Department of Chemistry, Indiana University, Bloomington, IN 47405, USA
Received on March 22, 1999; revised on September 3, 1999; accepted on September 9, 1999.
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Abstract |
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Key words: house mouse/mass spectrometry/oligosaccharide sequencing/pheromone-binding protein
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Introduction |
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Structurally, MUPs are acidic proteins (pI = 4.24.7) with an approximate molecular weight of 19 kDa (Duncan et al., 1988). More recently, their molecular heterogeneity in six mouse strains was assessed through accurate mass-spectrometric measurements (Robertson et al., 1996
), indicating the presence of at least 14 polypeptide species. MUPs belong to the lipocalin family (Flower, 1996
), sharing their advanced structure with the odorant-binding proteins of vertebrate olfactory tissues (Pelosi, 1994
). At least two groups (Kuhn et al., 1984
; Clark et al., 1985
) suggested glycosylation of certain MUPs. One protein has been tentatively identified (Clark et al., 1985
) as a minor N-glycosylated protein because of its characteristic sequence segment for N-glycosylation and a shift in electrophoretic migration after treatment with PNGase F. Whether or not coincidental, the putative pheromone transporters in the mouse and rat vomeronasal organ (VNO) mucosa (seemingly a receptive tissue for the pheromones) (Khew-Goodall et al., 1991
; Miyawaki et al., 1994
; Mechref et al., 1999
) are also N-glycosylated proteins. Since large molecules, such as proteins, are capable of entering the VNO structure in rodents (Wysocki et al., 1980
), a glycosylated MUP could serve an important transport function for the animals contacting directly the pheromone source, i.e., a urine mark.
In this report, we describe the isolation of the glycosylated MUP (seemingly the one described by Clark et al., 1985) and structural analysis of its glycans. Size-exclusion, ion-exchange chromatography, and lectin binding were used to isolate and purify this protein at a low-microgram level. The sequence determination of its oligosaccharides and linkage analysis at such a minute quantity of the glycoprotein material has become feasible by the recent development of the on-plate sequencing techniques in our laboratory (Mechref and Novotny, 1998a
,b; Huang et al., unpublished observations), utilizing endo- and exoglycosidase cocktails.
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Results and discussion |
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Ion-exchange chromatography, under gradient elution conditions, was used to display the separation of MUP isoforms (Figure 1, upper trace) and compare this elution profile with the glycoprotein isolate after lectin chromatography (lower trace). As seen in the figure inset, a mass spectrum (obtained through matrix-assisted laser desorption/ionization (MALDI) process) of the lectin-bound material features a major broad signal at the mass-to-charge (m/z) value of 21,383.5 Da. The observed mass is higher by 2377 Da than the mass predicted from cDNA sequence of the MUP-15 gene (19,007 Da) (Clark et al., 1985; Robertson et al., 1996
). While a band in SDSPAGE corresponding to this protein was observed (data not shown), both sensitivity and resolution of MALDI-mass spectrometry were superior to the gel electrophoresis.
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The mass spectrum of the N-glycans treated with N-acetylglucosaminidase is shown in Figure 3c. The signals at m/z 934.5, 1299.9 and 1665.5 are likely to originate from the biantennary structures, while 1706.8, 1503.1, and 1342.0 may be due to the tetraantennary entities. Alternatively, the m/z-values of 1138.5, 1299.9, 1503.1, and 1665.5 can originate from the above triantennary structures.
The glucosaminidase used in this study cleaves specifically ß(12) terminal N-acetylglucosamine linked to mannose. The tetraantennary structure has two N-acetylglucosamine residues that are ß(14) linked to mannose, and thus will not be cleaved by the action of glucosaminidase used in this study. Therefore, digestion of the tetraantennary compound with galactosidase and glucosaminidase ultimately results in the formation of a fragment possessing the core structure plus two N-acetylglucosamines that are ß(14)-linked to mannose (1342 m/z). However, a tetraantennary structure with a galactose residue which is ß(13)-linked to an N-acetylglucosamine being ß(14)-linked to mannose will give a MALDI signal of 1503.1 m/z. Conversely, if the ß(13)-linked galactose is linked to N-acetylglucosamine that is ß(12)-linked to mannose, then a MALDI signal at 1706 m/z should be observed.
Regarding the triantennary structure, such entity possesses one ß(14)-linked N-acetylglucosamine that would not be cleaved by the glucosaminidase used in this study. A triantennary structure treated with galactosidase and glucosaminidase should yield a MALDI signal at 1138 m/z, as one of the N-acetyglucosamine residues is ß(14)-linked to mannose and thus resistant to the action of glucosaminidase used in this study. Moreover, the presence of a galactose residue that is ß(13)-linked will give a MALDI signal of 1299 m/z, assuming this galactose residue is linked to N-acetylglucosamine being ß(14)-linked to the mannose residue. However, if the same ß(13)-linked galactose residue is attached to N-acetylglucosamine, which is ß(12)-linked to mannose, then a signal of 1503 m/z would be observed. A triantennary structure with two galactose residues that are ß(13)-linked to N-acetylglucosamine (one of which is ß(14)-linked to mannose) will give a MALDI signal of 1665.5 upon a simultaneous treatment with galactosidase and glucosaminidase.
It should be noted that no tri- or tetraantennary structures were observed directly in either the positive or negative ion mode prior to a treatment with neuraminidase. However, these structures became readily observable in the positive ion spectrum after treatment with neuraminidase. This is due to the fact that the structures existed originally as sialylated entities at very small amounts. The positive MALDI mass spectrometry is currently a more sensitive technique than its negative-ion counterpart. As evidenced in Figure 3a, tri- and tetraantennary structures clearly exist in this glycoprotein.
The structures identified in this work after desialylation are summarized in Scheme 01. The signals at m/z 934.5, 1138.5, and 1342.0 can be rationalized as containing just ß(14) linked galactose: biantennary a; triantennary e, and tetraantennary k, respectively. The m/z of 1665.5 could correspond to the biantennary structure d revealed by a simultaneous treatment with neuraminidase and ß-galactosidase, but also to triantennary structures i and/or j. The intense signal at m/z 1299.9 is most probably due to the biantennary b and/or c, containing one ß(13)-linked galactose. However, one of the triantennary structures containing a single ß(13)-linked galactose f could yield a product of identical m/z. The other triantennary structures with one ß(13)-linked galactose (g and h, and/or tetraantennary l and m) could also contribute to m/z 1503.0. The minute signal at m/z 1706.8 can be attributed to the remaining tetraantennary structures n and/or o.
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The recent advances in MALDI mass spectrometry of oligosaccharides have made it feasible to study the minute quantities of glycoproteins associated with chemical communication in mammals. The glycosylated mouse protein characterized here and the vomeromodulin glycan structures reported from this laboratory (Mechref et al., 1999) represent only microgram quantities of these materials. Future experiments can now be directed toward elucidating a role of glycosylation in pheromone binding and biological activity.
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Materials and methods |
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Methods
(1) Glycoprotein isolation and purification.
The mouse urine was subjected to size-exclusion chromatography on a Pharmacia Smart system (Piscataway, NJ), utilizing a Pharmacia HR 5/20 column packed with a Superdex 75 material. The column was eluted with a buffer containing 10 mM Tris (pH 7.8) and 0.15 M sodium chloride, at a flow-rate of 30 µl/min.
Lectin chromatography was performed on the system consisting of a Pharmacia P-3 peristaltic pump and an HR 5/5 column packed with 1 ml volume of ConA Superose. A binding mobile phase, consisting of 10 mM Tris buffer (pH 7.8) and 0.5 M sodium chloride, was pumped through the column at a flow rate of 200 µl/min. The same buffer containing 0.5 M -D-methylglucopyranoside was used for displacement.
(2) Analytical chromatography.
Anion-exchange chromatography was carried out on the Pharmacia Smart system equipped with a MiniQ PC 3.2/3 column, at a flow rate of 0.2 ml/min. A linear gradient elution from 0 to 0.25 M sodium chloride (over 30 min) was employed using 10 mM Tris buffer, pH 7.8.
(3) Electrophoresis.
Isoelectric focusing was performed on a T5C3 polyacrylamide gel containing 1.6% Biolyte 4/6 and 0.4% Biolyte 3/10, for 3 h, at 1000 V, using a Bio-Rad (Hercules, CA) Biophoresis horizontal cell. The gel was stained with a Bio-Rad Silver Stain Kit. SDSPAGE was carried out with a Bio-Rad Mini Protein apparatus in a T12C2.67 separation gel at 200 V. A Bio-Rad Silver Stain kit was used for visualization.
(4) Cleavage and sequencing of N-glycans.
Prior to the enzymatic release and sequencing of the N-glycans from glycosylated MUP, the glycoprotein was desalted using a Microconcentrator 10 from Amicon (Beverly, MA). Enzymatic release and sequencing of N-glycans were performed on the plate according to the procedure developed in our laboratory (Mechref and Novotny, 1998a). Approximately 1 µg quantities of the glycoprotein were deposited on the plate and reconstituted in 1 µl reaction buffer (10 mM sodium phosphate, pH 6.5). Typically, 30 mU PNGase F aliquots were added to each spot. For enzymatic sequencing, five glycoprotein spots were deposited on the MALDI plate, while different enzyme arrays were added to each spot. The first spot contained PNGase F (30 mU). PNGase F (30 mU) and neuraminidase (3 mU) were added to the second spot. PNGase F and neuraminidase from Newcastle disease virus were added to the third spot. PNGase F, neuraminidase, and ß-galactosidase (0.3 mU) were added to the fourth spot, while PNGase F, neuraminidase, ß-galactosidase, and N-acetyl-ß-D-glucosaminidase (0.3 mU) were placed on the fifth spot. The plate was then placed in a crystallization beaker containing water, while the jar was covered with parafilm and placed in the water bath at 40°C for 3 h. High-purity water, obtained from Stephens Scientific (Riverdale, NJ), was continuously added to the spots to prevent drying.
(5) MALDI/mass spectrometry instrumentation.
All MALDI-TOF mass spectra were acquired on a Voyager-DE RP BioSpectrometry Workstation (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm). The instrument can operate in both the reflector and linear modes, but the linear mode was used solely in this study. The spectra were acquired at 25 kV accelerating voltage with a delayed ion extraction. The delay time was set to 150 ns, in addition to the inherent instrument delay. The instrument was externally calibrated with the maltose ladder, encompassing the m/z values of the analyzed sample constituents. All acquired spectra were smoothed by applying the 19-point Savitzky-Golay procedure (Savitzky and Golay, 1964). The MALDI instrument utilizes a multichannel plate detector and, in order to preclude low-mass ions from saturating the detector, the ion gate was set at 800.
MALDI mass spectra of the cleaved or sequenced neutral N-glycans were acquired utilizing the arabinosazone matrix developed in this laboratory (Chen et al., 1997). The matrix using a mixture of DHB/spermine (Mechref and Novotny, 1998b
) was employed for acquiring the spectra of sialylated oligosaccharides (negative-ion mode). The arabinosazone matrix was prepared in ethanol at 10 mg/ml concentration. DHB/spermine was prepared by dissolving DHB crystals in 300 mM spermine aqueous solution in order to obtain a saturated DHB solution. The matrix solution was then desalted by adding a few cation-exchange resin beads (NH4+, ionic form), thus minimizing the sodium ions in the matrix. The samples were prepared by reconstituting the spots in 0.5 µl of water, followed by addition of 1 µl of the matrix solution. The spots were allowed to dry at room temperature.
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Acknowledgments |
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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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