Bile salt-stimulated lipase (BSSL) is an enzyme with a broad substrate specificity toward mono-, di-, and triacylglycerols; cholesterol esters; fat-soluble vitamins; and lipoamides (Bernbäck et al., 1990; Hui et al., 1993; Chen et al., 1994). The enzyme represents the major lipolytic activity in the milk and is also important in the energy uptake (Hernell and Olivecrona, 1974). BSSL is a glycoprotein which amounts to approximately 0.5-1.0% of the milk protein (Hernell and Olivecrona, 1974; Bläckberg and Hernell, 1981). The biological roles of BSSL glycans are speculative at present, since glycosylation does not seem to effect the main catalytic activity of this enzyme (Hansson et al., 1993; Bläckberg et al., 1995).
The glycoprotein consists of 722 amino acid residues, with one potential N-glycosylation site at Asn-187 at the N-terminus (Nilsson et al., 1990; Baba et al., 1991). Toward the C-terminus, however, the residues with potential O-glycosylation sites abound (Nilsson et al., 1990; Baba et al., 1991). While the amino acid sequence of BSSL appears identical to the pancreatic bile salt-dependent lipase (also known as pancreatic cholesterol esterase; Nilsson et al., 1990), the two enzymes differ in their molecular size, which could be due to differences in the degree and type of glycosylation. Glycosylation is known to vary with the tissue of protein expression (Rademacher et al., 1988). In the case of native BSSL, it has been even suggested (Hernell and Bläckberg, 1994) that the degree and type of glycosylation could vary among individuals and a month of lactation. Moreover, a difference in glycosylation between a native and recombinant BSSL was recently reported (Strömqvist et al., 1995).
We describe here the structural determination of the N-glycans isolated from 'averaged" BSSL (Bläckberg, et al., 1995). This structural characterization has been enabled through the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in conjunction with the action of specific exoglycosidases. The unique capabilities of MALDI mass spectrometry (MS) in carbohydrate analysis have gradually become evident during the recent years (Mock et al., 1991; Stahl et al., 1991; Harvey et al., 1994). The use of tandem mass spectrometry (MS/MS) has further made it feasible to analyze mixtures of neutral and sialylated oligosaccharides in one spectrum (Powell and Harvey, 1996).
In the work described here, the BSSL sample was first treated enzymatically to release N-linked oligosaccharides. These were further profiled for their approximate number and type by the established technique of high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and a newer technique of capillary electrophoresis (CE) using laser-induced fluorescence. Methylation of the sialic acid groups in the mixture of enzymatically released glycans, followed by MALDI-MS, revealed the existence of four main oligosaccharide components. The oligosaccharides were further sequenced using tandem MS (MS/MS) (Mechref et al., in press) with collision-induced dissociation (CID). Finally, five specific exoglycosidases were employed to confirm the sequences and yield the linkage forms of component monosaccharides.
HPAEC-PAD and CE separations: orientation experiments
In order to assess the glycoprotein's complexity and the extent of sialylation, we first used HPAEC-PAD (Figure
Figure 1. (a) HPAEC-PAD map of the N-glycans derived from BSSL(the utilized elution program is summarized in Table II), and(b) electropherogram of the ANTS-labeled BSSL N-glycans. Conditions: background electrolyte, 25 mM sodium phosphate, pH 2.5; capillary, 50 cm/80 cm × 50 µm i.d. fused-silica capillary; voltage, 20 kV. Peaks marked with arrows are due to the reagent blank while those marked with asterisks correspond to the labeled N-glycans.
The electrophoretic map of the fluorescently labeled BSSL oligosaccharides is shown in Figure
MALDI/MS of esterified and desialylated BSSL and fetuin N-glycans
A major problem encountered in the MALDI-MS of acidic oligosaccharides in the positive-ion mode is the loss of sialic acids and detection of multiple peaks resulting from the ion formation between the free acid and the salts. This fragmentation is known to decrease sensitivity of the MALDI/MS analysis for acidic oligosaccharides to the extent that, in some cases, the signal is totally lost. This problem was recently overcome by Powell and Harvey (Powell and Harvey, 1996) who acquired MALDI spectra of acidic oligosaccharides and gangliosides after methylation through the procedure of Norgard-Sumnicht et al. (Norgard-Sumnicht et al., 1995). This esterification step permits analysis of mixtures of neutral and acidic oligosaccharides in a single spectrum.
The MALDI mass spectrum of esterified N-linked oligosaccharides from BSSL is illustrated in Figure
Figure 2. MALDI mass spectra of esterified (a) and desialylated (b) N-glycans derived from BSSL. The m/z values correspond to monoisotopic masses of the sodiated ions. The brackets around the structures symbolize their tentative nature. Symbols: solid circles, mannose; open squares, N-acetylglucosamine; open squares, galactose; open triangles, methylated sialic acid; solid circles, fucose. CID-MALDI/MS of desialylated glycans
An attractive feature of a MALDI time-of-flight instrument with the reflectron geometry is its ability to provide the means of fragmentation for the analyzed oligosaccharides. In principle, this leads to the determination of sequence, branching and linkage forms (Spengler et al., 1994; Harvey et al., 1995; Lemoine et al., 1996; Penn et al., 1996; Mechref et al., in press). We have utilized this capability in providing the structural information regarding the sequence and branching in the two desialylated structures observed in Figure
The mass spectrum obtained through the collision-induced dissociation (due to the parent m/z 1663) is illustrated in Figure
Figure 3. CID-MALDI mass spectrum of m/z 1663 as the parent ion. Symbols as in Figure 2. The m/z values correspond to monoisotopic masses of the sodiated fragments.
The information deduced from the CID spectrum acquired for the m/z 1809 was even more revealing (Figure
Figure 4. CID-MALDI mass spectra of m/z 1809. Symbols as in Figure 2. The m/z values correspond to monoisotopic masses of the sodiated fragments.
To this point, we have determined the sequence and branching of the N-glycans derived from BSSL, but the absence of any cross-ring fragmentation in the CID spectra limited the determination of linkages. Fortunately, this information could be obtained through sequential enzymatic cleavages of the N-glycans using specific exoglycosidases. Enzymatic determination of sequence and linkages
Table I lists the enzymes that have been employed in verification of the monosaccharide sequences and determination of the linkages in BSSL oligosaccharides. A negative sign in this table indicates that no change in a MALDI spectrum was observed after the enzymatic digestion. The sialic acid linkage in the oligosaccharide structures was determined to be an [alpha]-2,6-type, as concluded from a negative action of the neuraminidase that is specific for cleavage at [alpha]-2,3-position, and a positive action of the neuraminidase cleaving both [alpha]-2,3- and [alpha]-2,6-linkages (Figure
Table I.
Incubation of the BSSL N-glycans with neuraminidase and [beta]-galactosidase caused a shift of ~324 m/z for the two signals observed upon a treatment of the N-glycans with neuraminidase alone (Figures
Treatment of the BSSL oligosaccharides with N-acetylglucosaminidase after desialylation and degalactosylation resulted in a shift of ~404 m/z , in addition to the 324 m/z shift observed from degalactosylation. This additional shift is due to the loss of two N-acetylglucosamine residues which are [beta]-1,2-linked (Figures
Moreover, the site of fucosylation was judged to be on the reducing-end GlcNAc, as based on the action of endoglycosidase F which cleaves specifically the N-linked oligosaccharides between the two reducing-end GlcNAc residues. The MALDI spectrum of desialylated BSSL oligosaccharides cleaved by endoglycosidase F showed a signal at m/z 1460.1, corresponding to a dibranched structure with one GlcNAc at the reducing end (Figure
Scheme 1 summarizes the structures of BSSL N-glycans as based on our determinations. The linkage and sequence of the first five monosaccharide residues were not determined enzymatically, since it is widely known that N-linked oligosaccharides generally contain a common pentasaccharide core consisting of three mannoses and two GlcNAc residues (the so-called 'mannose core").
Scheme 1. Structures of BSSL N-glycans.
Figure 5. MALDI mass spectra of the exoglycosidase digests. (a) Treatment with neuraminidase and [alpha]-1,3/4-fucosidase; (b) treatment with [beta]-galactosidase; (c) treatment with N-acetylglucosaminidase; (d) desialylated BSSL oligosaccharides cleaved by Endoglycosidase F. Symbols as in Figure 2. The m/z values correspond to monoisotopic masses of the sodiated ions.
The structures elucidated here are similar to those reported for another human bile-salt stimulated lipase that was isolated from pancreatic juice (Mas et al., 1993; Sugo et al., 1993). However, the N-glycans from the pancreatic lipase were more heterogeneous and their fucosylation was determined to be [alpha](1-6)- and [alpha](1-2)-types, linked to the innermost N-acetylglucosamine residue and a terminal galactose residue, respectively. Additionally, the structural determinations reported here are in general agreement with the very recent general studies (Landberg et al., 1997) aiming at comparison of the native and recombinant forms of the human milk BSSL.
Exoglycosidase
Specificity
Digestion of BSSL oligosaccharides
[beta]-d-galactosidase from Diplococcus pneumoniae
Hydrolyzes terminal galactose residues which are [beta]-1,4-linked to GlcNAc.
+
N-acetyl-[beta]-d-glucosaminidase from Diplococcus pneumoniae
Cleaves terminal N-acetylglucosamine residues that are [beta]-linked to oligosaccharides.
+
[alpha]-1,3/4-fucosidase from Streptomyces species
Cleaves [alpha]-1,3- and [alpha]-1,4-linked terminal fucose on N-linked oligosaccharides.
-
neuraminidase from Arthrobacter ureafaciens
Hydrolyzes terminal N- or O-acylneuraminic acids which are [alpha]-2,3- and [alpha]-2,6-linked to galactose, Hex, GlcNAc or N- or O-acetyl neuraminyl residues in oligosaccharides or colominic acid.
+
neuraminidase from Newcastle disease virus Hitchner B1 strain
Hydrolyzes very specifically terminal [alpha]-2,3-bound neuraminic acids of N- or O-glycosidically linked oligosaccharide chains in glycoproteins and glycolipids.
-
Fetuin from the fetal calf serum and neuraminidase (EC 3.2.1.18) from Arthrobacter ureafaciens were purchased from Sigma Chemical Co. (St. Louis, MO). The BSSL sample (from a pool of breast milk of over 100 individuals) was received from Astra Hässle AB (Umeå, Sweden), after being prepared according to the procedure reported by Bläckberg et al. (Bläckberg et al., 1995). N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum, a recombinant product from E.coli (EC 3.2.2.18), endoglycosidase F from Flavobacterium meningosepticum (EC 3.2.196), N-acetyl-[beta]-d-glucosaminidase from Diplococcus pneumoniae (EC 3.2.1.30), [beta]-galactosidase from Diplococcus pneumoniae (EC 3.2.1.23), neuraminidase (Sialidase) from Newcastle disease virus (EC 3.2.118) and [alpha]-1,3/4-fucosidase from Streptomyces species (EC 3.2.1.51) were all from Boehringer Mannheim (Indianapolis, IN). All common chemicals were received from Aldrich (Milwaukee, WI). The fluorescence labeling reagent, 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS), was purchased from Molecular Probes, Inc. (Eugene, OR).
Enzymatic cleavages
The N-linked oligosaccharides from bovine fetuin and BSSL were enzymatically released by PNGase F. The glycoproteins to be digested were reconstituted in 0.05 M sodium phosphate buffer (pH 7.5), followed by addition of PNGase F (2.0 U/1.0 mg glycoprotein). The samples were subsequently incubated for 24 h at 37°C.
The enzymatically released oligosaccharides were recovered by applying the digestion mixtures to C18 Sep-Pak cartridges (Waters Corp., Milford, MA) that were preconditioned with methanol, acetonitrile, and aqueous methanol (1:4 v/v methanol/water). The oligosaccharides were eluted with 3 ml of the aqueous methanol solution and then dried under a stream of nitrogen. In the case of endoglycosidase F, the BSSL sample (100 µg) was dissolved in 20 µl of 50 mM sodium acetate buffer (pH 6.0) and incubated with 100 mU of the enzyme at 37°C for 18 h. The oligosaccharides cleaved were eventually pooled as described above. Specificities of the exoglycosidases used in this study are summarized in Table I. All digestions were performed on the oligosaccharides released from 100 µg of BSSL. Monosaccharides were removed sequentially from the nonreducing end of BSSL oligosaccharides by incubation with specific exoglycosidases at different times.
Desialylation. The sialic acid residues of the N-glycans derived from fetuin and BSSL were liberated by reconstitution of the oligosaccharides released from 100 µg of a glycoprotein in 50 mM sodium phosphate buffer (pH 6.0), followed by the addition of neuraminidase from Arthrobacter ureafaciens, or Newcastle disease virus (~20 mU/ 1.0 nmol of substrate) and incubation for 12 h at 37°C.Degalactosylation. The samples desialylated, as described above, were treated with 20 mU [beta]-galactosidase from Diplococcus pneumoniae (in the same buffer) for 18 h at 37°C.Removal of N-acetylglucosamine. The desialylated and degalactosylated sample was desalted by passing through a fritted 1 ml syringe containing mixed DOWEX-50W (H+) and DOWEX 1 (OH-) resins. The eluent was lyophilized before reconstitution in 50 mM sodium citrate/phosphate buffer (pH 5.0) and incubation with 20 mU of N-acetyl-[beta]-d-glucosaminidase from Diplococcus pneumoniae for 18 h at 37°C.Treatment with [alpha]-1,3/4-fucosidase from Streptomyces species. BSSL oligosaccharides were treated with 20 mU neuraminidase and 20 mU [alpha]-1,3/4-fucosidase in 50 mM sodium phosphate buffer (pH 6.0), at 37°C for 18 h. In all cases, the enzymatic activity was stopped by heating the reaction microtubes for 5 min at 100°C prior to a mass-spectrometric analysis.Esterification of acidic oligosaccharides
The sialic acid residues of acidic oligosaccharides were esterified according to the procedure described by Powell and Harvey (Powell and Harvey, 1996). Briefly, the acidic oligosaccharides dissolved in water were applied to a short column (made from a fritted 10 ml plastic syringe) containing Dowex-50W resin (Sigma Chemical Co.) that had been preconditioned with 1.0 M sodium hydroxide and water. The eluent was then lyophilized and dissolved in dry dimethylsulfoxide (DMSO). Methyl iodide was added, and the mixture was thoroughly mixed and allowed to react at room temperature for 2 h. Finally, the reaction mixture was dried under a stream of nitrogen and desalted by passing through a short desalting column (made from a fritted 10 ml plastic syringe) containing a mixture of cation- and anion-exchange resins. The eluent was lyophilized and redissolved in deionized water.
Labeling of BSSL N-glycans
BSSL oligosaccharides were labeled with ANTS through reductive amination. The oligosaccharides, cleaved from 75 µg of BSSL, were dissolved in 2 µl 0.15 M ANTS in 3:17 propionic acid:DMSO and 2 µl 1.0 M sodium cyanoborohydride in DMSO. The reaction mixture was vortexed prior to incubation in a water bath at 40°C for 15 h. Propionic acid was employed to eliminate or minimize hydrolysis of the sialic acid groups. Prior to a CE analysis, the sample was diluted 100-fold with deionized water.
Sample and matrix preparation for MALDI/MS studies
Arabinosazone, a new matrix reported recently by our laboratory (Chen et al., 1997) was used exclusively for all MALDI/MS measurements. Esterified samples as well as the exoglycosidase digests were prepared for the MALDI analysis by spotting 1.0 µl of the test solution on a polished stainless-steel sample plate, followed by addition of 1.0 µl arabinosazone dissolved in ethanol/water (3:1 v/v) at a final concentration of 10 mg/ml and allowing the spot to dry at room temperature.
Instrumentation
MALDI-Time-of-Flight Mass Spectrometry. Mass spectra were acquired on a Voyager-DE RP Biospectrometry Workstation instrument (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm). The instrument operates both in the reflector and linear modes of detection and has the delay extraction capability. MALDI spectra were acquired at 25 kV accelerating voltage, while the low-mass gate was used to discard the ions with m/z values of less than 500. All acquired spectra were smoothed by applying a 19-point Savitzky-Golay smoothing routine (Savitzky and Golay, 1964). The instrument is equipped with a collision cell, with argon being used as the collision gas. For collision-induced dissociation (CID) spectra, the precursor ion was selected by the time ion selector, with a mass window of approximately 45 m/z. The CID spectra were acquired at 25 kV accelerating voltage, while the reflectron voltage was successively decreased by 10%, allowing the detection of certain fragments at a time. Generally, 10-12 segments were acquired at 100 scans for each segment, and the composite CID spectrum was a combination of all acquired segments, as generated by the instrument software.
HPAEC-PAD instrument and separation conditions. HPAEC-PAD mapping was performed using the Dionex Bio-LC System (Dionex, Sunnyvale, CA) equipped with a CarboPac PA-100 column (4.6 × 250 mm) working at a flow rate of 1 ml/min at ambient temperature. The pulse potentials and durations were 0.05 V/300 ms, 0.75 V/120 ms, and -0.70 V/60 ms, working at 300 nA full scale. The gradient utilized for acquiring the map is summarized in Table II.Table II.
Min | Eluent 1 (250 mM sodium hydroxide) | Eluent 2 (500 mM sodium acetate) | Eluent 3 (Deionized water) |
0 | 40% | 0% | 60% |
10 | 40% | 0% | 60% |
60 | 40% | 50% | 10% |
70 | 40% | 50% | 10% |
We thank Andrew Baker for his assistance with mass-spectrometric instrumentation, and Drs. Jörgen Vessman and Lennart Lundberg for their interest in this study. This study was supported by Grant GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services, and a grant-in-aid from Astra/Hässle, Mölndal, Sweden.
ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; BSSL, bile salt-stimulated lipase; CE, capillary electrophoresis; CID, collision-induced dissociation; DMSO, dimethylsulfoxide; GlcNAc, N-acetylglucosamine; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PNGaseF, N-glycosidase F.
1To whom correspondence should be addressed.
2Present address: Department of Chemistry, University of the United Arab Emirates,Al-Ain, United Arab Emirates