The aspartic protease produced extracellularly by the fungus Mucor pusillus (Arima et al., 1967) possesses relatively high milk-clotting activity along with low proteolytic activity. It is called Mucor rennin and widely used as milk coagulant in industrial cheese production. The structural gene of Mucor pusillus rennin encodes a prepro enzyme composed of 361 amino acid of the mature rennin and an additional NH2-terminal sequence of 66 amino acid residues (Tonouchi et al., 1986) enclosing the signal peptide and the pro-region. The gene was expressed by Yamashita et al., (1987) in Saccharomyces cerevisiae cells under the yeast GAL7 promoter control and a highly glycosylated form of rennin was excreted into the medium efficiently. Recently the Mucor pusillus rennin was also cloned in the methylotrophic yeast P.pastoris, and the mature protein was secreted reaching expression levels higher than ~1.5 mg/ml (unpublished observations).
The amino acid sequence of M.pusillus rennin contains three potential N-glycosylation sites located at the Asn79, Asn113, and Asn188. Commercial preparation of natural rennin contains only a few glycosidic moieties in its molecule. However, rennin secreted by recombinant yeast was highly glycosylated mainly with oligomannosidic N-glycans (Aikawa et al., 1990).
Recent studies have indicated that the number of mannose residues added to P.pastoris recombinant glycoproteins are mainly Man8GlcNAc2 and Man9GlcNAc2, being clearly smaller than the ones observed for glycoproteins expressed in Saccharomyces cerevisiae. However, in some cases larger oligosaccharides have also been described for recombinant proteins secreted from P.pastoris (e.g., HIV gp120) (Scorer et al., 1993). The presence of charged oligosaccharides was shown in the P.pastoris expressed recombinant kringle 2 domain of tissue-type plasminogen activator by Miele et al., (1997).
In the present study the N-glycosylation pattern of M.pusillus rennin expressed in recombinant P.pastoris cells was determined using a combination of the recently developed oligosaccharide two-dimensional profiling techniques (Quintero et al., 1998), enzymatic and chemical degradation and mass spectrometric techniques.
Structural characteristics of N-linked oligosaccharides on aspartic protease secreted by P.pastoris
The size of N-linked oligosaccharides present on the Mucor pusillus aspartic protease glycoprotein secreted from P.pastoris was examined using a combination of HPLC and FACE techniques. As described in Materials and methods, N-linked carbohydrates were removed by digestion with an endoglycosidase (PNGase F), derivatized with the fluorogenic compound ANTS and separated by size using HPLC and FACE. For HPLC analysis, the retention times of fractions were compared to that of the standard Man7GlcNAc2-ANTS oligomannoside. For FACE analysis, electrophoretic migration of a particular band was compared to that of a ladder of ANTS-malto-oligosaccharides run in a parallel lane. Both methods separated molecules primary by size and our analysis assumed that all N-linked carbohydrates observed were of the oligomannoside type composed of two GlcNAc plus an unknown number of mannose residues. Relative migration index (RMI) (reported as glucose units) and relative retention times (trMan7) showed a straightforward correlation to oligosaccharide size (Quintero et al., 1998). The contribution of a single Man residue in this methodology was previously reported as RMI = 0.7 and trMan7 = 0.09.
The HPLC profile generated by P.pastoris-secreted aspartic protease glycans, revealed that structures larger than Man9GlcNAc2 are present on its profile displaying an even distribution of sizes (Figure
Figure 1. (A) N-Glycan profile in NH2-HPLC column using ion suppression conditions of ANTS-oligosaccharide pools from recombinant aspartic protease. The peak marked with an asterisk corresponds to Man7GlcNAc2 used as internal standard. Man9GlcNAc2 oligomannoside peak was identified in the chromatogram. (B), Fluorophore assisted carbohydrate electrophoresis (FACE) of the isolated oligosaccharides from aspartic protease. The lane numbers correspond to the same HPLC fraction numbers. Lane a (ladder of ANTS-malto-oligosaccharide), used as molecular weight markers. Image was captured using a CCD camera by irradiating the gel with UV excitation light.
Table I.
Protein
Fraction
Rel. retention time (trMan7)
Rel. migration index (RMI) glucose units (GU)
Assignmenta
Aspartic
1
1.23
9.4
M9
protease
2
1.33
10.2
M10
3
1.40
10.9
M11
4
1.48
11.4
M12
5
1.57
12.1
M13
6
1.66
7.0; 7.9
?
7
1.79
9.0; 9.9
?
8
2.04
6.9
?
HPLC-fraction 6 was subjected to [alpha]1,2-mannosidase digestion giving very limited reaction products (results not shown). On another approach, direct alkaline phosphatase digestion of the same fraction-6 gave also no reaction products, excluding the presence of terminal phosphate groups. However, a combination of mild acid hydrolysis and subsequent alkaline phosphatase treatment was performed to this fraction. This reaction, previously described by Varki and Kornfeld, (1980), remove first phosphodiester bridged mannose residues followed by removal of the phosphate group.
The initial acid hydrolysis step gave rise to two bands in FACE at even lower relative migration indexes (RMI = 7.0 and 6.1) (Table II). The trMan7 was not determined after mild acid hydrolysis because of the strong interaction with the stationary phase of the reaction products. This first increase in electrophoretic migration suggests an increase on the overall charge or a size decrease of the oligosaccharide molecule. The reaction products were recovered from the gel and then treated with alkaline phosphatase showing an increase, of each band, in their relative migration indexes. A further HPLC analysis showed two peaks at lower retention times than the original single one corresponding to fraction 6 (Table II). These reaction products were assigned to Man10GlcNAc2 and Man11GlcNAc2 neutral oligomannosides (Figure
Figure 2. Plotting of the trMan7 and RMI values of standard oligomannosides from Man5GlcNAc2 to Man9GlcNAc2 (solid circles) and fraction 6 (solid squares) before and after the consecutive mild acid hydrolysis and alkaline phosphatase digestion. The arrow indicates the initial and final stage of the reactions.
Table II.
Glycoform peak 6
Before ah and ap
After ah
After ah and ap
trMan7
1.66
NDa
1.40
1.30
RMI
7.9
7.0
10.8
7.0
6.1
10.1
aND, Not determined.
Site specific glycosylation
Reduced and carboxymethylated protein was treated with the proteolytic enzyme Asp-N. The resulting proteolytic peptide mixture was separated by a rp-HPLC (C18) system (Figure
Figure 3. Separation in rp-HPLC (C18) system of proteolytic peptide mixture obtained after Asp-N digestion. Peptides were eluted with a linear gradient of 4% to 56% acetonitrile in 0.06% trifluoroacetic acid at a flow rate of 40 µl/min. The absorbance of the eluent was monitored at 214 nm. Signaled by arrows are the glycopeptides analyzed by MALDI-MS.
Figure 4. Amino acid sequence of aspartic protease. The potential N-glycosylation sites appears in bold and italics. Glycopeptides analyzed by MALDI-MS are underlined.
The MALDI-MS spectrum of fraction 10 showed a series of molecular ions with a mass increment of 162 Da, characteristic of the presence of oligomannosidic N-glycans (Man6-17GlcNAc2) attached to first glycosylation site (Asn79). Additionally, a series of molecular ions of approximately equal intensity were observed exhibiting a mass shift of 80 Da compared to the molecular ion series explained above. This indicates the presence of phosphorylated glycans confirming the results obtained by the methods described before (Figure
Figure 5. MALDI-MS spectra of (Figure 3, fraction 10) glycopeptide obtained by Asp-N digestion of the recombinant glycoprotein. 2,5-dihydroxybenzoic acid was used as UV-absorbing matrix. Measurements were performed on a Bruker REFLEX MALDI mass spectrometer using a N2 laser (337 nm). The ions were detected in a positive mode. The series signaled by asterisks corresponds to the neutral oligomannosides and signaled by arrows the charged (phosphorylated) species. At the bottom, the glycopeptide amino acid sequence, the arrow below the sequence shows the N-terminal sequence verified by automatic Edman degradation. Microscopy analysis
The P.pastoris strain expressing aspartic protease was harvested from molasses medium and biomass samples every 6 h (after induction) were taken for further characterization by electron microscopy. The same P.pastoris strain but expressing dextranase was used as negative control. Incubation with affinity purified polyclonal antibody to aspartic protease localized the recombinant protein at the periplasmic space suggesting that there is not intracellular retained protein (Figure
Figure 6. Transmission electron microscope view at immunocytochemical level showing in P.pastoris cell, at 72 h of induction with methanol. A polyclonal antibody to aspartic protease (1 mg/ml) was used. Influence of the culture medium on glycosylation profiles
Many authors have reported that differences in the cell culture conditions can profoundly affect the final glycosylation pattern of secreted recombinant glycoproteins (Watson et al., 1994; Gawlitzek et al., 1995). In order to evaluate the influence of different growth media on the variation of the glycoforms, aspartic protease was purified from the same P.pastoris strain cultured in two different media, a molasses-based medium (Roca et al., 1996) and an inorganic salts-based medium supplemented with whey. Pure aspartic protease was then enzymatically deglycosylated, and oligosaccharide pools derivatized with ANTS were separated by HPLC (Figure
Figure 7. NH2-HPLC profiles of oligosaccharide from aspartic protease harvested in molasses (A) and inorganic salt media (B). HPLC separation was achieved as described previously.
P.pastoris has become an important system for the synthesis and secretion of foreign proteins for academic and commercial uses (Romanos, 1992). The general characteristics of the N-linked oligosaccharides structures on heterologous proteins secreted by this methylotrophic yeast have been recently reported (Montesino et al., 1998). The Man8GlcNAc2 and Man9GlcNAc2 oligosaccharides are present in almost all the proteins analyzed, lower and higher structures were also detected from Man6-17GlcNAc2. Hypermannosylation of foreign proteins that is commonly observed in S.cerevisiae does not often occur in P.pastoris. Aspartic protease analyzed in this paper, however, showed the additional presence of phosphorylated oligosaccharides.
The presence of mannose 6-phosphate in yeast glycoproteins was demonstrated by Mill, (1966). Previous work reported phosphorylated N-linked oligosaccharides from S.cerevisiae glycoproteins (Hernández et al., 1989a) where mono- and di-phosphate fractions, were isolated mainly in the diester form (Hernández et al., 1989b). However, little is known about charged oligosaccharide structures on P.pastoris-secreted proteins.
MALDI-MS corroborated these results and furthermore showed site-specific glycosylation. Whereas in site I a mixture of neutral and charged oligomannosides (Man6-17GlcNAc2) was detected, site III mainly carried a less heterogeneous population of neutral oligosaccharides. Oligosaccharide phosphorylation of a P.pastoris-secreted protein was also reported by Miele et al., (1997), on the recombinant kringle 2-domain of tissue-type plasminogen activator (tPA).
Although the role of phosphate groups in yeast cells is unknown, it was postulated that the oligosaccharide[prime]s phosphorylation does not lead to protein targeting to the vacuole (Hercovics and Orlean, 1993). Particularly, we confirmed this pattern in P.pastoris by immunomicroscopy (Figure
When the same recombinant strain was grown in a salts-based medium, lesser amounts of phosphorylated glycans were detected, in comparison of the glycosylation pattern of the protein obtained from molasses-based medium (Figure
Electrophoresis reagents (electrophoresis grade) were from Bio-Rad Inc. (Richmond CA). ANTS-reagent was a gift kindly given by P.Jackson of Cambridge University. PNGase F was from BioLabs (Beverly, MA). Oxford GlycoSystems (Oxford, UK) supplied oligomannoside standards and ([alpha]1,2-mannosidase Aspergillus saitoi). Acetonitrile (lichrosolv) was from Riedel-de-Haem (Seelze, Germany). HPLC instrument was from Pharmacia (Pharmacia-LKB, Sweden). The Nucleosil 5 HPLC-NH2 column was purchased from Knauer (Germany). Ribonuclease B was from Sigma Chemical Co. (St. Louis, MO). DIG Glycan Differentiation Kit was bought from Boehringer (Mannheim, Germany). All other reagents were of analytical grade. Recombinant glycoprotein
Aspartic protease was obtained from the Center for Genetic Engineering and Biotechnology (CIGB, Cuba), BioTechnology Division. The glycoprotein was obtained from an integrative expression vector, using the yeast signal sequence (SUC2), the AOX1 promoter of P.pastoris, the terminator sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPT) gene and the HIS3 gene marker. Fermentation conditions
Fermentation in a molasses medium consisting of 10 mM (NH4)2HPO4, 6.8 mM (NH4)2SO4, 8.3 mM urea, and 4% total reducing sugars from cane molasses (Roca et al., 1996). Upon depletion of the carbon source, the AOX1 promoter was induced by adding 1% (v/v) methanol. When the culture began to acidify, the methanol-feeding rate was started at 2 g l-1 per h and was gradually increased up to 3.5 g l-1 per h.
Aspartic protease obtained from P.pastoris cells were also harvested in an inorganic salt-based medium supplemented with whey. One liter of fermentation medium contained: 40% of whey, 30 mM (NH4)2HPO4, 30 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 6.7 mM KCl, 12.5 mM urea, 3.5% (w/v) glycerol, and 5 g/l yeast extract. Additionally 4 ml PTM1 trace salts (38 mM cupric sulfate, 0.5 mM sodium iodide, 17.75 mM manganese sulfate, 0.83 mM sodium molybdate, 0.32 mM boric acid, 2.1 mM cobalt chloride, 147.75 mM zinc chloride, 233.8 mM ferrous sulfate, 0.2% (w/v) biotin and 0.5% (v/v) sulfuric acid) were added. Methanol induction was started by feeding methanol at a rate of 2 g per l-1 per h. The supernatant was harvested after total induction by methanol. Protein purification
The clear supernatant was dialyzed against 10 mM sodium acetate, pH 5.5. The crude protein was applied on FFQ Sepharose (Pharmacia Fine Chemicals, Sweden) column (145 × 20 mm) previously equilibrated with the same buffer. Aspartic protease was eluted with a linear gradient of 10 mM to 1 M sodium acetate pH 5.5 at a flow rate of 1 ml/min. The fractions collected were tested for enzymatic activity.
The purity of the protein preparation was greater than 95% and it was checked to be absent of any other glycoprotein-contaminant using the Dig Glycan Differentiation Kit (Boehringer, Germany). Endoglycosidase digestion of glycoproteins
About 250 µg of pure and desalted protein was resuspended in 5 µl deglycosylation buffer (0.5 M sodium phosphate, pH 7.5) and 5 µl denaturing buffer (5% (w/v) SDS, 10% (v/v) [beta]-mercaptoethanol). Denaturation was carried out at 100°C for 5 min. Nonidet NP-40 (5 µl) was added and then diluted with distilled water to a final protein concentration of 5 mg/ml. PNGase F (2 µl) was added and incubated for 2 h. at 37°C. Deglycosylated proteins were precipitated by adding three volumes of cold ethanol and leaving for 10 min at -20°C, followed by centrifugation at 3000 r.p.m. for 3 min. When required, the protein was redissolved in water, precipitated again, and the supernatants combined. Fluorophore labeling of oligosaccharides
The label was fulfilled according to Cremata et al., (1998). The oligosaccharides obtained either by a complete PNGase F digestion of glycoproteins or directly from a commercial source were derivatized according to Jackson, (1990). Oligosaccharides dried in a centrifugal vacuum evaporator (CVE) were reductively aminated with 5 µl of 0.15 M-ANTS in acetic acid (3:17, v/v) and 5 µl of 1 M sodium cyanoborohydride in dimethyl sulfoxide, and incubated for 16 h at 37°C. The reaction was diluted 10 time in water and dried in a CVE for 1 h. Fluorophore-assisted carbohydrate electrophoresis (FACE)
Polyacrylamide gels (30% w/v acrylamide-0.8% w/v bis-acrylamide) and running buffer were prepared according to Jackson, (1990). Electrophoresis was performed in a Mighty Small II SE 250 apparatus (Hoefer Scientific Instrument, San Francisco, CA) at 20 mA constant current. The run was stopped 10 min after the ANTS excess reached the bottom of the gel. Electrophoretic mobility was reported as relative migration index (RMI) values, as described by Stack and Sullivan, (1992), compared to a ladder of maltooligosaccharide-ANTS series obtained by [alpha]-amylase enzymatic hydrolysis of amylose and further derivatized with ANTS. HPLC running conditions
High performance liquid chromatography (HPLC) separations were carried out on a Nucleosil 5 NH2-column (4.5 × 250 mm) using a C18 cartridge pre-column. Buffer (A): glacial acetic acid (6% v/v) in a 70:30 (v/v) mixture of acetonitrile-water titrated to pH 5.5 with triethylamine. Buffer (B): glacial acetic acid (6% v/v) titrated to pH 5.5 with triethylamine. Gradient: 5% to 50% B buffer in 90 min at a flow rate of 0.7 ml/min. Fluorescence was measured using a Shimadzu RF 530 (Kyoto, Japan) detector at [lambda]exc. 353 nm and [lambda]em. 535 nm. All the retention times are calculated relative to Man7GlcNAc2 (trMan7) oligomannoside standard from Oxford GlycoSystems (Oxford UK). Oligosaccharide composition assignment
Oligomannoside composition analysis was done according to Cremata et al., (1998) and Quintero et al., (1998). The values of trMan7 and RMI of standard oligomannosides (Man5-9GlcNAc2) were plotted into a graph, resulting in a linear correlation between these two parameters. It could be assumed as equivalent structures when the behavior of the unknown oligosaccharide is similar to a standard oligomannoside. Sizes of bigger oligomannosides are determined considering the contribution of a single mannose residue in the two-dimensional graph (RMI = 0.7 and trMan7 = 0.09). Finally, a deviation from this straight line implies the presence of structural motives different to that of neutral oligomannoside oligosaccharides. Exoglycosidase digestion of oligosaccharide-ANTS derivatives
Purified oligosaccharide-ANTS derivatives from HPLC were dried in a CVE. Isolated glycans at a concentration of 15 µM were incubated with [alpha]1,2-mannosidase (from Aspergillus saitoi) at a final concentration of 0.5 mU/ml in 100 mM sodium acetate pH 5.0 at 37°C for 18 h. For HPLC analysis, samples were injected directly after enzymatic digestion. For FACE analysis samples were previously dried in a CVE and redissolved in 10 µl of 20% w/v glycerol. Mild acid hydrolysis of oligosaccharide-ANTS derivatives
HPLC-purified ANTS-oligosaccharide derivative (1 nmol) was dissolved in 300 µl of 0.01 N HCl and heated at 100°C for 30 min. Before treatment the pH was adjusted at 2.5. Samples were dried in a CVE and redissolved in 5 µl of glycerol:water (1:4 v/v) before being subjected to FACE analysis. Alkaline phosphatase treatment
Oligosaccharide electrophoresis bands from FACE separation, after a mild acid hydrolysis, were excised from the gel and recovered by immersing them in 300 µl of water for 3 h at 4°C. The solution was dried in CVE and redissolved in a reaction buffer (0.1 mM EDTA, 0.05 mM Tris-HCl, pH 8.5). Incubation with alkaline phosphatase (Boehringer Mannheim, Germany) was performed at 37°C for 16 h using 5 U of the enzyme. Reduction and S-carboxymethylation of the cystein residues
The procedure was done according to Charbonneau, (1989). The lyophilized protein was dissolved in reduction and carboxymethylation buffer (Tris-HCl 0.3 mM, pH 9.5, guanidinium chloride 6 M, and EDTA 0.03 M) at a concentration of 5 mg/ml. Dithiothreitol (DTT) equivalent to 20 times the number of cystein residues was added. Reaction was performed at 37°C for 2 h. After that an amount of iodoacetic acid equivalent to 2× DTT was added and incubated for 1 h at room temperature in the dark. The sample was desalted in a PD-10 column (Pharmacia Uppsala, Sweden) in bidistilled water. Proteolytic digestion
Denaturated protein (250 µg) was resuspended in 1% (w/v) bicarbonate buffer pH 8.0. Asp-N digestion was performed at 37°C for 4 h using an enzyme:substrate ratio of 1:50 (w/w). Separation of the digest of the aspartic protease
The digest was analyzed on a HPLC system using a reversed-phase Aquapore OD-300 C18 column (1.0 × 100 mm) Applied Biosystems 172A. Peptides were eluted with a linear gradient of 4% to 56% acetonitrile in 0.06% trifluoroacetic acid at a flow rate of 40 µl/min. The absorbance of the eluent was monitored at 214 nm and by mass spectrometry on a TSQ 700 triple quadrupol instrument equipped with a Finnigan electrospray ion source connected on line to the HPLC system. Matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-MS)
2,5-Dihydroxybenzoic acid was used as UV-absorbing matrix; 10 mg/ml 2,5-dihydroxybenzoic acid was dissolved in 10% (v/v) ethanol in water. For analysis by MALDI-MS the solutions of the reduced and carboxymethylated glycopeptides. One microliter of the sample was spotted onto the stainless steel target and dried at room temperature. The concentrations of the analyte mixture were ~10 pmol/µl. Measurements were performed on a Bruker REFLEX MALDI (Bruker-Franzen, Germany) mass spectrometry using a N2 laser (337 nm) with 3 ns pulse width and 107-108 W/cm2 irradiance at the surface (0.2 mm2 spot). Spectra were recorded at an acceleration voltage of 20 kV. Sequence analyses
Automatic amino acid sequence analysis of glycopeptides was performed using an Applied Biosystems Sequencer model 475A (Applied Biosystems, EUA). A PTH 120A analyzer was used for amino acid phenylthiohydantoins analyses. Electron microscopy
The yeast samples were fixed in 4% paraformaldehyde containing 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3 at 4°C for 2 h. Postfixed for 1 h in 1% OsO4 was done. Samples were rinse in 0.1 M phosphate buffer, pH 7.2 and dehydrated in increasing ethanol concentrations. After embedding in Spurr resin (Spurr, 1969), the blocks were sectioned with an ultramicrotome 2188 (LKB, Sweden) and the ultrathin sections were placed on 400 mesh grids without membrane. The samples were incubated with a polyclonal antibody (1 mg/ml) for 1 h. and then with a colloidal gold-protein A conjugated diluted 1/200 for 1 h (Amersham, England). The sections were stained and analyzed in a transmission electron microscope JEOL/JEM 2000 EX.
We thank Prof. P.Jackson from University of Cambridge for providing ANTS and Eduardo Ramos and Vladimir Martínez for support in fermentation. We are grateful to the cloning and expression staff of the BioIndustry Department.
Man, mannose; GlcNAc, N-acetyl glucosamine; AOX1, alcohol oxidase 1 gene; GAPT, glyceraldehyde-3-phosphate dehydrogenase gene; HIS3, histidine 3 gene; ANTS, 8-amine-1,3,6- naphthalene trisulfonic acid; FACE, fluorophore assisted carbohydrate electrophoresis; HPLC, high performance liquid chromatography; RMI, relative migration index; trMan7, relative retention time respect to Man7GlcNAc2; CVE, centrifugal vacuum evaporator; PNGase F, peptide-N4-(N-acetyl-[beta]-d-glucosaminil) asparagine amidase; MALDI/TOF-MS, time of flight matrix assisted laser-desorption ionization mass spectrometry; ESI-MS, electrospray mass spectrometer.
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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