Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn,Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany
Received on November 22, 2000; revised on January 16, 2001; accepted on March 12, 2001.
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Abstract |
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For the analysis of the disulfide bond structure, the protein was proteolytically digested and the resulting peptides were analyized by MALDI-MS. The analysis revealed three disulfide bonds (C91C137; C309C360; C534C551) and a free cysteine (C487). The analysis of the N-glycosylation was performed by tryptic digestion of the protein, isolation of glycopeptides by lectin chromatography and mass measurement before and after enzymatic deglycosylation. Carbohydrate structures were calculated from the mass difference between glycosylated and deglycosylated peptide. For ß-hexosaminidase B from human placenta, four N-glycans were identified and analyzed, whereas the recombinant protein expressed in SF21 cells carried only three glycans. In both cases the glycosylation belongs to the mannose-core- or high-mannose-type, and some carbohydrate structures are fucosylated.
Key words: hexosaminidase B/Sandhoff disease/disulfide bonds/MALDI-MS/N-glycans
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Introduction |
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Both subunits are synthesized as glycosylated precursor proteins that dimerize in the Golgi apparatus (Proia et al., 1984) and reach the lysosome by the mannose-6-phosphate pathway (Sonderfeld-Fresko and Proia, 1989
). The ß subunit contains five potential N-glycosylation sites. In a study using site-directed mutagenesis, it has been demonstrated that four of these are utilized (Sonderfeld-Fresko and Proia, 1989
). The glycans connected to three N-glycosylation sites in the mature lysosomal HexB have been analyzed by nuclear magnetic resonance (NMR) spectroscopy (O'Dowd et al., 1988
).
In the lysosome, the proteins are subjected to proteolytic processing. In the ß subunit, a part of the N-terminal sequence is removed along with two internal peptides, leading to a mature enzyme consisting of three peptides ßp, ßa, and ßb, which remain connected by disulfide bond linkages (Mahuran et al., 1988). Because the protein contains seven cysteine residues, a maximum of three disulfide bonds can be formed. The only one of these bonds which has been localized so far is the linkage C309C360, which connects the peptides ßa and ßb (Sagherian et al., 1993
). The connectivity of the remaining five cysteines is so far unknown. The importance of the disulfide bond connectivity for the enyzmatic function is indicated by the fact that a missense mutation that leads to the severe infantile form of Sandhoff disease causes an exchange of cysteine 534 to tyrosine (Kuroki et al., 1995
) and therefore results in the loss of one disulfide bond.
Here, we describe the complete analysis of the disulfide bond structure of HexB. Additionally the glycosylation is analyzed completely by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In respect to the glycosylation, the mature HexB isolated from human placenta (plHexB) is compared to the recombinant protein expressed in SF21 cells using the baculovirus expression system (bcHexB).
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Results |
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Identification of nontryptic peptides
The MALDI-MS of the tryptic digest of pure HexB showed several masses that could not be assigned to tryptic peptides but were present in all preparations measured. This was suprising because of the high purity of the protein as demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (data not shown). For identification, several of these peptides were isolated by RP-HPLC and sequenced by MALDIpost source decay (PSD). Their sequences correspond to HexB sequences, with at least one terminus produced by a nontryptic cleavage. During the course of the investigation, 30 such peptides were identified (Figure 3, Table IV). For reference, they were named X with a number indicating their position in the sequence. Their generation may be due to a contamination of trypsin by chymotrypsin as proposed by O'Dowd et al. (1988). But because all attempts to suppress this activity by using different preparations of trypsin, shorter incubation times, or less protease were unsuccessful, no final explanation for their existence can be given. Figure 4 shows the sequence and PSD spectra of three of these peptides (X7, X13, and X16), which contain N-glycosylation sites and are therefore of interest for the analysis of the glycoforms.
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Because both tryptic peptides which should be involved in this bond carry a N-glycosylation site (Figure 1) and several nontryptic cleavages were identified is this region (Figure 3), a heterogeneous mixture of disulfide bonded species had to be expected. To isolate this mixture from the tryptic digest, concanavalin A (Con A) chromatography was performed on the tryptic peptides produced from untreated plHexB. The eluate of the Con A column was separated by RP-HPLC. The RP-HPLC elution profile was compared to that of a sample treated identically but reduced prior to digestion. The elution profile of the untreated protein showed two signals that were not present in the profile of the reduced protein. In the fraction corresponding to one of these signals, a mixture of species with masses in the range of 70008500 Da was detected (data not shown). This corresponds to the mass range expected for glycosylated, disulfide bonded species containing C91 and C137. After deglycosylation by peptide N-glycanase F (PNGase F), the complexity of the mixture was reduced and the mass range was shifted to 40005000 Da. These species disappear on reduction, giving rise to a mixture of nontryptic peptides (Figure 5, Table V) that had been identified previously. It was possible to assign all masses detected in the mass range of 40005000 Da to disulfide-bonded species. Each of these was identified as a combination of nontryptic peptides, one of which contains C91 and the other contains C137 (Table V). One of the major disulfide-bonded species corresponds to the combination of X7 and X13. The PSD spectra that led to the identification of these two nontryptic peptides are shown in Figure 4. These results confirm the postulated existence of a disulfide bond between C91 and C137.
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The resulting glycoforms that were deduced from the spectra are presented in Table VI and summarized in Table VII. The carbohydrate connectivites displayed in Table VI are putative. Because MS does not give information on connectivites, structures were drawn in analogy to known N-glycosylation structures (Kornfeld and Kornfeld, 1985). For fucose-containing structures, a terminal
1,3-connected mannose was assumed, which is in accordance with the known specificities of fucosyltransferases in man and insect cells (Staudacher et al., 1999
).
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The major signal in the spectrum of this glycopeptide corresponds in mass to a nonfucosylated high-mannose type glycan (see Figure 6D). This glycan is described here for the first time.
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Discussion |
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The availability of the complete disulfide bond structure should make structure predictions by molecular modeling more reliable in the future and makes it possible to compare HexB to homologous proteins like the chitobiase (ChB) from Serratia marcencens. As the human hexosaminidases, this protein protein belongs to family 20 of glycosyl hydrolases. It is the only protein of this family for which a crystal structure exsists and shows a high homology to HexA and HexB in its central region, which contains the active site. A homology model for the active domain of HexA has been proposed on the basis of this structure (Tews et al., 1996a). Like HexB, ChB contains three disulfide bonds that are arranged sequentially along the sequence. The catalytic domain of both proteins contains one disulfide bond (C505C578 in ChB [Tews et al., 1996b
] and C309C360 in HexB). Although the positions of the cysteines are not exactly conserved, they show only a slight shift in their position and span a homologous sequence of similar length containing the catalytically active glutamate in both proteins (Glu540 in ChB [Tews et al., 1996a
], Glu 355 in HexB [Pennybacker et al., 1997
; Liessem et al., 1995
]).
The position of the remaining two disulfide bonds does not seem to be equivalent in ChB and HexB, though. Although ChB contains two disulfide bonds which span short sequences N-terminal to the catalytic domain, HexB contains just one bond in this region that spans a much longer sequence containing the processing site between the ßp- and the ßb-peptide. The disulfide bond at the very C-terminus of HexB does not find an equivalent in ChB either, because ChB does not contain any disulfide bonds C-terminal of the catalytic domain. Whether these two bonds stabilize regions of HexB that perform tasks specific for the human lysosomal hexosaminidases, for example the formation of dimers, can only be speculated on at the moment. A mutation that exchanges Cys534 to Tyr and therefore abolishes the C-terminal disulfide bond leads to a severe infantile form of Sandhoff disease with a complete loss of HexB activity (Kuroki et al., 1995). This demonstrates the importance of this disulfide bond for the function of human HexB.
The glycosylation of the protein purified from human placenta had been analyzed previously by O'Dowd et al. (1988) using NMR spectroscopy. This method allows an exact analysis of connectivities between sugars; the exact number of identical sugars with identical connectivites is hard to determine, though. This is not a problem with MALDI-MS. It is therefore not suprising that we detected a higher heterogeneity in the carbohydrate structures than was reported by O'Dowd et al. (1988)
. One major difference between our results and the glycan structures published by O'Dowd et al. (1988)
is the glycosylation on N327. In their study they reported only the unusal glycosylation by a single GlcNAc residue. This result was confirmed by our data, but we found an additional major glycan of the high-mannose type in this position. Moreover, the glycan structure of N84 was not determined by O'Dowd et al. (1988)
, because the ßp-peptide is lost in the purification procedure they used. This glycan was analyzed for the first time in the study presented here and was found to be a partially fucosylated high-mannose type glycan.
The combination of proteinchemical methods and MALDI-MS which has been used to determine the disulfide bond linkages and the glycosylation of HexB is extremely sensitive and especially suited for the analysis of complex mixtures. Although it has been used for similar purpose for some time (Yazdanparast et al., 1987; Carr and Roberts, 1986
), the special properties of the lysosomal protein HexB gave rise to some new problems that have been solved in the work presented here. The extreme stability of HexB against proteolytic enzymes made it necessary to use high concentrations of proteases. This led to an unspecific cleavage in addition to the expected specific proteolysis. Although this phenomenon had been described previously for HexB (O'Dowd et al., 1988
), it had never been analyzed in detail. We successfully identified the products of the unspecific cleavage by MALDI-PSD sequencing and were therefore able to determine the localization of the disulfide bonds and the glycan structures. These results may facililtate the analysis of posttranslational modification in lysosomal proteins by MALDI-MS in the future.
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Materials and methods |
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Cation exchange chromatography:
The flowthrough of the Octylsepharose chromatography was extensively dialyzed against ion exchange buffer (10 mM phosphate buffer, pH 6.0) and applied to a POROS HS column (4.6 x 10 mm, PerSeptive) at a flow rate of 10 ml/min. The column was washed with 15 column volume (CV) of ion exchange buffer. Elution was performed using a gradient of 01000 mM NaCl in 10 CV of ion exchange buffer and keeping up 1000 mM NaCl for an additional 3 CV. Fractions were tested for HexB activity using the synthetic substrate MUG as described (Pennybacker et al., 1996). Fractions containing HexB were collected and pooled.
Desalting by gel filtration:
The column (HiPrep Desalting 26/10, Pharmacia) was equilibrated in 5 CV of affinity buffer (10 mM phosphate, pH 6.0, 100 mM NaCl) at 15 ml/min. Fifteen milliliters of eluate of the cation exchange chromatography were applied to the column and eluted in 1.7 CV of affinity buffer.
Preparation of affinity gel:
Five grams of bromcyan-activated Sepharose 4 B (Sigma) were swollen in 1 mM HCl and washed with 1 L of 1 mM HCl. After removing the washing solution by suction filtration the gel was added quickly to a solution of 25 mg of 2-acetamido-N-(-aminocaproyl)-2-deoxy-ß-D-glucopyranosylamine (Sigma) in coupling buffer (100 mM NaHCO3/NaOH, pH 10.0). The mixture was shaken at 4°C over night. After removing the coupling solution by suction filtration the gel was washed several times with changes of coupling buffer and washing buffer (100 mM citrate buffer, pH 4.0) and was finally stored in affinity buffer.
Affinity chromatography:
A column containing 20 ml of affinity gel (18 mm x 80 mm) was equilibrated in affinity buffer and loaded with desalted eluate of the cation exchange chromatography (4 U/ml gel) at 2 ml/min. The column was washed with 2.5 CV of affinity buffer and elution was performed using 2.5 CV of 125 µM N-acetyl-ß-D-glucono-1,5-lactone (Toronto Research Chemicals) in affinity buffer.
Purification of recombinant HexB
HexB was expressed in SF21 cells as described for acid sphingomyelinase (Bartelsen et al., 1998) using the recombinant baculovirus described previously (Boose et al., 1990
). The medium was collected by centrifugation at 20,000 x g. The pH of the medium was set to 4.0 by addition of 1:10 volume of 200 mM citrate buffer, pH 4.0.
Cation exchange chromatography:
After filtration (0.4 µm) the medium was applied to a column (28 mm x 105 mm, CV = 65 ml) containing SP Sepharose fast flow (Pharmacia) at a flow rate of 20 ml/min. The column was washed with 3 CV of 20 mM citrate buffer, pH 4.0. Elution was performed at a flow rate of 10 ml/min using 1 CV of 10 mM phosphate buffer, pH 6.0, a gradient of 0500 mM NaCl in this buffer in 5 CV and an additional 3 CV of 500 mM NaCl in this buffer. The fractions containing HexB were collected and subjected to further purification. Desalting and affinity chromatography were performed as described for HexB from human placenta.
Tryptic digestion
Lyophilized modified trypsin was resuspended at a concentration of 1 µg/µl as described by the supplier. Trypsin was added at a concentration of 1:20 (w/w) to a solution of 50100 µM pure HexB in 25 mM NH4CO3. The mixture was incubated at 37°C overnight, and the digestion was stopped by freezing or by acidification with trifluoracetic acid (TFA).
Reduction
To a solution of protein or peptide in 25 mM NH4CO3 dithiothreitole was added to a concentration of 10 mM. The solution was incubated at 95°C for 15 min.
Alkylation with IAA
To a solution of protein or peptide in 25 mM NH4CO3 IAA was added to a concentration of 22 mM. The solution was incubated in the dark at room temperature for 15 min.
Isolation of glycopeptides
A column containing 100 µl of Con A Sepharose (Sigma) was equilibrated in loading buffer (50 mM phosphate buffer, pH 7.0, 200 mM NaCl) and the tryptic digest of 200 µg of HexB was added. The column was washed with 2 ml of loading buffer. Elution was performed using 1 ml of 10% -D-methylglucoside in loading buffer. The eluate was then subjected to RP-HPLC as described below.
RP-HPLC
RP-HPLC was performed on a Smart System (Pharmacia) using a Vertex column (2 x 250 mm) filled with Nucleosil C18120 µm material at a flow rate of 100 µl/min. TFA (0.1%) in water was used as solvent A, 0.06% TFA in 70% acetonitrile and 30% isopropanol as solvent B.
Tryptic peptides:
The column was equilibrated in 5% solvent B. After injection of the sample the column was washed for 15 min with 5% solvent B. Elution was achieved by a gradient of 5% solvent B to 88% solvent B in 55 min.
Glycopeptides:
The column was equilibrated in 5% solvent B. After injection of the sample the column was washed for 15 min with 5% solvent B. Elution was achieved by a gradient of 5% solvent B to 70% solvent B in 65 min.
Deglycosylation by PNGase F
The glycopeptide was isolated by RP-HPLC. The solvent was removed in a vacuum-concentration centrifuge and the peptide was resuspended in 20 µl of 25 mM NH4CO3. Two hundred units of PNGase F (New England Biolabs) were added and the mixture was incubated at 37°C for 2 h. Digestion was stopped by freezing or by acidification with TFA.
MALDI-MS
Matrix solutions:
-Cyano-4-hydroxycinnamic acid (Sigma): 10 mg/ml in 50% acetonitrile, 50% water containing 0.1% TFA. Sinnapinic acid (Sigma): 10 mg/ml in 40% acetonitrile, 60% water containing 0.1% TFA.
Sample preparation:
For RP-HPLC fraction, 1 µl of sample was mixed with 1 µl of matrix solution on the target and dried at room temperature. For digestion mixtures, 1 µl of sample was mixed with 0.5 µl of 5% TFA in water and 1 µl of matrix solution on the target and dried at room temperature.
Measurement:
Measurements were performed on a TOF Spec E (Micromass) at an acceleration voltage of 20 kV. An extraction voltage of 19.5 kV and a focus voltage of 15.5 kV were used. For linear measurements (above 4 kDa) pulse voltage was set to 1200 V, for reflectron measurements a pulse voltage of 2200 V was used. Measurements were performed at threshold laser energy. Calibration was achieved using commercially available peptides (Sigma).
PSD was performed as described by the supplier.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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