Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule

Norihisa Ishiwata, Koji Takio, Masahiko Katayama, Kiyoaki Watanabe, Koiti Titani, Yasuo Ikeda, Makoto Handa

Research output: Contribution to journalArticle

104 Citations (Scopus)

Abstract

To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G. I., Bliss, G. A., Newman, P. J., and McEver, R. P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747- 760). In contrast, platelet P-selectin reacted with both antibodies. S- Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.

Original languageEnglish
Pages (from-to)23708-23715
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number38
Publication statusPublished - 1994 Sep 23

Fingerprint

P-Selectin
Protein Isoforms
Molecules
Platelets
Blood Platelets
Peptides
lysyl endopeptidase
Plasmas
Plasma (human)
Antibodies
High performance liquid chromatography
Staphylococcus
Tail
Immune Sera
Digestion
Complementary DNA
High Pressure Liquid Chromatography
Rabbits

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ishiwata, N., Takio, K., Katayama, M., Watanabe, K., Titani, K., Ikeda, Y., & Handa, M. (1994). Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule. Journal of Biological Chemistry, 269(38), 23708-23715.

Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule. / Ishiwata, Norihisa; Takio, Koji; Katayama, Masahiko; Watanabe, Kiyoaki; Titani, Koiti; Ikeda, Yasuo; Handa, Makoto.

In: Journal of Biological Chemistry, Vol. 269, No. 38, 23.09.1994, p. 23708-23715.

Research output: Contribution to journalArticle

Ishiwata, N, Takio, K, Katayama, M, Watanabe, K, Titani, K, Ikeda, Y & Handa, M 1994, 'Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule', Journal of Biological Chemistry, vol. 269, no. 38, pp. 23708-23715.
Ishiwata N, Takio K, Katayama M, Watanabe K, Titani K, Ikeda Y et al. Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule. Journal of Biological Chemistry. 1994 Sep 23;269(38):23708-23715.
Ishiwata, Norihisa ; Takio, Koji ; Katayama, Masahiko ; Watanabe, Kiyoaki ; Titani, Koiti ; Ikeda, Yasuo ; Handa, Makoto. / Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 38. pp. 23708-23715.
@article{d612eb470e6d4524b1d461b39556841f,
title = "Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule",
abstract = "To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G. I., Bliss, G. A., Newman, P. J., and McEver, R. P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747- 760). In contrast, platelet P-selectin reacted with both antibodies. S- Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.",
author = "Norihisa Ishiwata and Koji Takio and Masahiko Katayama and Kiyoaki Watanabe and Koiti Titani and Yasuo Ikeda and Makoto Handa",
year = "1994",
month = "9",
day = "23",
language = "English",
volume = "269",
pages = "23708--23715",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "38",

}

TY - JOUR

T1 - Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule

AU - Ishiwata, Norihisa

AU - Takio, Koji

AU - Katayama, Masahiko

AU - Watanabe, Kiyoaki

AU - Titani, Koiti

AU - Ikeda, Yasuo

AU - Handa, Makoto

PY - 1994/9/23

Y1 - 1994/9/23

N2 - To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G. I., Bliss, G. A., Newman, P. J., and McEver, R. P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747- 760). In contrast, platelet P-selectin reacted with both antibodies. S- Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.

AB - To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G. I., Bliss, G. A., Newman, P. J., and McEver, R. P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747- 760). In contrast, platelet P-selectin reacted with both antibodies. S- Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.

UR - http://www.scopus.com/inward/record.url?scp=0028128695&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028128695&partnerID=8YFLogxK

M3 - Article

VL - 269

SP - 23708

EP - 23715

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 38

ER -