Glycobiology Advance Access originally published online on December 15, 2004
Glycobiology 2005 15(5):475-488; doi:10.1093/glycob/cwi022
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Glycobiology vol. 15 no. 5 © Oxford University Press 2004; all rights reserved.
Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure
2 Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Foundation, Bünteweg 15, 30559 Hannover, Germany; 3 Shimadzu Deutschland GmbH, Albert-Hahn-Str. 6-10, 47269 Duisburg, Germany; 4 Institute for Animal Breeding, Hoeltystr. 10, 31535 Neustadt, Germany; and 5 Department of Gynaecology and Obstetrics, Georg August University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany
1 To whom correspondence should be addressed; e-mail: edda.toepfer-petersen{at}tiho-hannover.de
Received on August 27, 2004; revised on November 30, 2004; accepted on December 4, 2004
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The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-ß-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-ß-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn84/93, Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn84/93. N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.
Key words: glycosylation sites / MALDI-TOF MS / N-linked glycan chains / zona pellucida
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Introduction |
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The zona pellucida (ZP) is the transparent extracellular matrix surrounding mammalian oocytes and preimplantation embryos that has several important functions during fertilization and early embryonic development. The ZP mediates species-selective recognition between both gametes, the oocyte and the spermatozoon (Töpfer-Petersen, 1999
); induction of the acrosome reaction, which enables sperm to penetrate the matrix; and prevention of polyspermy after fertilization and subsequent protection of the fertilized oocyte and the early embryo (Yanagimachi, 1994). The ZP matrix is composed of three glycoproteins encoded by the genes ZPA, ZPB, and ZPC that are orthologous throughout mammalian species (Harris et al., 1994; McLeskey et al., 1998). According to Spargo and Hope (2003), the ZPB subfamily includes three gene groups (ZPB1, ZPB2, and ZPB). Within the ZPB subfamily the mouse mZP1 glycoprotein is encoded by the group-level paralog ZPB1 (also assigned as ZP1 gene).
The ZP forms a viscous and elastic border between the oolemma and the innermost layer of the cumulus oophorus. The ZP glycoproteins (here called ZPA, ZPB or mZP1, and ZPC glycoproteins, according to their genetic origin) display a conserved domain structure. The newly defined ZP module spanning about 260 amino acids is shared by all ZP glycoproteins (Bork and Sander, 1992). Additionally, a trefoil domain, a cysteine-rich 45-amino-acid stretch with a 22-amino-acid signature sequence (Prosite, PDOC00024), is located N-terminally to the ZP module in the ZPB glycoprotein family (Bork, 1993).
Posttranslational modifications and processing of the ZP protein backbone are species-specific events resulting in heterogeneity of the attached oligosaccharides and in differing lengths of the polypeptide chains. These structural differences may point to a different organization of the 3D matrix architecture and to functional differences of the ZP glycoproteins within various species (Sinowatz et al., 2001; Töpfer-Petersen, 1999). The murine ZP matrix is formed by long filaments of periodically arranged heterodimeric units of ZPA (mZP2) and ZPC (mZP3) glycoproteins, which are randomly cross-linked by mZP1 dimers (Wassarman and Mortillo, 1991). Furthermore, in mouse and hamster the carbohydrate recognition signals and the ability to induce the acrosome reaction have been mapped to ZPC (mZP3), whereas mZP1 seems to play a role in maintaining the 3D structure (Wassarman and Litscher, 2001). In contrast, binding ability to porcine and rabbit spermatozoa is associated with the products of the ZPB genes (Prasad et al., 1996; Yurewicz et al., 1991). In pig, the ZPB (pZP3a) and ZPC (pZP3ß) glycoproteins reconstitute in about equimolar ratios to high-mass heteromultimeric complexes. Oligomerization of the glycoproteins seems to improve the presentation of the carbohydrate chains, thereby enhancing sperm binding capacity (Yurewicz et al., 1998). The ZPB/ZPC glycoprotein oligomers may therefore determine the assembly of the porcine ZP architecture. The ZPA glycoproteins have been shown to participate in postfertilization events. N-terminal processing at a conserved proteolytic clip at A168 and D169 and intra-/intermolecular disulfide bond formation seem to contribute to zona hardening and thus to the prevention of polyspermy (Bauskin et al., 1999; Iwamoto et al., 1999; Rath et al., 2004).
Because of the availability of material, information on the ZP oligosaccharides was first obtained in pig (Hokke et al., 1994; for review see Nakano et al., 1996; Nakano and Yonezawa, 2001; Takasaki et al., 1999). Recently the corresponding oligosaccharide structures have been also analyzed in cow (Amari et al., 2001; Ikeda et al., 2002; Katsumata et al., 1996) and mouse (Boja et al., 2003; Easton et al., 2000; Nagdas et al., 1994; Noguchi and Nakano, 1993). Porcine ZP glycoproteins carry a complex pattern of N (asparagine)- and O (serine/threonine)-linked oligosaccharides. This pattern shows enormous heterogeneity due to varying degrees of sialylation and sulfation (Hokke et al., 1994; Nakano and Yonezawa, 2001). N-linked complex oligosaccharide structures are basically similar in the three species hitherto studied in that they are fucosylated at the chitobiose core and contain bi-, tri-, and tetraantennae carrying tandemly arranged N-acetyllactosamine repeats. Oligosaccharide structures differ in their degrees of sialylation and sulfation, in their terminal structures, and in the nature of their major neutral N-glycans (Amari et al., 2001; Easton et al., 2000; Nakano and Yonezawa, 2001).
The oligosaccharide pattern of the three N-glycosylation sites of each porcine ZPB and ZPC glycoprotein and of bovine ZPA glycoprotein (Ikeda et al., 2002; Kudo et al., 1998; Yonezawa et al., 1999) have recently been characterized, as have the glycosylation sites of murine ZP glycoproteins (Boja et al., 2003).
Here we report the distribution of N-glycosylation sites and the oligosaccharide pattern of the porcine ZPA glycoprotein of an immature oocyte population as a contribution to the understanding of porcine ZP structure.
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Electrophoretic analysis of ZP glycoproteins
2D electrophoresis of solubilized ZP glycoproteins in the absence of reductive reagents led to the resolution of heterogenous glycoforms of the ZPB and ZPC proteins with molecular masses of between 60 and 45 kDa in the pI range of 3–7. The ZPA isoglycoproteins that exerted an apparent molecular mass of 120–95 kDa extended over a pI range of 3.5–7. Between 1000 and 1500 ZPs collected from immature oocytes gave distinct signals by silver staining (Figure 1a). Endo-ß-galactosidase treatment gave rise to a significant alkaline pI shift of the ZPB/ZPC region (pI 4–9), indicating the loss of most of the acidic portion of the glycan chains due to the loss of the sialylated and sulfated N-acetyllactosamine repeats, which elongate from the antennae of the N-glycans and from the O-glycans (Figure 1b). In contrast, ZPA was scarcely sensitive to endo-ß-galactosidase treatment. The protein concentrated in the range of pI 6–7. This result was accompanied by a minor mass shift after enzymatic treatment and indicates that the glycans of ZPA carry only a small proportion of lactosamine units.
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Probing of the immobilized endo-ß-galactosidase-treated ZP (EßGal-ZP) with lectins and using the biotin-avidin system with chemiluminiscence detection showed an overall Lens culinaris agglutinin (LCA) staining. This demonstrates the overall glycosylation with complex N-glycans in all ZP proteins (Figure 2). Under reducing conditions, a double band at 
25 kDa appeared that stained positively with LCA (Figure 2) but not with Amaranthus caudatus agglutinin (ACA) (data not shown). This indicates the presence of complex N-glycans but not of O-linked glycans in the 25-kDa peptide of ZPA.
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Sugar mapping analysis of N-linked glycan chains
In-gel deglycosylation of electrophoretically separated ZPA allowed the analysis of the glycan pattern of the native glycoprotein (Figure 3a, Table I). Alternatively, the probes were desialylated with neuraminidase and deglycosylated by endo-ß-galactosidase digestion before electrophoresis (Figure 3b, c). Sialylated oligosaccharides were not detected in matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) in the positive ion mode.The labile sulfate ions are known to fragment under normal conditions and are rarely detected (Fukuyama et al., 2002; Harvey, 1999). Therefore, mass mapping of the desialylated N-glycans was first performed (Figure 3b, Table I).
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A distinct signal at m/z 1253.8 correlated with the [M+Na] adduct of the high-mannose type glycan Hex2Man3GlcNAc2. The most prominent signals at m/z 1644.2 and m/z 1805.7 corresponded to the [M+Na] adducts of neutral biantennary N-glycans that carry one and two galactose residues at the nonreducing ends of the antennae. A minor signal at m/z 1481.2 corresponded to the agalacto-biantennary glycan. The signals at m/z 1684.9, m/z 2009.0, m/z 2212.4, and m/z 2374.1 were in agreement with the oligosaccharide structures HexNAc3Fuc1Man3GlcNAc2, Hex2HexNAc3Fuc1Man3GlcNAc2, Hex2HexNAc4Fuc1Man3 GlcNAc2, and Hex3HexNAc4Fuc1Man3GlcNAc2, respectively. After endo-ß-galactosidase treatment (Figure 3c, Table I) the relative intensity of these signals increased, and a novel signal at m/z 2049.1 corresponding to Hex1Hex NAc4Fuc1Man3GlcNAc2 appeared. This indicates the existence of nonrepeated and repeated tri- and tetraantennary structures in porcine ZPA. The signal at m/z 2170.2 with the structure Hex3HexNAc3Fuc1Man3GlcNAc2 was reduced in EßGal-ZP (m/z 2169.8) and can be attributed in part to the biantennary N-glycan with one N-acetyllactosamine unit. The signal at m/z 2535.7 with the summary structure of Hex4HexNAc4Fuc1Man3GlcNAc2 completely disappeared after partial deglycosylation, giving rise to the increased signal intensity of the bi- and triantennary glycans. The minor signals at m/z 2738.7, 2900.9, 3103.0, 3265.6, and 3472.6 corresponded to the N-acetyllactosaminylated N-glycans, Hex4HexNAc5Fuc1Man3GlcNAc2, Hex5HexNAc5Fuc1Man3 GlcNAc2, Hex5HexNAc6Fuc1Man3GlcNAc2, Hex6HexNAc6 Fuc1Man3GlcNAc2, and Hex6HexNAc7Fuc1Man3GlcNAc2, respectively. These signals completely disappeared after endo-ß-galactosidase treatment and may therefore be attributed to bi-, tri-, and tetraantennary glycans carrying different numbers of N-acetyllactosamine repeats (Figure 3, Table I). In the mass range of m/z 1200 to m/z 2500, the mass map of the unmodified glycans showed a pattern almost like to that of the desialylated glycans. The relative increase of signal intensity at m/z 1254.1 indicates that the dominant species existed in the native ZP as their asialo and their sialo isoforms, whereas the minor lactosamine-rich oligosaccharides seemed to be sialylated and thus defied analysis (Figure 3a, Table I).
Tandem mass spectrometry (MS/MS) of the signals proved the glycan nature of the corresponding oligosaccharides (data not shown) and confirmed their identity as belonging to the complex N-glycans (not shown; Harvey, 1999). However, the fragmentation pattern of the signal at m/z 1257 [M+Na] indicates a Hex5GlcNAc2 structure (Harvey, 1999) and provides support for the existence of a high-mannose type N-glycan in ZPA.
In-gel digestion and mass mapping of the deglycosylated protein made it possible to assign 80% of the fragments to the ZPA polypeptide (data not shown).
Identification of N-linked glycosylation sites
The tryptic digest of the untreated glycoprotein mixture was subjected to Concanavalin A agglutinin (Con A) affinity chromatography and the major high-performance liquid chromatography (HPLC) fractions of a Con A–binding pool were analyzed by N-terminal sequencing by automated Edman degradation. The fractions eluting at concentrations of 45% and 50% acetonitrile resulted in the N-terminal sequence HVSHGQSLILASQLIXVADPVT ... that corresponds to the tryptic peptide H245–K282 of ZPA, including the potential glycosylation site at N268. The fraction eluting at a concentration of 35% acetonitrile contained two N-terminal sequences: YGSYYXASDYPVVK, which corresponds to the amino acid stretch Y328–K341 of ZPB and PAGXLSILR, which refers to the amino acid stretch P121–R129 of the mature ZPC glycoprotein. The ZPC peptide represents 25% of the yield of the ZPB peptide. The un-identified amino acid (X) was related to the glycosylated N333 (ZPB) and N124 (ZPC). Both N-glycosylation sites have been shown mainly to carry biantennary complex N-glycans (Kudo et al., 1998; Yonezawa et al., 1999). Other fractions gave multiply unidentifiable sequences or no signals by Edman degradation.
A MS approach was used to identify other glycosylation sites. To facilitate the analysis by MALDI-TOF MS, the ZP glycoproteins were treated with endo-ß-galactosidase to reduce the glycan heterogeneity and acidicity. The tryptic digest of the reduced and alkylated ZP glycoproteins (EßGal-ZP) were subjected to LCA affinity chromatography, which recognizes 
1,6-fucosylated complex N-glycans (Kornfeld et al., 1981). Again, the lectin-binding glycopeptides were further separated by reverse-phase HPLC (Figure 4).
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Fractions were continuously collected and analyzed by MALDI-TOF MS to detect all glycopeptides. To corroborate the data, the fractions containing typical glycopeptide patterns were digested with N-glycosidase F to release the glycan portion, thus allowing analysis of the deglycosylated peptide by MALDI-TOF MS. Porcine ZPA glycoprotein contains six potential N-glycosylation sites in the mature protein with the consensus sequence (NXT/S), in which X was not proline. Identification of the isolated glycopeptides by ExpasyGlycoMod Tools (Cooper et al., 2001) was based on the main structures of endo-ß-galactosidase-treated fucosylated bi-, tri-, and tetraantennary complex N-glycans as reported (Noguchi and Nakano, 1992; Noguchi et al., 1992a,b). With this approach, four of six of the predicted asparagine residues at positions 268, 316, 323, and 530 of ZPA were found to be N-glycosylated (Table II). N-glycosidase F treatment of the glycopeptides led to the identification of the corresponding deglycosylated tryptic peptides H245–K282 at m/z 4084.5 (fraction 18) and L313–K321 at m/z 1114.3 (fraction 13). Fraction 18 eluted at the same acetonitrile concentration (50%) as the Con A–positive glycopeptide with the N-terminal sequence HVSHGQSLILASQLIXVADPVT. The glycopeptide pattern in fractions 5 and 7 corresponded to the peptides T322–K327 and I528–R531, which carry fucosylated biantennary N-glycans (Tables II, III). After N-glycosidase F treatment, the glycopeptide pattern disappeared, although the small peptides of m/zcalc 678.7 and m/zcalc 516.6 were not detected by MS. The glycopeptide I528–K537, exhibiting one missed cleavage, gave rise to the corresponding deglycosylated peptide at m/z 1184.1 (fraction 9). Concomitantly, the typical glycopeptide pattern disappeared. Furthermore, glycopeptides for ZPC and ZPB were also identified. Following enzymatic deglycosylation, peptides m/z 1445.4 (fraction 12) and m/z 2601.2 (fraction 13) appeared, corresponding to glycopeptides H117–R129 and A252–R274 in ZPC. For ZPB, the glycopeptide N203–R219 at m/z 1951.0 (fraction 19) was determined.
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In-gel digestion of electrophoretically separated EßGal-ZPA followed by lectin affinity chromatography and reverse-phase HPLC as well as by N-glycosidase F digestion of the glycopeptide fractions provides support for the fact that the asparagines at positions 84/93, 316, 323, and 530 were N-glycosylated (Table III). The glycosylation site N530 was again present as the small glycopeptide with the calculated peptide mass m/z 516.6 and as the glycopeptide containing one missed cleavage with the corresponding peptide I528–K537 (m/z 1184.1). The glycopeptide including the glycosylation site at N323 appeared as T322–K327 (Table III). The 25-kDa peptide generated by reduction of the disulfide bonds from ZPA was shown to stain for LCA but not for ACA, indicating the presence of fucosylated complex N-glycans (Figure 2). The tryptic glycopeptide I69–K97 includes the two putative N-glycosylation sites at positions 84 and 93 of the N-terminal peptide. In-gel digestion of the 25-kDa peptide and mass mapping demonstrated a complex glycopeptide pattern in the molecular range of m/z 4800–5400, indicating the existence of one, although heterogenous, glycosylated asparagine residue. Deglycosylation gave rise to a dominant signal at m/z 3359.5 and minor signals at m/z 3306.6 and m/z 3376.7, which correspond to the carboxymethylated, the noncarboxymethylated, and the acrylamidylated cysteine residue of the tryptic peptide I69–K97 (Table II). These data are supporting evidence of one glycosylation site at N84/93.
The combined data obtained by lectin affinity chromatography and in-gel digestion of components of ZPA glycoproteins showed that the putative glycosylation sites 3, 4, 5, and 6 at positions N268, N316, N323, and N530 were indeed glycosylated. Although one glycosylated asparagine was localised in the N-terminal 25-kDa peptide, this approach could not differentiate between glycosylation at the positions N84 and N93. In summary, five of six potential N-glycosylation sites of the porcine ZPA glycoprotein were found to be glycosylated.
N-glycan pattern of ZPA glycoprotein
To avoid interference with the glycopeptides of ZPB and ZPC, sugar mapping analyses of the glycans were almost calculated from the in-gel digests of electrophoretically separated ZPA with exception of the position N268 (Table III). All glycosylation sites of porcine ZPA carry biantennary glycan chains, although these were found to be preferentially localized at the asparagine residues at positions 316, 323, and 530 (Table III, Figure 5).
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A very minor portion of tri- and tetraantennary glycans was observed at position 323. The most dominant signals were found in all three positions for the biantennary glycan, with no or one hexose residue (HexNAc2Fuc1Man3GlcNAc2, Hex1HexNAc2Fuc1Man3GlcNAc2). The precise interpretation of the MS spectra of the large glycopeptides I69–K97 and H245–K282 was complicated because of glycan heterogeneity, the existence of differently carboxymethylated and/or acrylamidylated cysteine residues, and the incomplete elution of the large peptides from the gel. The glycan pattern reported for these positions may therefore not be equal to the subtotal (Table III). The N-terminal glycosylation sites N84/93 showed the most complex pattern of bi-, tri-, and tetraantennary glycans (Table III). In-gel deglycosylation of the endo-ß-galactosidase-treated terminal 25-kDa peptide supported evidence for this observation. Dominant signals were shown to be m/z 1485.4 and m/z 1647.9 corresponding to biantennary chains as well as m/z 1689.0, m/z 1851.3, and m/z 2014.7 corresponding to triantennary chains both with one, two, or three terminal N-acetylhexosamine residues. Minor components were fully galactosylated biantennary chains (m/z 1810.3), triantennary chains (m/z 2176.1), and the tetraantennary chains (m/z 2055.9, m/z 2218.0, and m/z 2381.6) carrying one, two, and three terminal galactose residues. The dominant occurrence of terminal N-acetlyhexosamine residues in all glycopeptides points to the existence of antennae carrying N-acetyllactosamine repeats that have been lost by enzymatic treatment but also those that were not lactosaminylated (Table III; also see Figure 1a, b). No high-mannose type chains were identified in the glycan map of the 25-kDa peptide. Furthermore, the MS spectra of the Con A–binding glycopeptides gave no evidence for their existence at the positions 316, 323, and 530. The most probable linkage site seems to be the asparagines residue at position 268.
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The mammalian ZP is an unique, highly organized 3D structure that protects the egg and the preimplantation embryo from physical damage; it also modulates sperm function and is involved in the prevention of polyspermic fertilization. Since 1986, when the first piglets were born after transfer of embryos produced in vitro, many laboratories have established and improved porcine in vitro techniques. However, polyspermic fertilization remains a major issue in porcine in vitro fertilization (Niemann and Rath, 2001
). Evidence is accumulating that the ZP also undergoes biochemical, structural, and functional alterations in the final maturation phase prior to fertilization, which may be impaired by in vitro ovum maturation procedures (Rath et al., 2004). Acidification of the ZP glycoproteins caused by modification of the oligosaccharide chains have been found to be a distinct biochemical feature of the maturation process (Rath et al., 2004). In pig, ZPA is a minor component of the ZP. The molar ratio of ZPA to ZPB to ZPC has been determined as 1:3:3 (Iwamoto et al., 1999). Therefore, we developed a MS approach to identify the as yet unknown glycosylation sites and the glycan pattern of porcine ZPA. The complete information on what may be termed porcine ZP glycomics will provide the basis for the study of the underlying mechanisms of maturation-dependent glycan alterations.
N-glycosylation sites in ZP glycoproteins
Mature ZPA is the largest glycoprotein of porcine ZP. A 330-amino-acid polypeptide elongates N-terminally from the ZP domain. Following fertilization, ZPA proteins are cleaved at the conserved proteolytic clip at A168 and D169 into the disulfide-bridged N-terminal 25-kDa polypeptide and the 65-kDa polypeptide including the ZP domain (Bauskin et al., 1999; Hasegawa et al., 1994; Iwamoto et al., 1999). Five of six glycosylation sites are shown in this work to be glycosylated. The potential glycosylation sites at positions N84 and N93 are localized in the N-terminal polypeptide within the same tryptic glycopeptide (I69–K97). Therefore glycosylation could not be assigned to one of the six potential sites. ZPA isolated from an immature oocyte population has been shown to be partially cleaved at the proteolytic clip and has been separated after reduction into its 25-kDa and 65-kDa units. N-terminal amino acid sequencing of the N-terminal polypeptide by Hasegawa et al. (1994) showed an unidentified amino acid (X) at position 84 and an asparagine residue at position 93, which point to glycosylation of the asparagine residue at N84. These data are corroborated by the fact that N84 is the unique glycosylated site that is conserved in the three species studied to date (Boja et al., 2003; Ikeda et al., 2002). Comparison of the potential N-glycosylation sites of the orthologous ZPA proteins indicates that the conserved sequoon N268AT is glycosylated only in pig and mouse (N264AT). The motif NRT within the ZP domain is glycosylated in pig (N530) and cow (N527). Murine ZP2 (ZPA) contains one position at N393 in the ZP domain. The other glycosylation sites—N316 and N323 in pig, N191 in cow, and N172, N184, and N217 in mouse—are not conserved.
The predicted N-glycosylation sites in the mature porcine ZPB and ZPC proteins are located in the ZP domain. All three potential positions of ZPB at N203, N220, and N333 were found to be glycosylated, indicating that the motifs N203VT and N220DS are also conserved in the bovine protein. Murine ZP1 carries a N-terminal polypeptide stretch that is processed in the mature porcine and bovine proteins. Three of four N-glycosylated asparagine residues are localized within this region, and one site is localized in the ZP domain at the nonconserved position N371. In porcine ZPC, three of four potential sites at N124, N146, and N271 carry glycan chains (Kudo et al., 1998; Yonezawa et al., 1999). The motifs N146VS and N271DS are conserved in the three species and have also been shown to be glycosylated in mouse; the motif N124 LS is conserved in cow. The additional three glycosylated sites in mouse are located within the C-terminal peptide that is processed in the mature bovine and porcine proteins (Boja et al., 2003; Hasegawa et al., 1994). The combined data indicate that the glycosylation sites are not strictly conserved in the orthologous proteins. At this stage of investigation there can only be speculation about the influence of different glycosylation patterns on the assembly of the ZP glycoproteins into its 3D architecture.
Glycan pattern of N-linked glycan chains
The structures of the N-glycans of porcine ZP glycoprotein mixture have been reported by Mori et al. (1998) and those of the pZP3 family representing the gene products of ZPB and ZPC by the group around Nakano (Noguchi and Nakano, 1992; Noguchi et al., 1992a,b). Both groups confirmed the membership of the porcine ZP N-glycans in the complex bi-, tri-, tetra-antennary type with an a-1,6 fucosylated trimannosyl core that in part carries linear N-acetyllactosamine repeats in the outer chains. Neutral and acidic components are present in an molar ratio of 1:2. The acidic forms are sialylated at the nonreducing end and/or sulfated at the C-6 position of the GlcNAc (SO3–6 GlcNAc) residues in the N-acetyllactosamine repeats. Some uncommon sulfate linkages and outer chains have been identified including SO3–6GlcNAc in nonrepeated antennae, and sulfation at the C-3 position of the reducing terminal GlcNAc residue, as has the presence of two isomeric sialyl linkages (Sia2,3- and Sia2,6-) in the nonrepeated antennae. Additionally, sulfated and nonsulfated forms of differently fucosylated structures appear to be expressed in the repeated outer chain moieties (Mori et al., 1998; Takasaki et al., 1999). The majority of the neutral and acidic N-glycans are biantennary structures (Mori et al., 1998; Noguchi and Nakano, 1992).
In-gel deglycosylation of the desialylated and native electrophoretically separated protein confirmed the dominant occurrence of fucosylated biantennary structures in their neutral and acidic forms in the ZPA glycoprotein (Figure 3, Table I). The amounts of oligosaccharides with repeated antennae were comparatively low. Their acidic nature is indicated by the fact that the corresponding signals did not appear in the glycan map of the native probe. The minor shifts in mass and pI of ZPA in 2D electrophoresis after endo-ß-galactosidase treatment support this observation (Figure 1a, b). The dominant signals for bi- and triantennary structures carrying only one or two terminal galactose residues, respectively, in the native and desialylated probes are indications of the distinct existence of oligosaccharides with exposed N-acetylglucosamine at their nonreducing termini.
The most remarkable difference between the previously reported data and the present work is the evidence for the existence of a high-mannose type N-glycan in ZPA. The groups around Mori (Mori et al., 1998; Takasaki et al., 1999) and Nakano for review, see (Nakano and Yonezawa, 2001) both reported the absence of a high-mannose type N-glycan in the porcine ZP, although it had been found in the bovine and murine counterparts (Boja et al., 2003; Ikeda et al., 2002). However, glycan mapping and MS/MS showed the occurrence of a pentamannosyl glycan in the ZPA glycoprotein, which probably can be assigned to the glycosylation site N268 of the molecule. A comparable glycan pattern was generated by the same analytic procedure for the protein band composed of ZPB/ZPC. However, as reported for the ZPB/ZPC protein mixture (Noguchi and Nakano, 1992; Noguchi et al., 1992a,b) no oligomannosyl glycan structures were identified (unpublished data), which indicates its specific presence in the ZPA glycoproteins.
The main characteristics of the porcine ZPA glycoprotein obtained from oocytes in a defined GVI stage were found to be the existence of high-mannose type glycans, the dominant occurrence of biantennary glycans with N-acetylglucosamine in the nonreducing terminus, and the particularly low oligosaccharide content with N-acetyllactosamine repeats. In vitro maturation of occytes from the GVI to the MII stage resulted in a significant acidic shift of about 0.8 to 1.3 pI units for the three glycoproteins when probed by 2D electrophoresis. Lectinological studies with the sialic acid–recognizing lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin II provide supporting evidence that the observed acidification may be mainly due to sulfation of the oligosaccharides (Rath et al., 2004). It remains to be established whether acidification may be accompanied by an increase in lactosaminylation. Investigations of the alteration of ZP oligosaccharides during oocyte maturation and the underlying mechanisms are in progress.
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Materials
Unless otherwise stated, chemicals were obtained from Merck (Darmstadt, Germany) and Sigma Chemical (Steinheim, Germany) and were of suitably high purity. Following enzymes: trypsin EC 3.4.21.4 [EC] , neuraminidase EC 3.2.1.18 [EC] , N-glycosidase F EC 3.5.1.52 [EC] , endo-ß-galactosidase EC 3.2.1.103 [EC] .
Isolation of ZP
ZP-encased oocytes were collected from ovaries of peripuberal gilts obtained from a local abattoir as described (Dunbar et al., 1980). Oocytes were isolated from frozen/thawed ovaries that were shredded in a meat grinder and then passed through nylon screens of decreasing pore size (2000–200 µm). ZP-encased oocytes of 120–180 µm were collected from an 80-µm nylon screen and further purified by Percoll (Amersham Biosciences, Freiburg, Germany) density centrifugation using a discontinuous gradient (40%, 20%, 10% Percoll in 0.9% sodium chloride, 1900 x g, 20 min). The oocytes were collected from the 10% Percoll interface. The oocyte fraction was homogenized and the ZPs were collected from a 42.5-µm nylon screen in water, lyophilized, and stored at –20°C until use. Aliquots of the collected oocyte populations were screened for nuclear status by Orcein staining (Hancock, 1958).
Partial deglycosylation of ZP glycoproteins
ZP glycoproteins were solubilized in 0.1 M ammonium acetate buffer, pH 5.7 (2 mg/ml as determined by amino acid analysis) and treated with 20 mU endo-ß-galactosidase (Escherichia freundii, Calbiochem, Bad Soden, Germany) overnight at 37°C. The samples were lyophilized and stored at –20°C for further analysis.
Electrophoresis and lectin blotting
Solubilized ZP glycoproteins were analysed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) on 10%, 12%, or 15% polyacrylamide gels under nonreducing and reducing conditions according to the protocol of Laemmli (1970). Alternatively, 2D electrophoresis was performed as recently described by Blase et al. (1998). ZPs were solubilized in water (72°C, 2 h) and lyophilized. About 1000 ZP equivalents were dissolved in 130 µl rehydration buffer consisting of 9 M urea, 2% 3-[(3-cholamidopropyl-)dimethylammonio]-1-propanesulfonate (Applichem, Darmstadt, Germany), 0.5% IPG buffer (Amersham Biosciences) and applied to 7-cm-long Immobiline dry strip (pH 3–10; Amersham Biosciences). Under reducing conditions the rehydration was supplemented with 20 mM dithiothreitol (DTT) (Applichem). Isoelectric focusing was carried out on a IPGphor Unit (Amersham Biosciences) at 20°C and 20,000 Vh (1 h 150 V; 1 h 500 V; 1 h 1000 V) following a period of rehydration (15 h 50 V). For the second dimension the strips were equilibrated for 20 min in 0.05 M Tris-chloride, pH 6.8, containing 2% SDS, 30% glycerol, and 6 M urea. In case reducing conditions were chosen, the strips were incubated for 15 min in equilibration solution with 65 mM DTT and for additional 15 min with 240 mM iodacetamide (Sigma). The strips were then placed onto 15% SDS–polyacrylamide gels and electrophoresis was carried out according to the protocol of Laemmli (1970). The 1D and 2D gels were either silver stained (Heukeshoven and Dernick, 1988) or blotted (2 h, 1 mA/cm2) onto polyvinylidiene fluoride membranes (Towbin et al., 1979).
For lectin labeling, membranes were blocked in Tris-buffered saline (TBS) (50 mM Tris–HCl, pH 7.5, containing 150 mM NaCl) containing 1% Tween overnight at 4°C and incubated with lectins (1–2 µg lectin/ml) in TBS/1% Tween for 0.5 h at room temperature. After four washings (in TBS/1% Tween for 10 min) lectin binding was developed with streptavidin peroxidase (1:200,000 in TBS/1% Tween, 30 min, at room temperature; Dianova, Hamburg, Germany). After eight washings in TBS/1% Tween for 5 min, lectin binding was visualized by chemiluminescence (Super Signal West Pico, Pierce, Rockford, IL) according to the manufacturer’s recommendations and exposed to X-ray films using an average exposure time of 2–10 s. For subsequent analysis, membranes were stripped after two washings in TBS/1%Tween with 3 M KSCN (1–6 days).
Tryptic digestion of the ZP glycoproteins
Unmodified or EßGal-ZP samples (1 mg/0.5 ml) were solubilized in 100 mM Tris–HCl, pH 8.6, containing 5.3 M guanidine hydrochloride and reduced under air protection with 12.5 mM DTT (30 min at 50°C). For carboxymethylation of the sulfhydryl groups, the reaction mixture was cooled to room temperature and incubated with 49 mM iodoacetamide (Sigma) for 30 min at room temperature. The reduced and carboxymethylated proteines were desalted using a NAP-5 column (Amersham Biosciences) in 20 mM NH4HCO3, pH 8.5, and digested with tosylphenylalanine chlormethane–treated trypsin (Sigma) at an enzyme to substrate ratio of 1:50 overnight at 37°C. The reaction mixture was heated for 5 min at 95°C to denature the enzyme and was then lyophilized.
In-gel digestion
In-gel digestion was performed substantially according to the protocol of Jensen et al. (1997). About 1 mg of EßGal-ZP was subjected to 10% polyacrylamide SDS gels and separated following the protocol of Laemmli (1970) under nonreducing or reducing conditions as described. The gels were stained with Coomassie blue (0.1% Coomassie R-250, 50% methanol, 5% acetic acid), destained in 30% methanol, and washed overnight in water. Alternatively, the gels were stained with Sypro Ruby (BioRad, Munich, Germany) following the manufacturer’s recommendation. The candidate bands were excised from the gel. Washing of the gel pieces and reduction and alkylation of the protein were carried out as described (Jensen et al., 1997). The sample was digested with sequencing grade trypsin (Boehringer, Mannheim, Germany) (protein to enzyme ratio 20:1) in 0.05 M NH4HCO3, pH 8.5, containing 5 mM CaCl2 overnight at 37°C. The peptides were extracted from the gel material under sonication (5 min) after incubation for 30 min with 33% acetonitrile and three times for 40 min with 60% acetonitrile containing 0.1% trifluoroacetic acid. After heating for 3 min at 95°C and lyophilization, the combined extracts were prepared for MS analysis or subjected to lectin affinity chromatography.
In-gel deglycosylation
In-gel digestion was performed as described in detail by Küster et al. (1997). Unmodified and endo-ß-galactosidase-treated ZP glycoproteins (340 µg) were desialylated (0.2 mU/µg ZP, neuraminidase, Roche, Germany) and subjected to 8% SDS–PAGE under nonreducing conditions for analysis of ZPA. For analysis of the 25-kDa N-terminal peptide of ZPA a 15% SDS–PAGE was used under reducing condition. Staining and destaining were carried out as described for in-gel digestion. The excised bands were washed twice with 20 mM NaHCO3, pH 7.0, for 30 min. The protein was then reduced with 2.8 mM DTT in 20 mM NaHCO3, pH 7.0, at 60°C for 30 min. Protein alkylation was performed with 6 mM iodacetamide for 30 min at 25°C in the dark. The gel pieces were washed for 60 min with 50% acetonitrile in 20 mM NaHCO3, pH 7.0, cut into smaller pieces of about 1 mm2; and completely dried in a SpeedVac (Christ, Osterode, Germany). To the dried gel pieces 15 U N-glycosidase F in 20 mM NaHCO3 (100 U/ml) were added; the digest covered with additional buffer and incubated at 37°C for 12–16 h. Glycans were extracted three times with water under sonication for 30 min and the aqueous extracts were combined. The oligosaccharides were desalted on small tip columns containing about 20 µl graphatised carbon (Alltech, Unterhaching, Germany) by elution with 25% acetonitrile containing 0.05% trifluoroacetic acid (Packer et al., 1998) and were concentrated before MS analysis.
Lectin affinity chromatography
Peptide mixtures of the EßGal-ZP or peptide mixtures obtained by in-gel digestions were resuspended in sample buffer (10 mM Tris–HCl, pH 7.2, containing 150 mM NaCl,1 mM phenylmethyl sulfonylfluoride, 0.025% sodium azide, 1 mM each of CaCl2 and MgCl2) and incubated for 2–3 h with suspended Con A–Sepharose (0.7 ml/mg protein; Pharmacia Biotech, Freiburg, Germany) or alternatively with LCA agarose (0.5–2.5 ml/mg protein; Alexis/Vector, Grünberg, Germany) under shaking in a biospin disposable chromatography column (BioRad). The flowthrough of the mini-column was collected and the sediment washed with about four column volumes of sample buffer to remove nonbinding peptides. The binding peptide fraction was then eluted by incubating the matrix with elution buffer (sample buffer containing 200 mM -methyl-mannopyranoside) for 1 h and washed with elution buffer (three column volumes).
The Con A–binding and LCA-binding fractions were further fractionated by reverse-phase HPLC on a Grom Sil 120 ODS-4HE column (150 x 2 mm, 3 µm particle size) eluting at 0.1 ml/min with a gradient of 0.1% trifluoroacetic acid in water (A) and acetonitrile (B), first isocratically (1% B) for 10 min, followed by a gradient of between 1% and 70% B for 70 min. Fractions (200 µl) were continuously collected for further analysis.
Deglycosylation of N-linked glycopeptides
Fractions containing glycopeptides were concentrated in a SpeedVac concentrator, adjusted to 30 µl with water, and treated with 2–3 U recombinant N-glycosidase F (Roche) overnight at 37°C. The samples were concentrated to 10–20 µl and prepared for MS analysis of the deglycosylated peptides.
MALDI-TOF MS analysis
Peptide samples were desalted on C18 zip tips (Millipore, Eschborn, Germany) by eluting with 50% and 80% acetonitrile containing 0.1% trifluoroacetic acid and directly spotted onto stainless steel targets and dried. Peptides and glycopeptide profiles were recorded with a Kratos MALDI-II Analytical Kompact (V5.2, Kratos Anatytical, Manchester, U.K.) after cocrystallisation with -cyano-4-hydroxycinnamic acid (Sigma) in a mixture of acetonitrile and 0.1% trifluoroacetic acid (70:30, v/v) using the sandwich method (Kussmann et al., 1997).
Released glycans were cocrystallized with 2,5-dihydroxybenzoic acid (Sigma) by mixing the sample solution with the same volume of the matrix. The mixtures were spotted onto stainless steel targets. Mass spectra were recorded using the positive ion mode at linear high power. Typically between 20 and 80 laser shots were added per spectrum.
MS/MS mass spectra were acquired on an AXIMA-QIT MALDI TRAP-TOF instrument (Kratos Analytical) described elsewhere (Ding et al., 1999). MALDI ions generated in the ion source are trapped and cooled using helium. The pressure in the trap is held at 4 x 10–3 Pa. The TOF mass analyzer is used to acquire spectra in both MS and MS/MS modes of operation. Prior to MS/MS analysis the trap is used to isolate a precursor ion by applying a filtered noise field waveform to the end cap electrodes. The window used for precursor ion isolation was set to a mass resolution of 250 (m/Dm, where m corresponds to 1000 u). To induce precursor ion fragmentation resonant excitation is applied to the end cap electrodes with a frequency matching the secular frequency of the ion of interest. Following decomposition, the product ions are extracted into the TOF for mass analysis. In both MS and MS/MS modes ions are pulsed into the TOF mass analyzer with an acceleration voltage of 10 kV. A microchannel plate is used as detector and acquisition is performed by a dedicated 1 GHz transient recorder integrated into the instrument. The TOF mass analyzer was externally calibrated using fullerite (Sigma) deposited directly onto the sample stage.
Amino acid analysis and N-terminal sequencing
Amino acid analyses were carried out employing an Alpha Plus amino acid analyser (Pharmacia) after sample hydrolysis with 6 M HCl for 24 h at 110°C in evacuated and sealed ampules. N-terminal amino acid sequencing analysis (Edman degradation) was carried out using an Applied Biosystem (Darmstadt, Germany) 477/120A sequencer.
Evaluation of glycopeptide and glycan structures
Glycopeptides and glycans were calculated by the ExpasyGlycoMod Tool (Cooper et al., 2001). The evaluation of glycopeptides was based upon the following amino acid sequences: SwissProt accession number P42099
[GenBank]
(ZPA), Q 07287 (ZPB), P42098
[GenBank]
(ZPC). Calculation of the glycan part of the endo-ß-galactosidase-treated glycopeptides was based on the main structures of the complex N-glycans as described (Noguchi and Nakano, 1992; Noguchi et al., 1992a,b).
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Acknowledgements |
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The excellent technical assistence of Mrs. C. Hettel and Mrs. C. Kochel is gratefully acknowledged. The authors wish to thank Dr. McAllister for critical English reading of the manuscript. This study was supported by a Grant-in-Aid for scientific research from the German Research Foundation (DFG) (E.T.P., D.R., and H.W.M.).
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Abbreviations |
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ACA, Amaranthus caudatus agglutinin; Con A, Concanavalin A agglutinin; DTT, dithiothreitol; HPLC, high-performance liquid chromatography; LCA, Lens culinaris agglutinin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; ZP, zona pellucida
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