Tài liệu Báo cáo khoa học: Localization of N-linked carbohydrate chains in glycoprotein ZPA of the

Localization of N-linked carbohydrate chains in glycoprotein ZPA

of the bovine egg zona pellucida

Keiichi Ikeda1

, Naoto Yonezawa1,2, Keita Naoi1

, Toshiyuki Katsumata3

, Seizo Hamano4 and

Minoru Nakano1,2

1

Graduate School of Science and Technology and 2

Department of Chemistry, Chiba University, Japan; 3

College of Liberal Arts

and Science, Tokyo Medical and Dental University, Chiba, Japan; 4

Animal Bio-Technology Center,

LivestockImprovement Association, Tokyo, Japan

The zona pellucida, a transparent envelope surrounding the

mammalian oocyte, consists of three glycoproteins, ZPA,

ZPB and ZPC, and plays a role in sperm–egg interactions. In

bovines, these glycoproteins cannot be separated unless the

acidic N-acetyllactosamine regions of the carbohydrate

chains are removed by endo-b-Galactosidase digestion.

Endo-b-Galactosidase-digested ZPB retains stronger sperm￾binding activity than ZPC. It is still unclear whether ZPA

possesses significant activity. Recently, we reported that

bovine sperm binds to Man5GlcNAc2, the neutral N-linked

chain in the cow zona proteins. In this study, we investigated

the localization of the sperm-ligand active high-mannose￾type chain and the acidic complex-type chains in bovine

ZPA. Three N-glycopeptides of ZPA, containing an N-gly￾cosylation site at Asn83, Asn191 and Asn527, respectively,

were obtained from endo-b-Galactosidase-digested ZPA. Of

these glycosylation sites, only Asn527 is present in the ZP

domain common to all the zona proteins. The carbohydrate

structures of the N-linked chains obtained from each

N-glycopeptide were characterized by two-dimensional

sugar mapping analysis, while considering the structures of

the N-linked chains of the zona protein mixture reported

previously. Acidic complex-type chains were found at all

three N-glycosylation sites, while Man5GlcNAc2 was found

at Asn83 and Asn191, but there was very little of this sperm￾ligand active chain at Asn527 in the ZP domain of ZPA.

Keywords: glycoprotein; N-linked carbohydrate chain;

sperm ligand; zona pellucida; ZP domain.

The mammalian oocyte is coated with a transparent matrix

called the zona pellucida. This matrix plays various roles in

the early phase of fertilization: species-specific sperm

binding, blocking polyspermy, and protecting the embryo

until implantation [1,2]. The zona is composed of three

glycoproteins, called ZPA, ZPB and ZPC in the order of the

size of their cDNAs [3], and their carbohydrate chains are

responsible for species-specific sperm-zona binding [1,4].

In the pig, the carbohydrate structures of O-linked chains

of zona protein mixture [5,6] and the acidic and neutral

N-linked chains of ZPB/ZPC mixture [7,8] are well charac￾terized. Sperm-binding activity has been ascribed to the

O-linked chains of the zona protein mixture [9] or to the

neutral N-linked chains obtained from the ZPB/ZPC

mixture [8]. We have shown that the triantennary/tetraan￾tennary chains of the ZPB/ZPC mixture possess greater

activity than the diantennary chains [10]. The triantennary/

tetraantennary chains of ZPB are localized at Asn220 in the

N-terminal region [10]. Moreover, the isolated N-terminal

peptide of ZPB including Asn220 has sperm-binding activity

[11]. Yurewicz et al. [12] showed that ZPB and ZPC form

heterocomplexes that have sperm-binding activity, but that

monomeric ZPB or ZPC does not exhibit this activity.

These results suggest that ZPC contributes to the expression

of the sperm-binding activity of the neutral N-linked chain

of ZPB. In ZPC, the triantennary/tetraantennary chains are

mainly localized at Asn271 in the C-terminal region [13].

The sperm-binding activity of porcine ZPA, a minor

component, has not been assessed because its purification

is difficult.

The N-linked chains of the bovine zona protein mixture

include neutral (23%) and acidic (77%) chains [14]. We

have determined the structures of the neutral chain and the

core regions of the acidic chains [14]. The acidic chains are

diantennary, triantennary and tetraantennary, fucosylated

complex-type chains with a tandem N-acetyllactosamine

repeat in the nonreducing regions to which the main sialic

acids are attached. The neutral chain is a high-mannose￾type chain with five mannose residues (Man5GlcNAc2).

This high-mannose-type chain exhibits sperm-binding

activity and inhibits in vitro fertilization [15].

The native bovine zona proteins cannot be separated into

their three components because their molecular masses are

almost identical (74 kDa) and their carbohydrate chains are

very heterogeneous [16]. When they are digested with endo￾b-Galactosidase to remove the acidic N-acetyllactosamine

repeat, the three components become separable [16–18].

Sperm-binding activity is mainly ascribed to ZPB, while

Correspondence to M. Nakano, Department of Chemistry,

Faculty of Science, Chiba University, 1-33, Yayoi-cho,

Inage-ku, Chiba-shi, Chiba, Japan 263-8522.

Fax: + 81 43 2902874, E-mail: [email protected]

Abbreviations: BrCN, cyanogen bromide; LCA, Lens culinaris

agglutinin; ConA, Canavalia ensiformis agglutinin.

Enzymes: b-Galactosidase (EC 3.2.1.23); keratan-sulfate

endo-1,4-b-Galactosidase (EC 3.2.1.103); sialidase (EC 3.2.1.18);

trypsin (EC 3.4.21.4); N-glycanase (EC 3.5.1.52)

(Received 22 April 2002, revised 21 June 2002, accepted 12 July 2002)

Eur. J. Biochem. 269, 4257–4266 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03111.x