MS/MS spectrum at m/z 720.3 shows both b- and y- (bn, n = 2–12; yn, n = 2–11) fragments were dominant fragment ions. The inset spectra (B) from m/z 930 to 940 and from (C) m/z 1044 to 1054 show fragments b9 (m/z 934.3) and b10 (m/z 1049.2), respectively. This result confirms the presence of -Asp- instead of -Asn- in the sequence (mass difference of 114.9 Da between -Met and -Met-Asp).

 
Monosaccharide composition analysis
The glycans derived from the recombinant FucT VII produced in CHO cells were analyzed as alditol acetates using gas–liquid chromatography/mass spectrometry (Savage et al., 1986Go). It was demonstrated that the protein contains fucose, galactose, mannose, N-acteylgalactosamine, N-acetylglucosamine, and N-acetylneuraminic acid, in a ratio as shown in Table I. The ratio of monosaccharides suggests complex type N-linked glycans, with the majority of structures consisting of sialylated, fucosylated, biantennary carbohydrate chains, in addition to a few O-linked glycans.


View this table:
[in this window]
[in a new window]
 
Table I. Monosaccharide composition of recombinant FucT VII produced in CHO cells. Numbers are expressed as nmol monosaccharide/200 µg glycoprotein
 
Identification of disulfide-bonded Cys residues
FucT VII has a total of six Cys residues, the four highly conserved Cys residues shown in Figure 1, and two closely spaced residues (Cys211 and Cys214) located in the middle of the catalytic domain. Potentially free Cys residues in FucT VII were modified with a biotinylated form of iodoacetamide, whereas those involved in disulfide bonds were reduced and alkylated. Peptide fragments were generated with trypsin or chymotrypsin, separated by high-performance liquid chromatography (HPLC), and characterized by ESI/MS analysis.

None of the peptide fragments was found to be modified by the biotin probe, however two peptides (AA 205–223 and 317–329), 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 76–82) 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 53–71) 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 Cys211–Cys214 and a tryptic peptide containing Cys318–Cys321. Figure 5 shows the full scan mass spectrum of the tryptic disulfide containing peptide (AA 317–329) 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 = 8–12; yn, n = 2–6), confirming the peptide sequence with a disulfide bond (AA 317–329).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. The full scan mass spectrum of the tryptic peptide AA 317–329 containing the disulfide linked residues Cys 318 and Cys 321 at m/z 795.1. The inset of zoom scan from m/z 794 to m/z 798 demonstrates that m/z 794.9 is the doubly charged monoisotopic peak for the tryptic disulfide-containing peptide. The ion at m/z 1137.4 is a doubly charged ion for a peptide derived from trypsin.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 8. The MS/MS spectrum from m/z 1252.4 contains dominant b-terminus ions (bn, n = 9–12) and y6. The presence of b-type fragments at m/z positions corresponding to masses of 2 less than would be predicted for the peptide with free Cys residues supports the assignment of a sequence for a disulfide-containing peptide.

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6. The corresponding MS/MS spectrum at m/z 795 shows both b- and y- (bn, n = 8–12; yn, n = 2–6) dominant fragment ions, confirming the sequence and the existence of disulfide linkage within the peptide (AA 317–329).

 
Figure 7 shows the full scan mass spectrum of the disulfide containing (Cys211–Cys214) chymotryptic peptide AA 211–222. The inset zoom scan shows a dominant singly charged ion at m/z 1252.4. This measured (M+H)+ = 1252.4 Da is very close to the calculated (M+H)+ = 1252.5 Da of the peptide (AA 221–222) with a disulfide linkage between Cys211 and Cys214. The MS/MS spectrum of m/z 1252.4 (Figure 8) is dominated by b-terminus ions (bn, n = 4–12 but not n = 7), and y6; thus the sequence of this disulfide-containing peptide was unambiguously confirmed by the MS/MS analysis.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. The full scan mass spectrum of the chymotryptic peptide AA 211–222 containing the disulfide-linked residues Cys 211–Cys 214 at m/z 1252.4. The inset of zoom scan from m/z 794 to m/z 798 demonstrates that m/z 1252.4 is the singly charged monoisotopic peak for the tryptic disulfide-containing peptide.

 
Because the peptide containing the other Cys (Cys76) residue is close to a potential N-linked glycosylation site (Asn81) in FucT VII and no information concerning the structure of the oligosaccharide is available, it was difficult to detect the third disulfide-containing peptide (Cys68–Cys76). Although it should be possible to detect a disulfide-containing chymotryptic peptide without the N-linked site (cleavage at Phe56, Tyr71, and Leu78 to obtain the disulfide bond-containing peptides AA 57–71 and AA 72–78), we were unable to detect it. We speculate that this was due to the presence of the oligosaccharide that prevented cleavage of the peptide bond. Consistent with this hypothesis, we were able to identify peptides containing the disulfide-linked Cys residues following PNGase F treatment of FucT VII. Figure 9 shows the full scan mass spectrum of the disulfide-containing peptides (AA 57–71 and AA 72–78) obtained from FucT VII, which was treated with PNGase F prior to chymotryptic digestion. The inset zoom scan spectrum clearly shows that the peak at m/z 830.4 is the monoisotopic peak of a triply charged ion (a 0.3-Da difference between the monoisotopic peak and the peak containing 13C). Thus, the measured (M+H)+ of the peptide is equal to 2489.2 Da, which matches the calculated (M+H)+ = 2489.2 Da predicted for the disulfide-linked peptide. The MS/MS spectrum (Figure 10) of the triply charged ion at m/z 830.4 is more complicated to assign than those of singly and doubly charged ion because singly, doubly, and triply charged fragments are all present in the MS/MS spectrum. However, the dominant fragments observed (yn, n = 5, 8–12, and 14; and b15 fragments) for the Cys68-containing peptide (AA 57–71) in combination with the y*7 and b*6 fragments of the Cys76-containing peptide (AA 72–78) in the spectrum strongly support the assignment of this disulfide-bonded (Cys68–Cys76) peptide.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 9. The full scan mass spectrum of the chymotryptic peptides (AA 57–71 and AA 72–78) obtained from FucT VII pretreated with PNGase F. The inset zoom scan spectrum demonstrates that the peak at m/z 830.4 is the monoisotopic peak of a triply charged ion. Ions detected at m/z 661.7 and 991.7 are unidentified peaks.

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 10. The MS/MS spectrum generated from the ion at m/z 830.4 (peptides AA 57–71 and AA 72–78) shows mixed doubly and triply charged fragments dominated the spectrum. The inset illustrates the assignment of most of the major fragments, supporting the sequence of the disulfide-containing (Cys68–Cys76) peptides.

 
Fold recognition/threading and homology modeling
Several possible protein folds were returned when a search for FucT VII was done with the 123D fold-recognition program. Visual inspection of the highest-ranking three folds (PDB entries 1onr, 1eny, and 1iow) showed that the positions of the conserved Cys residues of the aligned FucT VII would not allow disulfide bond formation for the three adjacent Cys pairs on the basis of their mutual distances in these structures. The 123D fold recognition program ranked the ({alpha}/ß)8-barrel fold of Escherichia coli dihydrodipicolinate synthase (1dhp) as the fourth most likely fold for FucT VII. Because Breton et al. (1996)Go have presented a model for FucT IV based on the crystal structure of bacteriophage T4 ß-glucosyltransferase (2bgu), this structure was also evaluated as a model for FucT VII. The final alignments used for homology modeling of the FucT VII catalytic domain with either 2bgu or dihydrodipicolinate synthase (1dhp) are shown in Figure 11. The alignment of FucT VII with 2bgu shows that two ß-sheets are deleted in the FucT VII model and that an insertion is positioned near the C-terminus. There is only limited correspondence between the secondary structure elements observed in 2bgu and those predicted for FucT VII. A better correspondence between predicted and observed secondary structure is found for the alignment between FucT VII and 1dhp, even though some regions predicted to form ß-sheets are still aligned with {alpha}-helices in the known structure. In the model based on the 2bgu fold, the first N-terminal Cys68 could not be mapped onto the available structure. It is located at the start of a ß-strand in which its partner, C76, also is located (see Figure 11).




View larger version (57K):
[in this window]
[in a new window]
 
Fig. 11. Sequence alignments used for homology modeling of the catalytic domain of FucT VII. The top panel shows the final alignment against the sequence of ß-glucosyltransferase (taken from PDB entry 2bgu). The bottom panel shows the final alignment with E. coli dihydrodipicolinate synthase (PDB entry 1dhp). For the crystal structures of 2bgu and 1dhp the observed secondary structure is indicated with circles for {alpha}-helical segments and triangles for ß-sheets. For the FucT VII sequence, the predicted secondary structure elements are indicated in a similar fashion.

 
Due to the low degree of sequence identity between FucT VII and either 2bgu or 1dhp, it is difficult to obtain accurate models for FucT VII. Nevertheless, low-resolution models may offer some insight into the possible placement of the conserved Cys residues in the 3D context of the {alpha}- and ß-fold of 2bgu (Figure 12, left side) and the ({alpha}/ß)8-barrel of 1dhp (Figure 12, right side). As shown in Figure 12 (right side), the 1dhp fold allows for a much closer proximity of covalently bonded Cys residues, than the 2bgu model does. In our model, the distances between the Cß-atoms of Cys68–Cys76, Cys211–Cys214, and Cys321–Cys328 are 4.6, 6.2, and 6.5 Å, respectively. Two of the Cys pairs (68–76 and 211–214) are located in loop regions. The third pair is located at the N-terminus of the last {alpha}-helix in the model, after which the 1dhp structure no longer provides a scaffold for FucT VII. The Cß–Cß distances are within the range of 4.0–7.5 Å reported by Richardson (1981)Go. In the case of the 2bgu fold, these distances are > 14, 11.3, and 7.2 Å, respectively.



View larger version (82K):
[in this window]
[in a new window]
 
Fig. 12. Position of conserved Cys residues in two structural models for the catalytic domain of FucT VII. The left panel shows the model based on the 2bgu structure as a ribbon with the conserved Cys residues as Van der Waals spheres. The nucleotide binding site is indicated by UDP in similarity to its binding in ß-glucosyltransferase in the 2bgu structure. The right panel provides a similar representation of the model based on the 1dhp structure.

 
The structure of the recently crystallized bovine ß4-galactosyltransferase I (Gastinel et al., 1999Go) presents a new fold, which was not part of the database used for threading in this study. Because this protein is a member of the glycosyltransferase family, the suitability of this fold for FucT VII was examined as well. By aligning the amino acid sequence of ß4-GalT with the human FucTs, using Clustal W, the putative positions of the FucT VII Cys residues could be mapped to the GalT fold. However due to the low sequence identity, the alignments obtained were treated as providing only a rough estimate. The first Cys pair of FucT VII aligned with residues just before the N-terminus of the crystal structure (starting at residue Leu131), therefore its position could not be evaluated from the current crystal structure. The second Cys pair aligned with a loop region surrounding Asp260 of GalT, which connects two ß-sheets. In this instance, the Cys residues could be positioned to form a disulfide bond, but this depends strongly on the exact local alignment. The larger part of this region is in an extended conformation that excludes formation of disulfide bonds. The third Cys pair, finally, is aligned with an extended region near Tyr380, causing the Cys Cß-atoms to be more than 10 Å apart. The formation of a disulfide bond in this region of the GalT structure seems therefore very unlikely. In conclusion, the ß4-GalT fold appears to be different from that of the fucosyltransferases.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cloning of human FucTs has provided information on the predicted amino acid sequences and potential N-linked glycosylation sites. Subsequent studies have gathered information on the functional significance of some of the amino acids in the primary sequence and have verified that the FucTs are decorated with oligosaccharides at some of the Asn residues. Thus, Lowe and colleagues (Weston et al., 1992aGo) demonstrated that FucT III contains N-linked oligosaccharides and we have demonstrated that at least one of the four potential N-linked sites in FucT V is not glycosylated when the enzyme is expressed in COS cells (Nguyen et al., 1998Go). We (Nguyen et al., 1998Go; Vo et al., 1998Go), Lowe and colleagues (Legault et al., 1995Go), and Oriol and colleagues (Dupuy et al., 1999Go) have also identified specific amino acids that either affect acceptor substrate specificity or lie in or near the binding site for GDP-Fuc. Additionally, a segment of the amino acid sequence of all FucTs cloned to date (originating from mammals, chickens, nematodes, and bacteria) contains a set of highly conserved residues (Breton et al., 1998Go) and we have demonstrated that one of these (Lys255 of FucT V) is important for catalytic activity (Sherwood et al., 1998Go). Based on the results obtained to date, it is possible to propose that certain segments of the primary sequence of FucTs must fold in a manner that brings them close to one another. For example, the identification of amino acids at both the N- and C-termini of the catalytic domain of FucT III and V, which affect acceptor substrate specificity, suggests that these two segments of the amino acid sequence must be brought together in the native protein (Vo et al., 1998Go). Folding of proteins is determined in part by the occurrence of disulfide bridges between Cys residues throughout the molecule. These disulfide bridges are required to maintain stable protein geometry. Only recently have studies been done to determine the distribution of free Cys residues and disulfide-linked Cys residues in glycosyltransferases (Yen et al., 2000Go; Holmes et al., 2000Go; Li et al., 2000Go).

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 (Cys68–Cys76, Cys211–Cys214, and Cys318–Cys321). 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 {alpha}7 receptor (Servent et al., 1997Go).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 13. Possible models of FucT VII structures based on different disulfide bonding patterns.

 
Determination of disulfide linkages by protein chemical modification can result in an incorrect assignment. For example, the original disulfide bond analysis of bovine ß4-GalT (Yadav and Brew, 1991Go; Wang et al., 1994Go) was challenged when the crystal structure of this enzyme was solved recently (Gastinel et al., 1999Go). Surprisingly, Gastinel (1999)Go identified two disulfide bridges in the GalT structure, Cys134–Cys176 and Cys247–Cys266, in contrast to a single disulfide bond between Cys134 and Cys247 proposed previously (Yadav and Brew, 1991Go; Wang et al., 1994Go). We (Yen et al., 2000Go) have recently shown that the discrepancy between the disulfide bonding assignments from chemical versus crystal structure is due to the propensity for thio-disulfide exchange reactions to occur in proteins with both disulfide-bonded and free Cys residues. Our procedure (Yen et al., 2000Go), which was designed to ensure that free Cys residues are alkylated as the protein is denatured and thus minimizes thio-disulfide exchange from occurring, verifies the disulfide bond pattern identified in the crystal structure of ß4-GalT (Gastinel et al., 1999Go). In the case of FucT VII, there are no free cysteines; thus, exchange reactions are unlikely to occur. Finally, in contrast to the methods used in the earlier chemical study of ß4-GalT (Yadav and Brew, 1991Go), we have directly demonstrated the disulfide bond pattern by sequencing the peptide pairs. Because our methods have been validated with a series of proteins containing both free and disulfide-bonded cysteine residues, we expect that the results presented here will be substantiated when a crystal structure is solved for FucT VII.

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 ({alpha}/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)Go. The ({alpha}/ß)8-barrel fold is observed in a number of glycosyl hydrolases, amylases, and other carbohydrate processing or binding proteins (Davies and Henrissat, 1995Go) and was also found in the fold-recognition searches performed by Breton et al. (1996)Go.

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 {alpha}3-motifs (Breton et al., 1998Go) 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 ({alpha}/ß)8-barrel model, close to where the active sites of other carbohydrate processing enzymes are located (Davies and Henrissat, 1995Go). These are residues Glu62, Asp146, Asp200, Asp235, and Glu239 of FucT VII.

Our results indicate that an ({alpha}/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 ({alpha}/ß)8-barrel fold seems more likely than the {alpha}- and ß-type fold of ß-glucosyltransferase proposed by Breton et al. (1996)Go. Although our model is of low resolution and likely to be incorrect in details, it strongly suggests a fold consisting of alternating {alpha}-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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Unlabeled GDP-fucose was purchased from Boehringer Mannheim (Indianapolis, IN). GDP-[14C]Fuc (250 Ci/mol) was purchased from New England Nuclear Corp. (Boston, MA) and diluted with the unlabeled nucleotide sugar to obtain a specific radioactivity of 5 Ci/mol. Iodo-LC-biotin was purchased from Pierce Chemical Co. PNGase F and chymotrypsin were purchased from Boehringer Mannheim and trypsin was obtained from Worthington Biochemical Corp. (Lakewood, NJ). All other chemicals were obtained from commercial sources and were of the highest purity available.

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., 1997Go).

Modification of Cys residues
Forty micrograms of FucT VII (4 mg/ml in 0.2 M Tris–HCl, 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., 1986Go). 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)Go. 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., 1996Go). The ({alpha}/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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. L. N. Gastinel of the Architecture et Function des Macromolecules Biologiques laboratorium of the CNRS in Marseille, France, for kindly making the coordinates of bovine ß4-galactosyltransferase I available to us prior to our submission to the PDB. We thank Carolien Koeleman for the analysis of the monosaccharide composition. This research was supported by the Technology Foundation STW, applied science division of NWO, and the technology program of the Ministry of Economic Affairs Grant # 349-4211 to D.H.J. and D.H.v.d.E. and by the National Science Foundation Grants MCB-9513722 and MCB-9816780 to B.A.M., a National Center for Research Resources (Research Infrastructure in Minority Institutions) Grant P20 RR11805 with funding from the Office of Research on Minority Health to B.A.M.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
AA, amino acid; 2bgu, ß-glucosyltransferase; 1dhp, dihydrodipicolinate synthase; ESI, electrospray ionization; FucT, fucosyltransferase; GalT, galactosyltransferase; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Alexandrov, N., Nussinov, R., and Zimmer, R. (1996) Fast protein fold recognition via sequence to structure alignment and contact capacity potentials. In Hunter, L., and Klein, T.-E, (eds.), Pacific Symposium on Biocomputing ’96, World Scientific Publishing Co., Singapore, pp. 53–72.

Breton, C., Oriol, R., and Imberty, A. (1996) Sequence alignment and fold recognition of fucosyltransferases. Glycobiology, 6, vii–xii.[Medline]

Breton, C., Oriol, R., and Imberty, A. (1998) Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology, 8, 87–94.[Abstract/Free Full Text]

Davies, G., and Henrissat, B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure, 3, 853–859.[ISI][Medline]

De Vries, T., Srnka, C.A., Palcic, M.M., Swiedler, S.J., Van den Eijnden, D.H., and Macher, B.A. (1995) Acceptor specificity of different length constructs of human recombinant {alpha}1, 3/4-fucosyltransferases. Replacement of the stem region and the transmembrane domain of fucosyltransferase V by protein A results in an enzyme with GDP-fucose hydrolyzing activity. J. Biol. Chem., 270, 8712–8722.[Abstract/Free Full Text]

De Vries, T., Palcic, M.P., Schoenmakers, P.S., Van den Eijnden, D.H., and Joziasse, D.H. (1997) Acceptor specificity of GDP-Fuc:Gal ß1, 4GlcNAc-R {alpha}3-fucosyltransferase VI (FucT VI) expressed in insect cells as soluble, secreted enzyme. Glycobiology, 7, 921–927.[Abstract]

Dupuy, F., Petit, J.M., Mollicone, R., Oriol, R., Julien, R., and Abderrahman, M. (1999) A single amino acid in the hypervariable stem domain of vertebrate {alpha}1, 3/4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis. J. Biol. Chem., 274, 12257–12262.[Abstract/Free Full Text]

Gastinel, L.N., Cambillau, C., and Bourne, Y. (1999) Crystal structures of the bovine ß4-galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose. EMBO J., 18, 3546–3557.[Abstract/Free Full Text]

Goelz, S.E., Hession, C., Goff, D., Giffiths, B., Tizard, R., Newman, B., Chi Rosso, G., and Lobb, R. (1990) ELFT: A gene that directs the expression of an ELAM-1 ligand. Cell, 63, 1349–1356.[ISI][Medline]

Holmes, E.H., Yen, T-Y., Thomas, S., Joshi, R.K., Nguyen, A., Long, T., Gallet, F., Maftah, A., Julien, R., and Macher, B.A. (2000) Human {alpha}3/4 Fucosyltransferases: Characterization of highly conserved cysteine residues and N-linked glycosylation sites. J. Biol. Chem., 275, 24237–24245.[Abstract/Free Full Text]

Kaneko, M., Kudo, T., Iwasaki, H., Ikehara, Y., Nishihara, S., Nakagawa, S., Sasaki, K., Shiina, T., Inoko, H., Saitou, N., and Narimatsu, H. (1999) {alpha}1, 3-fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX. FEBS Lett., 452, 237–242.[ISI][Medline]

Koszdin, K.L., and Bowen, B.R. (1992) The cloning and expression of a human {alpha}1, 3-fucosyltransferase capable of forming the E-selectin ligand. Biochem. Biophys. Res. Commun., 187, 152–157.[ISI][Medline]

Kukowska-Latallo, J.F., Larsen, R.D., Nair, R.P., and Lowe, J.B. (1990) A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group {alpha}(1-3/4)FT. Genes Dev., 4, 1288–1303.[Abstract]

Kumar, R., Potvin, B., Muller, W.A., and Stanley, P. (1991) Cloning of a human {alpha}(1, 3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on chinese hamster ovary cell transfectants. J. Biol. Chem., 266, 21777–21783.[Abstract/Free Full Text]

Legault, D.J., Kelly, R.J., Natsuka, Y., and Lowe, J.B. (1995) Human {alpha}(1, 3/1, 4)-fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment. J. Biol. Chem., 270, 20987–20996.[Abstract/Free Full Text]

Li, J., Yen, T-Y., Allende, M.L., Joshi, R.K., Cai, J., Pierce, W.M., Jaskiewicz, E., Darling, D.S., Macher, B.A., and Young, W.W. (2000) Disulfide bonds of GM2 synthase homodimers: Antiparallel orientation of the catalytic domains. J. Biol. Chem., in press.

Lowe, J.B., Kukowska-Latallo, J.F., Nair, R.P., Larsen, R.D., Marks, R.M., Macher, B.A., Kelly, R.J., and Ernst, L.K. (1991) Molecular cloning of a human fucosyltransferase gene that determines expression of the LewisX and VIM-2 epitopes but not ELAM-1 dependent cell adhesion. J. Biol. Chem., 266, 17467–17477.[Abstract/Free Full Text]

Maly, P., Thall, A., Petryniak, B., Rogers, C.E., Smith, P.L., Marks, R.M., Kelly, R.J., Gersten, K.M., Cheng, G., Saunders, T.L., and others (1996) The {alpha}(1, 3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell, 86, 643–653.[ISI][Medline]

Natsuka, S., Gersten, K.M., Zenita, K., Kannagi, R., and Lowe, J.B. (1994) Molecular cloning of a cDNA encoding a novel human leukocyte {alpha}1, 3-fucosyltransferase capable of synthesizing the sialyl LewisX determinant. J. Biol. Chem., 269, 16789–16794.[Abstract/Free Full Text]

Nguyen, A.T., Holmes, E.H., Whitaker, J.M., Ho, S., Shetterly, S., and Macher, B.A. (1998) Human {alpha}1, 3/4-fucosyltransferases. I. Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis. J. Biol. Chem., 273, 25244–25249.[Abstract/Free Full Text]

Oriol, R., Mollicone, R., Cailleau, A., Balanzino, L., and Breton, C. (1999) Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria. Glycobiology, 9, 323–334.[Abstract/Free Full Text]

Reguigne-Arnould, I., Couillin, P., Mollicone, R., Faure, S., Fletcher, A., Kelly, R.J., Lowe, J.B., and Oriol, R. (1995) Relative positions of two clusters of human {alpha}-L-fucosyltransferases in 19q (FUT1-FUT2) and 19p (FUT6-FUT3-FUT5) within the microsatellite genetic map of chromosome 19. Cytogenet. Cell Genet.,71, 158–162.[ISI][Medline]

Richardson, J. (1981) The anatomy and taxonomy of protein structure. Adv. Protein Chem., 34, 167–339.[Medline]

Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) Expression cloning of a novel {alpha}1, 3-fucosyltransferase that is involved in biosynthesis of the sialyl LewisX carbohydrate determinants in leukocytes. J. Biol. Chem., 269, 14730–14737.[Abstract/Free Full Text]

Savage, A.V., Koppen, P.L., Schiphorst, W.E.C.M., Trippelvitz, L.A., Van Halbeek, H., Vliegenthart, J.F.G., and Van den Eijnden, D.H. (1986) Porcine submaxillary mucin contains {alpha}2-3- and {alpha}2-6 linked N-acetyl- and N-glycolylneuraminic acid. Eur. J. Biochem., 160, 123–129.[Abstract]

Servent, D., Winckler-Dietrich, V., Hu, H.Y., Kessler, P., Drevet, P., Bertrand, D., and Menez, A. (1997) Only snake curaremimetic toxins with a fifth disulfide bond have high affinity for the neuronal {alpha}7 nicotinic receptor. J. Biol. Chem., 272, 24279–24286.[Abstract/Free Full Text]

Sherwood, A.L., Nguyen, A.T. Whitaker, J.M., Macher, B.A., and Holmes, E.H. (1998) Human {alpha}3/4-fucosyltransferases. III. A Lys/Arg residue located within the {alpha}3-FucT motif is required for activity but not substrate binding. J. Biol. Chem., 273, 25256–25260.[Abstract/Free Full Text]

Vo, L., Lee, S., Marcinko, M.C., Holmes, E.H., and Macher, B.A. (1998) Human {alpha}1, 3/4-fucosyltransferases. II. A single amino acid at the COOH terminus of FucT III and V alters their kinetic properties. J. Biol. Chem., 273, 25250–25255.[Abstract/Free Full Text]

Wang, Y., Wong, S.S., Fukuda, M.N., Zu, H., Liu, Z., Tang, Q., and Appert, H.E. (1994) Identification of functional cysteine residues in human galactosyltransferase. Biochem. Biophys. Res. Commun., 204, 701–709.[ISI][Medline]

Weston, B.W., Nair, R.P., Larsen, R.D., and Lowe, J.B. (1992a) Isolation of a novel human {alpha}(1, 3)fucosyltransferase gene and molecular comparison to the human Lewis blood group {alpha}(1, 3/1, 4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct substrate specificity’s. J. Biol. Chem., 267, 4152–4160.[Abstract/Free Full Text]

Weston, B.W., Smith, P.L., Kelly, R.J., and Lowe, J.B. (1992b) Molecular cloning of a fourth member of a human {alpha}(1, 3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the LewisX, sialyl LewisX, and difucosyl sialyl LewisX epitopes. J. Biol. Chem., 267, 24575–24584.[Abstract/Free Full Text]

Yadav, S.P., and Brew, K. (1991) Structure and function in galactosyltransferase. Sequence locations of {alpha}-lactalbumin binding site, thiol groups, and disulfide bonds. J. Biol. Chem., 266, 698–703.[Abstract/Free Full Text]

Yen, Y-T., Joshi, R.K., Yan, H., Seto, N.O.L., Palcic, M.P., and Macher, B.A. (2000) Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. J. Mass Spectrom., 35, 990–1002.[ISI][Medline]