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Associations of protein 4.2 with band 3 and ankyrin

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Abstract

Protein-protein and protein-lipid interactions are thought to play the vital role in maintenance and deformation of red blood cell (RBC) membrane. Protein 4.2, a 76-KDa peripheral protein, binds to the cytoplasmic domain of band 3 (CDB3) and also interacts with ankyrin in RBCs. In order to explore the characteristics of protein 4.2–CDB3–ankyrin interactions, three protein 4.2-derived recombinant proteins encompassing amino acid residues 31–200, 1–300, and 187–260 respectively were expressed in Escherichia coli. Their interactions with CDB3 and ankyrin were investigated by using Far-Western blot and pull-down assay. The results showed that the CDB3-binding site of protein 4.2 is located in the region of residues 200–211 and the ankyrin-binding site is located in the region of residues 187–200 of protein 4.2. Our findings also suggested that the ankyrin D34 domain can interact directly with protein 4.2. The proper tertiary structures of these protein 4.2 fragments are essential for protein 4.2–ankyrin interaction. Meanwhile, ankyrin can enhance the interaction between protein 4.2 and CDB3.

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Abbreviations

RBC:

red blood cell

P4.2:

protein 4.2

CDB3:

cytoplasmic domain of band 3

Ni-NTA:

nickel-nitrilotriacetic acid

BCA:

bicinchoninic acid

HRP:

horseradish peroxidase

IPTG:

isopropyl β-D-thiogalactopyranoside

DTT:

dithiothreitol

GST:

glutathione S-trasferase

TRX:

thioredoxin

OG:

n-octyl-β−D-glucoside

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Authors and Affiliations

  1. Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, 200433, China

    Yang Su, Yu Ding, Ming Jiang, Weihua Jiang, Xiaojian Hu & Zhihong Zhang

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  1. Yang Su
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  2. Yu Ding
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  3. Ming Jiang
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  4. Weihua Jiang
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  6. Zhihong Zhang
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Correspondence to Zhihong Zhang.

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Su, Y., Ding, Y., Jiang, M. et al. Associations of protein 4.2 with band 3 and ankyrin. Mol Cell Biochem 289, 159–166 (2006). https://doi.org/10.1007/s11010-006-9159-x

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  • DOI: https://doi.org/10.1007/s11010-006-9159-x

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