Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides

Yehia Mechref1, Lukas Zidek, Weidong Ma and Milos V. Novotny2

Department of Chemistry, Indiana University, Bloomington, IN 47405, USA

Received on March 22, 1999; revised on September 3, 1999; accepted on September 9, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 References
 
A minor component of the major urinary protein complex of the house mouse was chromatographically isolated and ascertained to be a previously suspected glycoprotein. Using highly sensitive mass-spectrometric techniques for sequencing and linkage analysis, the N-linked oligosaccharides of this glycoprotein were characterized. They were determined to be of the complex type with a wide heterogeneity. The heterogeneity was due to both the degree of sialylation and the presence of galactose residues in either ß(1–3) or ß(1–4) linkages. The biantennary structures were the most pronounced glycans, while tri- and tetraantennary entities were minor.

Key words: house mouse/mass spectrometry/oligosaccharide sequencing/pheromone-binding protein


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 References
 
An agglomerate of closely related polypeptides, known collectively as the major urinary protein (MUP) (Finlayson et al., 1965Go), has long been recognized as a developmentally interesting aspect of the house mouse. Among the rodents excreting large quantities of urinary proteins, the mouse MUP complex is known to be the product of a multigene family (Bishop et al., 1982Go; Hainey and Bishop, 1982Go; Clissold et al., 1984Go; Al-Shawi et al., 1992Go) that shows considerable dependence on hormone levels. Male mice excrete much larger quantities of MUP than females, but androgen treatment can induce in females the formation of MUP levels comparable to males. Although much remains to be elucidated concerning the exact biological role of MUP, considerable discussion has recently centered around the suggestions (Novotny et al., 1980Go; Robertson et al., 1993Go) that this protein complex may act as a slow-release and/or protecting molecular entity for transmission of the mouse pheromone ligands (Novotny et al., 1999) for chemical communication.

Structurally, MUPs are acidic proteins (pI = 4.2–4.7) with an approximate molecular weight of 19 kDa (Duncan et al., 1988Go). More recently, their molecular heterogeneity in six mouse strains was assessed through accurate mass-spectrometric measurements (Robertson et al., 1996Go), indicating the presence of at least 14 polypeptide species. MUPs belong to the lipocalin family (Flower, 1996Go), sharing their advanced structure with the odorant-binding proteins of vertebrate olfactory tissues (Pelosi, 1994Go). At least two groups (Kuhn et al., 1984Go; Clark et al., 1985Go) suggested glycosylation of certain MUPs. One protein has been tentatively identified (Clark et al., 1985Go) 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., 1991Go; Miyawaki et al., 1994Go; Mechref et al., 1999Go) are also N-glycosylated proteins. Since large molecules, such as proteins, are capable of entering the VNO structure in rodents (Wysocki et al., 1980Go), 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., 1985Go) 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, 1998aGo,b; Huang et al., unpublished observations), utilizing endo- and exoglycosidase cocktails.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 References
 
To isolate the minor, glycosylated protein from the MUP complex, we used two chromatographic steps: (1) fractionation on a Superdex 75 column (according to molecular size); and (2) lectin chromatography on a Concanavalin A column. Eleven 50 µl male urine aliquots had to be processed through step (1) to accumulate a sufficient amount of proteinaceous material for a recovery of the glycosylated MUP through the lectin binding. Based on a rough estimate through absorbance at 280 nm, ~5 µg quantity was recovered in two passes through the lectin column. The isolated protein was more acidic than other MUPs, as judged from its behavior during isoelectric focusing (pI = 4.0; data not shown). A low pI-value is expected when a glycoprotein features sialic acid residues (see below).

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., 1985Go; Robertson et al., 1996Go). While a band in SDS–PAGE 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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Fig. 1. Anion-exchange chromatogram of the isolated MUP components (upper trace) and the Concanavalin A bound fraction (lower trace); inset is the mass spectrum of the isolated glycoprotein, as indicated with asterisk. For conditions, see Materials and methods.

 
Both positive and negative ion spectra were acquired by MALDI-time-of-flight (TOF) mass spectrometry from the glycans released through the enzymatic cleavage by N-glycanase. The negative-ion spectrum depicted in Figure 2 suggests tentatively the presence of complex-type N-linked oligosaccharides. The m/z-values of 1930.4, 2223.4, and 2517.6 observed in the negative-ion mode suggest the existence of mono-, di-, and trisialylated biantennary structures, respectively. A trisialylated biantennary structure is feasible only if one of the sialic acid linkages is of the {alpha}(2–3) type. In fact, a simultaneous sample treatment with PNG-ase F and the neuraminidase from Newcastle disease virus (which selectively cleaves {alpha}(2–3) linked sialic acid residues) resulted in disappearance of the m/z = 2517.6 signal, verifying the {alpha}(2–3) linkage (data not shown). Moreover, since no signals in the positive-ion mode were observed following this treatment, multiple {alpha}(2–3) linkages can be ruled out. It is noteworthy that adding the average mass of the most intense signals in Figure 2 to the expected molecular mass of the MUP-15 polypeptide (Duncan et al., 1988Go) yields the sum close to the measured mass of the isolated glycoprotein.



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Fig. 2. Negative-ion MALDI mass spectrum of N-glycans. Solid squares, N-acetylglucosamine; open squares, galactose; open circles, mannose; open triangles, {alpha}(2–6) linked neuraminic acid; and solid triangles, {alpha}(2–3)-linked neuraminic acid.

 
The mass spectrum of the glycoprotein sample treated simultaneously with PNGase F and neuraminidase from Arthrobacter ureafaciens is shown in Figure 3a. One major and two minor signals are clearly observed. The larger peak at m/z 1663.8 corresponds to a biantennary structure, while the less intense signals are due to triantennary and tetraantennary entities.



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Fig. 3. MALDI mass spectra of the N-glycans treated with PNGase F and neuraminidase in (a); PNGase, neuraminidase and ß-galactosidase in (b); and PNGase F, neuraminidase ß-galactosidase and N-acetyl-ß-D-glucosaminidase in (c). Symbols as in Figure 2.

 
Enzyme arrays were utilized to determine the type of linkages associated with the above-described structures. A spectrum resulting from a simultaneous treatment with neuraminidase and ß-galactosidase is shown in Figure 3b. The chosen galactosidase is known to be specific for the cleavage of galactose residues that are ß(1–4)-linked to N-acetylglucosamine. If all of the galactose residues in all N-glycans were ß(1–4)-linked to N-acetylglucosamine, three new signals would be expected after a treatment with the enzyme array. In fact, eight signals became observable after the treatment, suggesting the additional presence of galactose residues that are ß(1–3)-linked. The m/z-value at 1340.5 corresponds to a biantennary structure that has both galactose residues in a ß(1–4) linkage. A signal at 1502.5 can be observed only if one of the galactose residues attached to the biantennary structure is ß(1–3)-linked, while the signal at 1664.6 can only be the result of both galactose residues in a biantennary structure being ß(1–3)-linked. Signals at m/z 1908.3 and 1749.6 can only be observed if a tetraantennary structure has one (ß(1–3)-linked galactose residue, or three similarly linked galactose residues, respectively. The remaining signals observed in Figure 3b originate from the triantennary structures with one or two ß(1–3)-linked galactose residues. Thus, it appears that the heterogeneity of MUP N-glycans is not only due to a different degree of silylation, but also due to different galactosidic linkages.

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(1–2) terminal N-acetylglucosamine linked to mannose. The tetraantennary structure has two N-acetylglucosamine residues that are ß(1–4) 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(1–4)-linked to mannose (1342 m/z). However, a tetraantennary structure with a galactose residue which is ß(1–3)-linked to an N-acetylglucosamine being ß(1–4)-linked to mannose will give a MALDI signal of 1503.1 m/z. Conversely, if the ß(1–3)-linked galactose is linked to N-acetylglucosamine that is ß(1–2)-linked to mannose, then a MALDI signal at 1706 m/z should be observed.

Regarding the triantennary structure, such entity possesses one ß(1–4)-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(1–4)-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(1–3)-linked will give a MALDI signal of 1299 m/z, assuming this galactose residue is linked to N-acetylglucosamine being ß(1–4)-linked to the mannose residue. However, if the same ß(1–3)-linked galactose residue is attached to N-acetylglucosamine, which is ß(1–2)-linked to mannose, then a signal of 1503 m/z would be observed. A triantennary structure with two galactose residues that are ß(1–3)-linked to N-acetylglucosamine (one of which is ß(1–4)-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(1–4) 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(1–3)-linked galactose. However, one of the triantennary structures containing a single ß(1–3)-linked galactose f could yield a product of identical m/z. The other triantennary structures with one ß(1–3)-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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Scheme 1. Desialylated N-glycan structures of the glycosylated MUP.

 
In conclusion, the MUP glycoprotein exhibits a highly heterogeneous glycosylation pattern. The glycan heterogeneity seems to be expressed in both the degree of sialylation and the linkages of terminal galactose residues. The predominant oligosaccharides are bi- or trisialylated biantennary glycans whose penultimate residue (next to a sialyl substitution) can be attached to C3 or C4 of an N-acetylglucosamine. The distribution of ß(1–3) and (1–4) linkages appears variable. This isolated glycoprotein seems to be the predicted MUP-15 gene product (Clark et al., 1985Go) glycosylated at the only available glycosylation site (Asp 44) (Kuhn et al., 1984Go).

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., 1999Go) 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 References
 
Materials
Urine was collected from ICR/A1b male mice (our own colony, started originally from the Sprague-Dawley stock; Indianapolis, IN) and stored frozen prior to analysis. Thawed urine samples were passed through a 0.2 µm filter prior to further processing. Neuraminidase (EC 3.2.1.18) from Arthrobacter ureafaciens, spermine (N,N'-bis[3-aminopropyl]-1,4-butanediamine tetrahydrochloride) and 2,5-dihydroxybenzoic acid (DHB) were obtained from Sigma Chemical Co. (St. Louis, MO). N-Glycosidase F (PNGase F) from Flavobacterium meningosepticum, a recombinant protein from E.coli (EC 3.2.2.18), N-acetyl-ß-D-glucosaminidase from Diplococcus pneumoniae (EC 3.2.1.30), ß-galactosidase from Diplococcus pneumoniae (EC 3.2.1.23) and neuraminidase from Newcastle disease virus (EC 3.2.118) were received from Boehringer Mannheim Corp. (Indianapolis, IN). The conventional chemicals used in this study were purchased from Aldrich (Milwaukee, WI). The lectin ConA Superose was received from Pharmacia (Piscataway, NJ). The Silver Stain Kit for electrophoresis was purchased from Bio-Rad Laboratories (Hercules, CA).

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 {alpha}-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. SDS–PAGE 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, 1998aGo). 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, 1964Go). 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., 1997Go). The matrix using a mixture of DHB/spermine (Mechref and Novotny, 1998bGo) 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 References
 
This research was supported by Grants GM24349 from the National Institute of General Medical Sciences and DC02418 from the National Institute of Deafness and Communicative Disorders, U.S. Department of Health and Human Services.


    Footnotes
 
1 Present address: Department of Chemistry, Faculty of Science, United Arab Emirates University, P.O. Box 17551, Al-Ain, UAE Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 References
 
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