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The Protein Journal

, Volume 31, Issue 1, pp 8–14 | Cite as

Isolation of Galectin-1 from Human Platelets: Its Interaction with Actin

  • M. M. González
  • L. Yoshizaki
  • C. Wolfenstein-Todel
  • N. E. Fink
Article

Abstract

Galectins are a family of animal lectins defined by their β-galactoside-binding specificity and a consensus sequence in their carbohydrate-recognition domain. Galectin-1 (Gal-1) is expressed as a non-covalently linked homodimer present in a variety of tissues. Here we describe its isolation from human platelets by a procedure involving ionic exchange chromatography and affinity chromatography on lactose-agarose. Platelet Gal-1 co-purifies with actin, forming an actin-Gal-1 complex which does no dissociate even after treatment with sodium dodecyl sulfate. The presence of both proteins was confirmed by Western blot and by trypsin digestion followed by mass spectrometry identification. By hemagglutination assays we studied the response of recombinant Gal-1/actin, mixed and pre-incubated in different proportions, and then tested against neuraminidase treated rabbit red blood cells. The complex formation was confirmed by confocal microscopy, showing that both proteins co-localised in resting platelets as well as in thrombin-activated ones. These results suggest that endogenous Gal-1 forms an intracellular complex with monomeric actin and that, after platelet activation, Gal-1 could play a role in the polymerization-depolymerization process of actin, which concludes in platelet aggregation.

Keywords

Actin Aggregation Galectin Mass spectrometry Platelets Confocal microscopy Hemagglutination 

Abbreviations

ACN

Acetonitrile

BSA

Bovine serum albumin

CDR

Carbohydrate recognition domain

CM

Confocal microscopy

DEAE

Diethylaminoethyl cellulose

E64

Cysteine proteinase inhibitor

EDTA

Ethylenediaminetetraacetic acid

FITC

Fluorescein isothiocyanate

MEPBS

Mercaptoethanol phosphate buffered saline

PBS

Phosphate buffered saline

PGE1

Prostaglandin E1

Plth

Human platelet

PMN

Polymorphonuclear neutrophils

PMSF

Phenyl methanesulfonyl fluoride

PRP

Platelet enriched plasma

rGal

Recombinant galectin

RP-HPLC-MS

Reversed phase-high performance liquid chromatography–mass spectrometry

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tr

Thrombin

TRICT

Tetramethylrhodamine isothiocyanate

Notes

Acknowledgments

This study was supported by grants from the Universidad Nacional de La Plata, Universidad de Buenos Aires and CONICET. Mass determinations were performed in the LANAIS-PRO Facility (UBA-CONICET).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • M. M. González
    • 1
  • L. Yoshizaki
    • 2
  • C. Wolfenstein-Todel
    • 2
  • N. E. Fink
    • 1
  1. 1.Departamento de Ciencias Biológicas, Facultad de Ciencias ExactasUniversidad Nacional de La PlataLa PlataArgentina
  2. 2.Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, U.B.A.Buenos AiresArgentina

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