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

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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.

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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

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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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Authors and Affiliations

  1. Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, 1900, La Plata, Argentina

    M. M. González & N. E. Fink

  2. Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, U.B.A., Junín 956, Buenos Aires, Argentina

    L. Yoshizaki & C. Wolfenstein-Todel

Authors
  1. M. M. González
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  2. L. Yoshizaki
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  3. C. Wolfenstein-Todel
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  4. N. E. Fink
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Correspondence to N. E. Fink.

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González, M.M., Yoshizaki, L., Wolfenstein-Todel, C. et al. Isolation of Galectin-1 from Human Platelets: Its Interaction with Actin. Protein J 31, 8–14 (2012). https://doi.org/10.1007/s10930-011-9367-4

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