Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus

Isam Khalaila1,2,3, Jasna Peter-Katalinic3, Clarence Tsang4, Catherine M. Radcliffe4, Eliahu D. Aflalo5, David J. Harvey4, Raymond A. Dwek4, Pauline M. Rudd4 and Amir Sagi5

3 Institute for Medical Physics and Biophysics University of Münster, Robert-Koch-Str. 31 D-48149, Münster, Germany; 4 Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK; 5 Department of Life Sciences and the Institute for Applied Biosciences, Ben Gurion University, P.O. Box 653, Beer Sheva, Israel

Received on February 17, 2004; revised on May 26, 2004; accepted on May 27, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycosylation is of importance for the structure and function of proteins. In the case of vitellin (Vt), a ubiquitous protein accumulated into granules as the main yolk protein constituent of oocytes during oogenesis, glycosylation could be of importantance for the folding, processing and transport of the protein to the yolk and also provides a source of carbohydrate during embryogenesis. Vt from the crayfish Cherax quadricarinatus is synthesized as a precursor protein, vitellogenin (Vg), in the hepatopancreas, transferred to the hemolymph, and mobilized into the growing oocyte via receptor-mediated endocytosis. The gene sequence of C. quadricarinatus shows a 2584-amino-acid protein with 10 putative glycosylation sites. In this study a combined approach of lectin immunoblotting, in-gel deglycosylation, and mass spectrometry was used to identify the glycosylation sites and probe the structure of the glycan moieties using C. quadricarinatus Vg as a model system. Three of the consensus sites for N-glycosylation—namely, Asn152, Asn160 and Asn2493—were glycosylated with the high-mannose glycans, Man5–9GlcNAc2, and the glucose-capped oligosaccharide Glc1Man9GlcNAc2.

Key words: N-glycan / oligosaccharides / site of glycosylation / vitellogenin / vitellin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Vitellin (Vt), is the major yolk protein in crustaceans, as in all oviparous animals. Vt is accumulated in yolk bodies in the oocytes during vitellogenesis, a process that has been well characterized in oviparous vertebrates and insects and lately in crustaceans. Vt later serves the developing embryo as an important source of proteins, lipids, and carbohydrates. As early as 1967, crustacean Vt was shown to be a high-density glycolipoprotein (Wallace et al., 1967Go) containing a high percentage of lipids, invariably conjugated to carotenoid pigments. Other studies have shown that crustacean Vt is a high-density lipoprotein (HDL) complex (Meusy and Payen, 1988Go).

In the decapod Cherax quadricarinatus, vitellogenin (Vg), the precursor of Vt, is produced in the hepatopancreas of vitellogenic females (Abdu et al., 2002Go). Vg is a high-molecular-weight lipoglycocarotenoprotein composed of several subunits with similar biochemical and immunological characteristics to Vt (Chang et al., 1994Go; Derelle et al., 1986Go; Kerr, 1969Go; Lee and Watson, 1994Go; Meusy, 1980Go). The partial amino acid sequencing of Vt subunits has allowed degenerate oligonucleotide primers to be produced and then used for complete cDNA isolation and sequencing of C. quadricarinatus Vg. The cDNA sequence indicates that Vg is composed of 2584 amino acids (Abdu et al., 2002Go). Furthermore, northern analysis of the Vg cDNA indicated a single 8000-nucleotide mRNA band present only in the hepatopancreas of vitellogenic females, suggesting that in C. quadricarinatus, Vg is produced in the hepatopancreas and transported to the maturing oocyte via the hemolymph (Abdu et al., 2002Go). Several proteins, with molecular weights of 86, 177, and 196 kDa, have been specifically found in the hemolymph of secondary vitellogenic C. quadricarinatus females (Yehezkel et al., 2000Go), and it has been suggested that these proteins are recognized and transported to the growing oocyte through receptor-mediated endocytosis (Warrier and Subramoniam, 2002Go), a process known to be related to the glycan content of the mobilized protein. Glycosylation has been shown to be involved in a number of functional roles, including protection of proteins from proteases (Marinaro et al., 2000Go), assistance in protein folding, targeting of proteins to specific locations, and regulation of protein activity (Helenius and Aebi, 2001Go). Thus vitellogenesis in C. quadricarinatus is a unique model for studying glycans and their roles in protein folding, processing, and transport, because the target protein is produced at an extra-ovarian site, released to the hemolymph, and taken up by the oocyte through receptor-mediated endocytosis.

The protein and lipid components of Vt have been characterized in both invertebrate and vertebrate systems (de Chaffoy de Courcelles and Kondo, 1980Go; Fyffe and O'Connor, 1974Go; Ohlendorf et al., 1977Go; Raikhel and Dhadialla, 1992Go; Tirumalai and Subramoniam, 1992Go, 2001Go), but very little information is available on the carbohydrate components of this major yolk protein, despite the structural and functional importance of these units. Many studies have been performed on insect glycoprotein due to the wide use of insect cell systems to produce glycosylated recombinant proteins. Although these studies suggested the presence of an N-glycosylation pathway in insect cells similar to that seen in mammalian cells, the most frequent structure of insect N-glycans is the paucimannosidic glycan, Man3GlcNAc2(±Fuc), not found in mammalian cells (Jarvis and Finn, 1995Go). Structural analysis of the glycan moiety of insect Vt revealed high-mannose oligosaccharides (Raikhel and Dhadialla, 1992Go). Using radiolabeling and chromatography, lipovitellin from the crustacean Emerita asiatica was shown to contain five different O-linked oligosaccharides and four different N-linked oligosaccharides (Tirumalai and Subramoniam, 2001Go), but no structural details of these glycan moieties are available. In contrast, the sequence of C. quadricarinatus Vg shows 10 putative N-glycosylation sites with the consensus sequence Asn-X-Ser/Thr (Abdu et al., 2002Go). In this study, the sites of glycosylation and glycan structure have been elucidated in Vg from the hemolymph and Vt from the eggs of C. quadricarinatus using a combined approach based on lectin immunoblotting, enzymatic in-gel deglycosylation, exoglycosidase sequencing, and chromatographic and mass spectrometric analysis.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lectin blotting of Vg and Vt proteins
Five and three major proteins could be found in the hemolymphatic and ovarian HDL fraction respectively, with apparent molecular weights of 86, 96, 177, 196, and 208 kDa in the hemolymph and 75, 86, and 96 kDa in the ovary (Figure 1A). Excluding the 75-kDa protein, all of the proteins were shown to exhibit a strong interaction with Galanthus nivallis agglutinin (GNA), which recognizes N-glycans and/or O-linked mannoses (Figure 1B), but no interactions were observed with the other lectins used, Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), peanut (Arachis hypogaea) agglutinin (PNA), and Dature stramonium agglutinin (DSA), which recognize specificly sialic acid linked {alpha}(2-6) to galactose, sialic acid linked {alpha}(2-3) to galactose, galactose ß(1-3) N-acetylgalactosamine and galactose ß(1-4)GlcNAc, respectively, (data not shown).



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Fig. 1. Ovarian (OV) and hemolymph (HEM) HDL from C. quadricarinatus separated on 7% SDS–PAGE. (A) Coomasie blue–stained SDS–PAGE; (B) blot probed with GNA, specific for high-mannose glycan chains.

 
Mass spectrometry analysis of Vg proteins and glycosylation site
Before the carbohydrate moiety could be obtained, the identity of the subunits of the HDL fractions 86, 177, and 196 kDa from the hemolymph and 86 kDa from the ovary (Figure 1A) were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the tryptic digest as deduced from the C. quadricarinatus Vg cDNA (accession number AF306784). All the proteins were found to correspond to specific regions of the Vg gene. Subsequently, all the corresponding regions included potential consensus N-glycosylation sites. The LC-MS/MS analysis of the 86-kDa protein revealed 9.7% coverage of the 2584 amino acids deduced from Vg cDNA. Coverage of 18.3% and 7.6% have been obtained from the LC-MS/MS analyses of 177 kDa and 196 kDa proteins, respectively.

In the LC-MS analysis of the tryptic digest of the electroeluted intact 86-kDa Vg, a series of quadruply charged molecular ions were found. The most intense peak at m/z 1692.464+ could be assigned to the glycopeptide 140–169 amino acid residues (calculated mass = 3034.48) glycosylated at Asn152 and Asn160 with Hex18(HexNAc4) moieties (calculated = 3729.18) that together give a mass of [6763.66 + 4H]/4 = [m/z 1691.92]4+ (Figure 2A). The spacing between the other peaks (162/4) corresponded to hexose. Thus the quadruply charged ions at m/z 1651.9, 1611.4, 1570.9, 1530.4, and 1489.9 corresponded to Hex17(HexNAc4), Hex16(HexNAc4), Hex15(HexNAc4), Hex14(HexNAc4), and Hex13(HexNAc4), respectively. After peptide N-glycosidase F (PNGase F) treatment and simultaneous digestion with trypsin and chymotrypsin, a shift of two mass units was observed in the doubly charged parent ion at m/z 1253.0 from the peptide containing amino acids 146–169 (calculated m/z 1252.1), and a shift of one or two mass units was observed in the peptide fragments bearing one or both originally glycosylated Asn residues, respectively. This shift was caused by conversion of Asn to Asp in the deglycosylation reaction (Figure 2B and Table I). In the tryptic digest of the PNGase F–treated 196-kDa Vg, a doubly charged ion at m/z 917.46 was observed by LC-MS corresponding to amino acids 2493–2508 (calculated m/z 916.94) (Table I); LC-MS/MS confirmed the sequence and the transition of Asn2493 to Asp (Table I).



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Fig. 2. LC-MS/MS for the glycosylation sites of C. quadricarinatus Vg. (A) Electrospray mass spectrum of the intact glycopeptide 140–169 amino acid residues, glycosylated at N152 and N160, numbers above the peaks represent [M + 4H]4+ of different number of hexoses. Hex represent the mass difference of a hexose between the peaks, adjacent peaks at higher mass than the main peaks are the sodiated adducts. (B) LC-MS/MS spectrum of the doubly charged ion at m/z 1253.0 of the glycopeptide 146–160 amino acid residues after PNGase F treatment; the transition of Asn to Asp acid is shown in the fragment profile. Molecular representations of the sugars are as indicated in the legend to Figure 4.

 

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Table I. Assignment of molecular ions of Vg peptides before and after PNGase F treatment

 


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Fig. 4. NP HPLC chromatogram of the glycans released from the 86-kDa band from the SDS–PAGE gel showing the oligomannose series Man5–9GlcNAc2 and Glc1Man9GlcNAc2. The lower chromatogram shows the JBM digest of the glycan pool. The products of the digestion are Man1GlcNAc2 and Glc1Man4GlcNAc2. Molecular representations of the sugars are included. The individual monosaccharides are represented as follows: open square, glucose; open circle, mannose; dotted line, {alpha} linkage; solid line, ß linkage. The linkage positions of the oligosaccharides are represented by the angle of the line linking adjacent monosaccharides. The sugar on the left is linked via C1 to ring carbons C2 (180°), C3 (225°), C4 (270°), or C5 (315°) of the sugar on the right.

 
MS of the N-glycans
Monoisotopic [M + Na]+ ions at m/z 1257.4, 1419.5, 1581.5, 1743.5, 1905.5, and 2067.6 were detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS in the N-linked oligosaccharides from the 86-kDa Vg subunits from the hemolymph and the ovary treated with PNGase F (Figure 3 and Table II). The 162-Da spacing of the ions is indicative of hexose units, and thus different hexose-type structures [Hex5–10HexNAc2] could be deduced from the measured monoisotopic masses (Table II). The N-linked oligosaccharide released by PNGase F from both the ovarian and hemolymphatic 86-kDa subunit gave the highest signal intensity compared to the N-glycan mixture released from 177- and 196-kDa Vg subunits (Figure 3). The [M + Na]+ ion at m/z 1905.6 [Hex9HexNAc2] gave rise to the most intense peak within the spectrum of the 86-kDa Vg subunit (Figure 3A and Table II). In the spectra of oligosaccharides released from the hemolymphatic 177- and 196-kDa proteins, the ion at m/z 1905.6 was almost completely dominant.



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Fig. 3. MALDI mass spectra of oligosaccharides released from C. quadricarinatus Vg subunits by PNGase F treatment in gel. Molecular weights correspond to the monoisotopic masses of the [M + Na]+ ions. Derived compositions are shown in the inset table. Hex: Hexose and HexNAc: N-acetylhexosamine. (A) Spectrum of N-oligosaccharides released from the 86-kDa Vg protein. (B) Spectrum of N-oligosaccharides released from 177-kDa Vg protein. (C) Spectrum of N-oligosaccharides released from 196-kDa Vg protein.

 

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Table II. Structure details of the glycans [M + Na]+ of C. quadricarinatus 86-kDa Vg

 
Exoglycosidase sequencing of oligosaccharides
The N-glycans released from the protein bands by in-gel digestion with PNGase F were labeled with the fluorophore 2-aminobenzamide (2AB) by reductive amination (Bigge et al., 1995Go). The chromatogram obtained by normal-phase high-performance liquid chromatography (NP HPLC, Figure 4, upper chromatogram) shows the intact glycan pool of the 86-kDa band with a series of oligomannose sugars, Man5–9HexNAc2, and the monoglucosylated oligomannose, Glc1Man9HexNAc2. Glycan structures were assigned from the glucose unit (GU) values of the intact glycan pool combined with data from digestions with the exoglycosidases jack bean {alpha}-mannosidase (JBM) and glucosidase II (glcII). JBM cleaves the nonreducing terminal mannose {alpha} 1-2, 3, and 6 linkages, and glcII cleaves the nonreducing terminal glucose {alpha} 1-3 mannose linkage. Digestion with JBM is shown in the lower chromatogram in Figure 4, in which Man5–9HexNAc2 is digested to Man1 HexNAc2 (GU 2.65) and Glc1Man9HexNAc2 is digested to Glc1Man4HexNAc2 (GU 5.99). Figure 5 shows the intact glycan pool (upper chromatogram) together with the digestion with glcII (lower chromatogram) in which peak 6, Glc1Man9HexNAc2 (GU 10.16) is completely digested to Man9HexNAc2 (GU 9.52). The peak areas of the glycans in the enzyme digests correlated with those of the undigested glycan pool. The assignments were confirmed by MALDI MS of the complete glycan pool (Table II).



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Fig. 5. NP HPLC chromatogram of the glycans released from the 86-kDa band from the SDS–PAGE gel showing the oligomannose series Man5–9GlcNAc2 (peaks 1–5) and Glc1Man9GlcNAc2 (peak 6). The lower chromatogram shows the glucosidase II digest of the glycan pool where Glc1Man9GlcNAc2 has been digested to Man9GlcNAc2. Molecular representations of the sugars are as indicated in the legend to Figure 4.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycosylation is important for protein structure and function. In the case of Vt, the glycan moieties also serve as a carbohydrate source; in fact, this protein is a major source of carbohydrate, protein, and lipid for the developing embryo (Lee et al., 1997Go). In C. quadricarinatus Vt originates from a precursor Vg, which is produced in the hepatopancreas (Abdu et al., 2002Go) and secreted to the hemolymph. We have confirmed that Vg from the hemolymph is posttranslationally modified by N-linked oligosaccharides. Furthermore, lectin blotting and exoglycosidase sequencing have identified the glycan moieties as those of the high-mannose type. The oligomannose moieties ranged from Man5GlcNAc2 to Man9GlcNAc2, which is similar to those of crayfish Astacus leptodactylus hemocyanin (Tseneklidou-Stoeter et al., 1995Go) and other glycosylated proteins from invertebrates, such as the deep-sea tube worm Riftia pachyptila hemoglobins (Zal et al., 1998Go), the larval serum protein of Drosophila melanogaster (Williams et al., 1991Go), and the storage glycoprotein arylphorin of lepidopteran insects (Kim et al., 2003Go; Ryan et al., 1985Go). Thus the N-glycan processing pathways seem to be conserved within Arthropoda.

In insects, glucose and mannose trimming of the N-linked Glc3Man9GlcNAc2 core is possible, and the partially trimmed Glc1Man9GlcNAc2 oligosaccharide structure has also been found in the N-linked glycan of C. quadricarinatus Vg. Furthermore, recent results show that the glucose-capped oligomannose glycans, such as Glc1Man9GlcNAc2, are conserved in glycoproteins from many species, including hemoglobins from R. pachyptila (Zal et al., 1998Go), the insect storage protein arylphorin (Kim et al., 2003Go), immunoglobulins from egg yolk of hen or Japanese quail (Matsuura et al., 1993Go; Ohta et al., 1991Go), glycoprotein from the egg jelly coat of the starfish Asterias amurensis (Endo et al., 1987Go), ovarian vitellogenic substances of Asterias rubens (De Waard et al., 1987Go), and membrane protein gp63 of the parasite Leishmania (Funk et al., 1997Go). The latter authors have suggested that the presence of monoglucosylated oligosaccharides is due to the low activity or low level of glucosidase II enzyme (Funk et al., 1997Go). Kim et al. (2003)Go have gone further and have given additional examples for storage proteins, such as arylphorin with monoglucosylated N-linked oligosaccharides, mainly Glc1Man9GlcNAc2, and have suggested a role for this unique structure in storage proteins. All of these mature glycoproteins, including C. quadricarinatus Vg, contain glucose-capped oligosaccharides and are large hydrophobic proteins. Hence it is possible that protein size or hydrophobicity could be linked to the unique glucose-capped oligomannose.

The deduced sequence of C. quadricarinatus Vg is composed of 2584 amino acid residues, from which a mass of 292 kDa for the unmodified protein is calculated. Within this molecule we confirm that of the 10 putative N-glycosylation sites Asn152 and Asn160 from the 86-kDa and Asn2493 from the 177- and 196-kDa subunits are glycosylated (Table I). The exact role of this glycosylation is not yet known. However, it is well known that glycans play a pivotal role in protein folding (Imperiali and O'Connor, 1999Go; Wormald and Dwek, 1999Go), oligomerization, quality control, sorting, and transport in the endoplasmic reticulum and Golgi apparatus (Helenius and Aebi, 2001Go). In protein folding, glycosylation alters the conformational preferences close to the glycosylation site, which leads to more compact conformations (Wormald and Dwek, 1999Go). More compact conformations may restrict the activity of glucosidase II and mannosidases, thereby producing an abundance of high-mannose and glucose-capped structures.

The lectins calreticulin and calnexin interact with the glycan moieties of substrate glycoproteins that have been trimmed by glucosidases I and II to the monoglucosylated form (Hammond et al., 1994Go; Spiro et al., 1996Go). Such interactions can lead to correctly paired disulfide bonds (Huppa and Ploegh, 1998Go). Thus Glc1Man9GlcNAc2 may have a similar function in Vg. It is possible that glycosylation of Vg has an important role in folding and subunits assembly to achieve the mature protein in the hemolymph and ovary. In our model organism, Vg is secreted to the hemolymph and, because glycosylation is known to increase solubility of proteins (Jaenicke, 1991Go), N-glycans could have a significant role in keeping this large, hydrophopic protein in the hemolymph to improve its transport to the ovary. Vg uptake into the oocytes is known to be mediated by receptors (Raikhel and Dhadialla, 1992Go; Warrier and Subramoniam, 2002Go). In this respect, the glycan moiety might play a role in recognition and receptor-mediated endocytosis. On uptake into the oocytes, it might also have a role in packaging and compacting the Vt in yolk bodies. The hypotheses concerning the roles of N-glycan in Vg processing need to be clarified experimentally.

This study of the N-glycosylation of Vg has demonstrated the presence of Glc1Man9GlcNAc2 for the first time in Crustacea. The present study focused on N-glycans of Vg; nevertheless, to complete the sequence and assembly of Vg subunits and their sites of glycosylation, future work on O-glycosylation is needed. Our study of Vg oligosaccharides during vitellogenesis provides a first step in understanding the cascade of assembly, recognition, and transport of Vg, its packaging in the egg and utilization during embryogenesis in crustaceans.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
PNGase F (EC 3.5.1.52), trypsin, and chymotrypsin, sequencing grade, were obtained from Roche Diagnostics GmbH (Mannheim, Germany). JBM (EC 3.2.1.24) was obtained from Glyko (Upper Heyford, Oxfordshire, UK). Glucosidase II was prepared in the Glycobiology Institute (Oxford); its activity, which was measured by the hydrolysis of [14C]glucose-labeled Glc2Man9GlcNAc2 (Karlsson et al., 1993Go), was determined as 5.7% min–1 of labeled glucose.

HDL purification
The HDL fractions from C. quadricarinatus secondary vitellogenic ovaries and hemolymph were isolated as described by Abdu et al. (2000)Go and Yehezkel et al. (2000)Go, respectively.

Structural characterization by lectins
HDLs from C. quadricarinatus vitellogenic ovaries and hemolymph (5 µg and 15 µg, respectively) were separated on 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and proteins were electroblotted onto a nitrocellulose membrane before blotting with GNA, SNA, PNA, DSA, and MAA from the digoxigenin (DIG) glycan differentiation kit (Roche Diagnostics). The membrane was then incubated with anti-DIG conjugated with alkaline phosphatase, and 5-bromo-4-chloro-3-indolylphosphate was used to determine the presence of the alkaline phosphatase, with enzyme catalysis resulting in a dark color.

N-linked glycan analysis and exoglycosidase sequencing
N-linked glycans were released from five gel bands of the purified HDLs with apparent molecular weights of 208, 196, 177, 95, and 86 kDa according to the method described by Küster et al. (1997)Go as modified by Radcliffe et al. (2002)Go. Briefly, the HDLs were reduced with 45 mM dithiothreitol for 10 min at 70°C, before alkylation with 100 mM iodoacetamide at room temperature. Fourteen micrograms of alkylated HDLs were separated on 6% SDS–PAGE and stained with Coomassie blue R-250. After destaining with 5% (v/v) methanol/7% (v/v) acetic acid, individual protein bands were excised from the gel. The gel pieces were washed twice with 1 ml acetonitrile followed by 1 ml 20 mM NaHCO3, pH 7.0. Subsequently, the gel pieces were dehydrated with acetonitrile and dried by vacuum centrifugation (SpeedVac). N-glycans were released with PNGase F, labeled with 2AB by reductive amination, and separated by NP HPLC, using a low-salt buffer system (Guile et al., 1996Go). The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to produce a dextran ladder from which the retention times of the individual glycans were converted into GU. These GU values were compared with a database of experimental values to obtain preliminary assignments for the glycans. The HPLC profiles of the glycans from all the gel bands showed the same mannose sugars in similar proportions, but because the release from the 86-kDa band produced the most glycans, this sample was chosen for enzyme digestions. The assignments were confirmed by NP HPLC, following digestion of a 2AB-labeled glycan pool with JBM and glucosidase II and by MALDI MS of an unlabeled glycan pool.

Glycopeptide analysis: in-solution or in-gel digestion of Vg
Intact or deglycosylated in-gel Vg proteins (see earlier description) were digested with trypsin 12.5 ng/µl in 5 mM (NH4)HCO3, pH 7.5, overnight at 37°C. The resultant peptides were extracted from the gel pieces by incubation with 25 mM (NH4)HCO3; the buffer was transferred to a separate vial and exchanged with 5% formic acid and then with acetonitrile. All incubations were performed in a volume of 200 µl for 30 min using a sonication bath; the supernatants were combined and the solvents were removed using a SpeedVac.

HDLs were separated on 7% SDS–PAGE and detected by imidazole-zinc staining. After development, the gel was rinsed with water for 30 s and incubated in 0.2 M imidazole, 0.1% SDS solution for 15 min. The imidazole-SDS solution was discarded, and the gel was stained with 0.2 M zinc sulfate until the gel background became white. Detected bands corresponding to Vg subunits were excised, and the proteins were electroeluted overnight with the Bio-Rad (Hercules, CA) model 422 electroeluter using 25 mM Tris buffer containing192 mM glycine and 0.1% SDS. The electroeluted protein was then precipitated at 4°C overnight with 20% trichloroacetic acid and centrifuged for 15 min at 14,000 rpm, and the trichloroacetic acid supernatant was discarded. The pellet was washed with ice-cooled acetone, centrifuged, and dried. The protein was resuspended in 30 µl 20 ng/µl trypsin solution in 25 mM (NH4)HCO3, pH 7.5, containing 10% acetonitrile. The solution was incubated overnight at 37°C with continuous shaking. At the end of the trypsin digestion the samples were dried in a SpeedVac. Pellets from in-gel or in-solution digestion were twice resuspended in pure water and dried to reduce the (NH4)HCO3 concentration. Before LC-MS analysis, the tryptic digests were dissolved in 10 µl 5% acetonitrile containing 0.05% formic acid.

MALDI-TOF MS
Positive ion reflection MALDI-TOF mass spectra were acquired using Bruker Reflex III instrument (Bruker Daltonik, Bremen, Germany) equipped with delayed extraction. For exoglycosidase sequencing, spectra were recorded with a Micromass TofSpec 2E reflectron-TOF mass spectrometer (Waters-Micromass, Manchester, UK) fitted with delayed extraction and a nitrogen laser (337 nm). The acceleration voltage was 20 kV, the pulse voltage was 3200 V, and the delay for the delayed extraction ion source was 500 ns. Aqueous solution (0.5 µl) of the sample was added to the matrix solution (0.5 µl of a solution of 2,5-dihydroxybenzoic acid in acetonitrile [10 mg/ml]) on a stainless steel target plate and dried at room temperature before recrystallization from ethanol.

Electrospray ionization MS/MS and LC-MS/MS analysis
Conventional and tandem MS were performed on an orthogonal hybrid quadrupole time-of-flight mass spectrometer (Q-TOF, Waters-Micromass) fitted with a Micromass Z-spray ion source. Tandem MS was performed by collision-induced dissociation at low energy, using argon as a collision gas. The collision energy was adjusted to that appropriate to the mass of the ions bring fragmented, typically between 25 and 45 eV. Data acquired by the Q-TOF mass spectrometer were processed with a MassLynx data system. LC-MS and LC-MS/MS were used to analyze the peptides and glycopeptides of Vg subunits obtained after tryptic digests. An LC-Packings Ultimate nano-HPLC system equipped with the LC-Packings, Dionex PepMap C-18 column (75 µm internal diameter, 15 cm length) was interfaced with the Q-TOF mass spectrometer. Chromatographic separations were achieved using a linear gradient elution of 5–42.5% acetonitrile (0.04% formic acid) over 60 min at 200 nl/min. All buffers were prepared from HPLC-grade solutions.


    Acknowledgements
 
We thank Dr. Claire S. Allardyce for editing the manuscript. This study was supported in part by grants from DFG (KE 206/17-1), BSF (2000116), and a Minerva Stiftung Postdoctoral Research Fellowship to I.K. The TofSpec mass spectrometer was purchased with a grant from the Biotechnology and Biological Sciences Research Council.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: isam.khalaila{at}epfl.ch

2 Present address: Ecole Polytechnique Fédérale de Lausanne, EPFL-BCH-LCOM, CH-1015 Lausanne, Switzerland Back


    Abbreviations
 
2AB, 2-aminobenzamide; DIG, digoxigenin; DSA, Dature stramonium agglutinin; GNA, Galanthus nevallis agglutinin; GU, glucose unit; HDL, high-density lipoprotein; HPLC, high-performance liquid chromatography; JBM, jack bean {alpha}-mannosidase; LC, liquid chromatography; MAA, Maackia amurensis agglutinin; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; NP, normal phase; PNA, peanut (Arachis hypogaea) agglutinin; Q-TOF, quadrupole time-of-flight; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SNA, Sambucus nigra agglutinin; Vg, vitellogenin; Vt, vitellin


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Abdu, U., Yehezkel, G., and Sagi, A. (2000) Oocyte development and polypeptide dynamics during ovarian maturation in the red-claw crayfish Cherax quadricarinatus. Invert. Reprod. Develop., 37, 75–83.[ISI]

Abdu, U., Davis, C., Khalaila, I., and Sagi, A. (2002) The vitellogenin cDNA of Cherax quadricarinatus encodes a lipoprotein with calcium binding ability, and its expression is induced following the removal of the androgenic gland in a sexually plastic system. Gen. Comp. Endocrinol., 127, 263–272.[CrossRef][ISI][Medline]

Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., and Parekh, R.B. (1995) Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal. Biochem., 230, 229–238.[CrossRef][ISI][Medline]

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