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.
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 120 kDa 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 (FSG120KA: 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.
Ohbayashi
H
H
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Mantoku
T
T
Yamamoto
T
T
Nomura
K
K
Suzuki
N
N
D020101:Acrosome Reaction; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018076:DNA, Complementary; D000939:Epitopes; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012617:Sea Urchins / Q000502:physiology*; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D018076:DNA, Complementary; D000939:Epitopes; D006023:Glycoproteins; D047908:Intracellular Signaling Peptides and Proteins; C117534:p120 FSG protein, Hemicentrotus pulcherrimus