Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus

Hideyuki Ohbayashi, Tsuyoshi Mantoku, Takehiro Yamamoto, Kohji Nomura, Norio Suzuki

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction- inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide- containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG 120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bP) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.

Original languageEnglish
Pages (from-to)641-650
Number of pages10
JournalDevelopment Growth and Differentiation
Volume40
Issue number6
DOIs
Publication statusPublished - 1998
Externally publishedYes

Fingerprint

Hemicentrotus
Acrosome Reaction
Sea Urchins
Proteins
Complementary DNA
Amino Acid Sequence
fucose-sulfate glycoconjugate
Peptides
DNA Primers
Sodium Dodecyl Sulfate
Disulfides
Trypsin

Keywords

  • Acrosome reaction
  • Cysteine-rich protein
  • Fucose sulfate glycoconjugate
  • Sea urchin egg jelly

ASJC Scopus subject areas

  • Anatomy
  • Developmental Biology
  • Cell Biology

Cite this

@article{278d244d2f604a029285f61ba6cebeba,
title = "Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus",
abstract = "A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction- inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide- containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG 120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bP) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40{\%} is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.",
keywords = "Acrosome reaction, Cysteine-rich protein, Fucose sulfate glycoconjugate, Sea urchin egg jelly",
author = "Hideyuki Ohbayashi and Tsuyoshi Mantoku and Takehiro Yamamoto and Kohji Nomura and Norio Suzuki",
year = "1998",
doi = "10.1046/j.1440-169x.1998.00394.x",
language = "English",
volume = "40",
pages = "641--650",
journal = "Development, growth & differentiation",
issn = "0012-1592",
publisher = "Wiley-Blackwell",
number = "6",

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TY - JOUR

T1 - Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus

AU - Ohbayashi, Hideyuki

AU - Mantoku, Tsuyoshi

AU - Yamamoto, Takehiro

AU - Nomura, Kohji

AU - Suzuki, Norio

PY - 1998

Y1 - 1998

N2 - A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction- inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide- containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG 120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bP) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.

AB - A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction- inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide- containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG 120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bP) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.

KW - Acrosome reaction

KW - Cysteine-rich protein

KW - Fucose sulfate glycoconjugate

KW - Sea urchin egg jelly

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U2 - 10.1046/j.1440-169x.1998.00394.x

DO - 10.1046/j.1440-169x.1998.00394.x

M3 - Article

C2 - 9865974

AN - SCOPUS:0031789996

VL - 40

SP - 641

EP - 650

JO - Development, growth & differentiation

JF - Development, growth & differentiation

SN - 0012-1592

IS - 6

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