B. Venerando

C. Tringali

F. D\u2019Avila

G. Tettamanti

L. Anastasia

N. Papini

English

F. D’Avila

AIR Universita degli studi di Milano

SODIUM CARBONATE TREATMENT CAUSES THE RELEASE OF ACIDIC SIALIDASE PRESENT ON HUMAN ERYTHROCYTE MEMBRANESD’Avila F., Tringali C., Papini N., Anastasia L., Venerando B. INTRODUCTION AND METHODSSialidases are enzymes widely distributed in nature that play a key role in the metabolism of sialoglycoconjugates. It is becoming evident that the partial desialylation of senescent erythrocyte membrane sialoglycoconjugates constitutes a primary or preliminary signal for erythrophagocytosis. The removal of sialic acid from the sialoglycoconjugates is assumed to be promoted by the sialidases present on the membranes. The presence of two sialidases in human erythrocytes has been previously reported. One acts optimally at acidic pH (4.2-4.7) and the other at neutral pH. In order to identify the exact nature of these two sialidases, we treated the erythrocyte membranes with 0,1 M sodium carbonate buffer pH 11,5. This treatment is one of the methods used to discriminate between peripheral and integral cell membrane proteins. We analyzed the solubilized and non-extractable proteins by western blot and by sialidase activity analysis.    RESULTS AND CONCLUSIONSThe sodium carbonate treatment led to the release of 50% acidic sialidase into the supernatant, whereas the neutral sialidase remained totally in the pellet. Our results suggest that the acidic enzyme is a peripheral protein. The released enzyme has been analyzed also using polyclonal anti-Neu1 antibodies. Our data reveal that Neu1 sialidase is present on the erythrocyte membranes and that the alkaline treatment causes the partial release of this sialidase from the membranes. Neu1 is usually located in a multienzymatic complex in the lysosomes of mammalian cells, but using antibodies against the other components of the complex, we observed that Neu1 is alone on the erythrocyte membrane.

Sodium carbonate treatment causes the release of acidic sialidase present on human erythrocyte membranes

SODIUM CARBONATE TREATMENT CAUSES THE RELEASE OF ACIDIC SIALIDASE PRESENT ON HUMAN ERYTHROCYTE MEMBRANE1F. D’Avila, 1C. Tringali, 1N. Papini, 1,2L. Anastasia, 1,2G. Tettamanti, 1,2B. Venerando 1 Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università degli Studi di Milano, Milano, Italia2  IRCCS Policlinico San Donato, San Donato Milanese, ItaliaSialidases or neuraminidases (EC 3.2.1.18) are enzymes widely distributed in nature from viruses to vertebrates. They play a key role in the metabolism of sialoglycoconjugates. In particular, they are able to remove sialic acid residues from gangliosides, sialoglycoproteins and sialoligosaccharides. It is becoming more and more evident that the partial desialylation of senescent erythrocyte membrane sialoglycoconjugates constitutes a primary or preliminary signal for erythrophagocytosis. The removal of sialic acid from the sialoglycoconjugates is assumed to be promoted by the sialidases present on the membranes. The presence of two sialidases in human erythrocytes has been previously reported. One acts optimally at acidic pH (4.2-4.7) and the other at neutral pH. In order to identify the exact nature of these two sialidases and the enzyme-membrane leaflet interaction, we incubated the erythrocyte membranes with different solutions. We obtained interesting results when we treated the membranes with 0,1 M sodium carbonate buffer pH 11,5. In literature it is reported that sodium carbonate treatment is one of the methods used to discriminate between peripheral and integral cell membrane proteins (Fujiki et al., 1982). The sodium carbonate treatment led to the release of 50% acidic sialidase into the supernatant, whereas the neutral sialidase remained totally in the pellet. Our results suggest that only alkaline pH is responsible for the release of this sialidase from the membrane and that the enzyme is a peripheral protein. Then we analyzed the stability of the acidic sialidase released after the treatment: the enzymatic activity decreases by 40% after 24 h at 4°C. The enzyme looses 25% of its activity after the first freeze-thaw cycle, while seems to be stable after the other five freeze-thaw cycles. The released enzyme has been analyzed also using polyclonal anti-Neu1 antibodies. The western blot analyses reveal that Neu1 sialidase is present on the erythrocyte membranes and that the alkaline treatment causes the partial release of this sialidase from the membranes. This is the first evidence of the presence on erythrocyte membranes of Neu1 sialidase, an enzyme that is usually located in a multienzymatic complex in the lysosomes of mammalian cells. So we investigated if the multienzymatic complex was present on erythrocyte membranes using antibodies against the other  components of the complex. Our results suggest that Neu1 sialidase is alone on the erythrocyte membrane and it does not need the other complex components to expound its enzymatic activity.Fujiki et al., (1982), The Journal of Cell Biology 93, 97-102

Sodium carbonate treatment causes the release of the acidic sialidase present on human erythrocyte membranes

https://air.unimi.it/bitstream/2434/50300/1/abstract%20SIB%202008%20Francesca%20D%27Avila.doc

Sodium carbonate treatment causes the release of the acidic sialidase present on human erythrocyte membranes

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