Localization of neutral N‐linked carbohydrate chains in pig zona pellucida glycoprotein ZPC

Yonezawa, Naoto; Fukui, Norio; Kudo, Katsuyasu; Nakano, Minoru

doi:10.1046/j.1432-1327.1999.00095.x

Cited by 25 publications

(18 citation statements)

References 27 publications

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“…3. Localization of the neutral carbohydrate chains at three Nglycosylation sites of ZPC [Yonezawa et al, 1999]. The amino acids are numbered relative to the translation initiation Met (residue 1) [Harris et al, 1994].…”

Section: Zpcmentioning

confidence: 99%

“…It is possible that the carbohydrate chains linked to Asn203 contribute to the sperm-egg binding, since Asn203 and Asn220 are situated at the position close to each other. [Yonezawa et al, 1999] Some part of intact acidic chains of ZPC might become neutral by digestion with endo-ß-galactosidase as in ZPB. Even after the digestion a considerable proportion of the acidic chains is still acidic at all three sites.…”

Section: Localization In Zpbmentioning

confidence: 99%

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Localization of Sperm Ligand Carbohydrate Chains in Pig Zona pellucida Glycoproteins

Nakano

Yonezawa

2000

Cells Tissues Organs

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Neutral N-linked carbohydrate chains from pig ZPB/ZPC mixture are shown to possess sperm ligand activity. Of these complex-type chains, triantennary/tetraantennary chains exhibit the activity stronger than that of diantennary chains. Intact ZPB and ZPC cannot be separated from each other unless acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-β-galactosidase digestion. The endo-β-galactosidase-digested ZPB and its N-terminal fragment of 111 residues retain the sperm ligand activity. Three glycopeptides, having one Asn residue to which the carbohydrate chain is linked, are obtained by lysylendopeptidase digestion of the heat-solubilized zonae containing intact ZPB, and lysylendopeptidase and chymotryptic digestions of endo-β-galactosidase-digested ZPB. On the basis of sugar mapping analysis of the N-linked chains from these glycopeptides and comparison with the carbohydrate structures of the main intact neutral N-linked chains of ZPB/ZPC, the triantennary and tetraantennary chains are shown to be localized mainly at Asn220 of ZPB, whereas diantennary chains are present on all the three N-glycosylation sites (Asn203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm-egg binding. ZPC has been shown to support the expression of sperm ligand activity of ZPB. Three glycopeptides, each having one of the N-glycosylation sites, are obtained by tryptic digestion of endo-β-galactosidase-digested ZPC. Triantennary and tetraantennary chains are found mainly at Asn271 of ZPC, whereas diantennary chains are present at all three N-glycosylation sites (Asn124, Asn146 and Asn271). Thus, the localization of triantennary and tetraantennary chains in ZPC is different from that in ZPB.

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Section: Zpcmentioning

confidence: 99%

Section: Localization In Zpbmentioning

confidence: 99%

Localization of Sperm Ligand Carbohydrate Chains in Pig Zona pellucida Glycoproteins

Nakano

Yonezawa

2000

Cells Tissues Organs

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“…In human and mouse, FUT activity resides in the plasma membrane and is retained after capacitation (Ram et al, 1989;Cardullo et al, 1989;Tulsiani et al, 1990). Carbohydrate moieties, sialyl and non-sialyl N-acetyllactosamines recognized by FUT are present on the zona pellucida of boar (Mori et al, 2000), mouse (Dell et al, 2003;Chalabi et al, 2006) and pig (Yonezawa et al, 1999). Oligosaccharides or artificial carbohydrate chains with N-acetyllactosamine terminals inhibit the binding of spermatozoa to the mouse oocyte (Litscher et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Chiu

Chung

Koistinen

et al. 2007

Journal of Cell Science

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Journal of Cell Science 34 suggest that the glycodelin receptor(s) and zona pellucida glycoprotein receptor(s) are closely related. The objectives of this study were to identify the receptor of glycodelin-A in human spermatozoa and to characterize its interaction with human zona pellucida. Results Fucosyltransferase 5 (FUT5) is a sperm surface glycodelin-A binding proteinThe results of identification of glycodelin-A-bound sperm surface protein(s) are shown in Figs 1, 2. Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3Ј-dithioproionate (Sulfo-SBED)-conjugated glycodelin-A was covalently bound to spermatozoa after photoactivation.Treatment of the glycodelin-A-sperm surface protein complexes with dithiothreitol (DTT; reducing conditions) resulted in reduction of the azo linkage and transfer of the biotin label from glycodelin-A to the sperm proteins. Thus, biotinylated glycodelin-A (Fig. 1A,B, lane 1) showed a single biotin band with molecular size ~30 kDa under non-reducing conditions, which was lost after DTT treatment (Fig. 1A,B, lane 2). One major biotinylated complex with size ~93 kDa appeared after incubation of glycodelin-A (Fig. 1A,B, lane 3) with the sperm extract. Treatment with DTT revealed a major biotinlabeled complex of size~65 kDa (Fig. 1A,B, lane 4). Some minor bands of smaller sizes were also seen. By contrast, glycodelin-A did not interact with fibroblasts and nonspecifically bound glycodelin-A was removed by washing of the cells after incubation. This is evidenced by the absence of a band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Fig. 1A,B, lane 6). Deglycosylated glycodelin-A did not interact with human spermatozoa (data not shown).Mass-spectrometry (MS) analysis of the tryptic digests and a database search identified the ~65 kDa glycodelin-A bound protein as FUT5 (Table 1). The six peptide sequences showed >90% match to the MS spectra and confirmed the protein identity at high confidence level. Two of the identified peptides, SFSWALAFCK and TELVAWAVSNWKPDSAR, were unique to FUT5 among fucosyltransferase 3 (FUT3), FUT5 and fucosyltransferase 6 (FUT6). The identity of the protein was confirmed by western blot analysis using the anti-FUT5 antibody, N-18 (Fig. 1C). The immunoreactivity was associated with bands of sizes ~93 and ~65 kDa, corresponding to the glycodelin-A-FUT complex in non-reducing conditions (Fig. 1C, lane 3) and FUT in reducing conditions (Fig. 1C, lane 4), respectively. Presence of FUT protein in human spermatozoaRecombinant FUT proteins were expressed in inclusion bodies and constituted for approximately 50% of the total protein of the transformed Escherichia coli. The yield of soluble protein was approximately 30-66 mg/l of culture. The expected sizes of recombinant FUT3, FUT5 and FUT6 are 51, 58 and 61 kDa, respectively. These recombinant FUTs were used to determine the specificities of the anti-FUT antibodies against different regions of the enzymes.Owing to the high sequence similarity (~90%) at the Cterminals, th...

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“…The amino acid sequences of the O-glycosylated regions are highly conserved within the ZPB and ZPC protein families. It was recently demonstrated [Yonezawa et al, 1999;Nakano and Yonezawa, 2001] that neutral N-linked carbohydrates from pig ZPB/ZPC mixture possess sperm ligand activity. Of these complex-type chains, triantennary/tetraantennary chains exhibit a stronger binding activity compared to that of diantennary chains.…”

Section: Carbohydrate Structures Of Zona Glycoproteinsmentioning

confidence: 99%

Biosynthesis and Expression of Zona pellucida Glycoproteins in Mammals

Sinowatz

Kölle

Töpfer‐Petersen

2000

Cells Tissues Organs

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The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three glycoproteins (ZPA, ZPB, ZPC), products of the gene families ZPA, ZPB and ZPC that have been found to be highly homologous within mammalian species. Most data on the structure and function of the ZP are obtained from studies in mouse. New data from pig and other domestic animals, however, indicate that the mouse model does not hold for all other species. Whereas in the mouse ZPB is the primary sperm receptor, in the pig ZPA has been shown to possess receptor activity. Contrary to the mouse, where the growing oocyte is the only source of zona glycoproteins, in domestic animals these proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play also a role in granulosa cell differentiation. In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins (oviductins) that become closely associated with the ZP of the ovulated oocyte. Once bound to the ZP, oviductin molecules could act as a protective layer around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains.

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Localization of neutral N‐linked carbohydrate chains in pig zona pellucida glycoprotein ZPC

Cited by 25 publications

References 27 publications

Localization of Sperm Ligand Carbohydrate Chains in Pig Zona pellucida Glycoproteins

Localization of Sperm Ligand Carbohydrate Chains in Pig Zona pellucida Glycoproteins

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Biosynthesis and Expression of Zona pellucida Glycoproteins in Mammals

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