Glycoconjugate Journal

, Volume 12, Issue 3, pp 258–267

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span

  • Daniela Bratosin
  • Joel Mazurier
  • Henri Debray
  • Myriam Lecocq
  • Benoni Boilly
  • Catherine Alonso
  • Magdalena Moisei
  • Cecilia Motas
  • Jean Montreuil
Glycoconjagate Journal

Abstract

Comparing the properties of ‘young’ and senescent (‘aged’) O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a β-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows:
  1. (1)

    Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes.

     
  2. (2)

    RCA120 as well asErythrina cristagalli andErythrina corallodendron lectins specific for terminal β-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation.

     
  3. (3)

    The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by α-2,6-sialyltransferase of fluorescent or radioactiveN-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively.

     
  4. (4)

    Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal β-galactose specific macrophage lectin.

     
  5. (5)

    Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to thein vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages

     

Keywords

Senescent erythrocytes lectins flow cytofluorimetry sialic acid erythrophagocytosis macrophages endogeneous galactolectin 

Abbreviations

BSA

bovine serum albumin

CMP-NeuAc

cytidine monophosphateN-acetylneuraminate

CSB

cell sialylation buffer

EDTA

ethylene diamine tetraacetic acid

FITC

fluoresceinyl isothiocyanate

9-FITC-NeuAc

9-fluoresceinyl-N-acetylneuraminate

NeuAc

N-acetylneuraminic acid

PAGE

polyacrylamide gel electrophoresis

PBS

Dulbecco's phosphate buffer saline solution

PMSF

phenylmethyl-sulfonyl fluoride

RBC

red blood cells

SCA

Senescent Cell Antigen

SDS

sodium dodecyl sulfate

SFG

senescent factor glycopeptides

Lectins

AAA

Aleuria aurantia agglutinin

DSA

Datura stramonium agglutinin

ECA

Erythrina cristagalli agglutinin

GNA

Galanthus nivalis agglutinin

LCA

Lens culinaris agglutinin

LFA

Limax flavus agglutinin

LPA

Limulus polyphemus agglutinin

MAA

Maackia amurensis agglutinin

PNA

peanut agglutinin

RCA

Ricinus communis agglutinin

SNA

Sambucus nigra agglutinin

UEA-I

Ulex europeus agglutinin-I

WGA

Wheat germ agglutinin

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Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • Daniela Bratosin
    • 1
  • Joel Mazurier
    • 2
  • Henri Debray
    • 2
  • Myriam Lecocq
    • 2
  • Benoni Boilly
    • 3
  • Catherine Alonso
    • 2
  • Magdalena Moisei
    • 1
  • Cecilia Motas
    • 1
  • Jean Montreuil
    • 2
  1. 1.Institute of Biochemistry of the Romanian AcademyBucharestRomania
  2. 2.Laboratoire de Chimie Biologique (UMR 111 du CNRS)Université des Sciences et Technologies de LilleVilleneuve d'AscqFrance
  3. 3.Laboratoire de Biologie du DéveloppementUniversité des Sciences et Technologies de LilleVilleneuve d'AscqFrance

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