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Purification of the NF2 Tumor Suppressor Protein from Human Erythrocytes

Published online by Cambridge University Press:  02 December 2014

Hitesh K. Jindal ,
Kazumi Yoshinaga ,
Pil-Soo Seo ,
Mohini Lutchman ,
Patrick A. Dion ,
Guy A. Rouleau ,
Toshihiko Hanada  and
Athar H. Chishti
Hitesh K. Jindal
Affiliation:
Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, U.S.A
Kazumi Yoshinaga
Affiliation:
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
Pil-Soo Seo
Affiliation:
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
Mohini Lutchman
Affiliation:
Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, U.S.A
Patrick A. Dion
Affiliation:
Center for the Study of Brain Diseases, CHUM Research Center- Notre Dame Hospital, Montreal, QC, Canada
Guy A. Rouleau
Affiliation:
Center for the Study of Brain Diseases, CHUM Research Center- Notre Dame Hospital, Montreal, QC, Canada
Toshihiko Hanada
Affiliation:
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
Athar H. Chishti*
Affiliation:
Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, U.S.A Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
*
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, 60612, U.S.A.
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Abstract

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Background:

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane.

Methods:

Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a ~70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins.

Results:

These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately ~41-65,000 molecules of NF2 protein per erythrocyte.

Conclusion:

We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

Résumé:

RÉSUMÉ:Contexte:

La neurofibromatose de type 2 (NF2) est une maladie dominante autosomique qui prédispose au développement de tumeurs au niveau des nerfs crâniens et des nerfs spinaux. La protéine codée par le gène suppresseur de tumeurs NF2, connue sous le nom de Merlin/Schwannomin, fait partie de la superfamille des protéines 4,1 impliquées dans l’interface entre le cytosquelette et la membrane plasmatique.

Méthodes:

Lors de l’extraction sélective des protéines associées à la membrane cellulaire d’érythrocytes plasmatiques (fantômes d’hématies) au moyen d’une solution dont la force ionique est faible, la majeure partie de la protéine NF2 demeure associée aux vésicules inversées dépourvues de spectrine-actine. L’analyse par buvardage Western a démontré la présence d’un polypeptide de ˜70 kDa dans la membrane plasmatique erythrocytaire. L’élimination quantitative de la protéine NF2 des vésicules inversées a été effectuée au moyen d’iodure de potassium 1,0 M, un traitement qui extrait les protéines membranaires périphériques fortement liées.

Résultats:

Ces résultats sont compatibles avec un nouveau mode d’association de la protéine NF2 à la membrane érythrocytaire qui est distinct des interactions membranaires connues au sujet des protéines 4,1. Plusieurs stratégies de purification fondées sur ces propriétés biochimiques ont été élaborées pour isoler la protéine NF2 native des fantômes d’hématies humaines. Nous avons quantifié approximativement ˜41-65 000 molécules de protéine NF2 par érythrocyte en utilisant la protéine NF2 purifiée et la protéine NF2 recombinante comme standards internes.

Conclusion:

Ces données sont compatibles avec la présence de la protéine NF2 dans la membrane des érythrocytes humains. L’identification de la protéine NF2 dans la membrane des érythrocytes humains permettra de découvrir de nouveaux modes d’interactions de la protéine NF2 dans les cellules de mammifères au moyen de techniques puissantes de biochimie et de génétique érythrocytaire.

Type
Original Articles
Information
Canadian Journal of Neurological Sciences , Volume 33 , Issue 4 , November 2006 , pp. 394 - 402
Copyright
Copyright © The Canadian Journal of Neurological 2006

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