Peripheral myelin protein 22 kDa and protein zero: domain specific trans-interactions

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The peripheral myelin proteins P0 and PMP22 are associated in preparations of compact myelin and in cell cultures coexpressing both molecules. The mechanism of this interaction, however, still needs to be unravelled. We have established three different (cell–cell, cell–protein, protein–protein based) assay systems using retrovirally transduced HeLa cells that overexpressed either PMP22 or P0 and purified GST fusion oligopeptides of PMP22 and P0 to detect domain-specific interactions between these proteins. The results revealed that PMP22 and P0 are involved in both trans-homophilic and trans-heterophilic interactions. Moreover, the data clearly indicate that the heterophilic trans-interaction is mediated through the second loop of PMP22, while the first loop is involved in homophilic trans-interaction of PMP22 proteins. Both modes of interaction are due to direct protein–protein binding. In addition, we demonstrate that disease-related point mutations of P0 resulted in a decreased adhesion capability correlating with the severity of the respective disease phenotype.

Introduction

The myelin sheath of peripheral nervous system (PNS) is generated by Schwann cells, from which the myelin radiates and wraps around adjacent axons (Arroyo and Scherer, 2000). Peripheral myelination requires the synthesis of large quantities of membrane lipids and a specific set of proteins that play key roles in the structure and function of the myelin sheath. Two important molecules of peripheral myelin are the protein zero (P0), which makes up approximately 50% of all myelin proteins (Greenfield et al., 1973), and the peripheral myelin protein 22 kDa (PMP22), which comprises 2–5% of total PNS myelin (Pareek et al., 1993).

The genes encoding P0 and PMP22 have been well characterized, and numerous mutations associated with these genes have been identified in hereditary demyelinating peripheral neuropathies in mice and human, such as Charcot–Marie–Tooth disease (CMT), Dejerine–Sottas Syndrome (DSS), Congenital Hypomyelination (CH) and Hereditary Neuropathy with Pressure Palsies (HNPP), respectively (for review, see De Jonghe et al., 1997, Müller, 2000, Suter and Snipes, 1995). Genotype–phenotype correlations revealed that different mutations cause phenotypes with varying degrees of disease severity (De Jonghe et al., 1997, Nelis et al., 1999, Warner et al., 1996). Furthermore, duplication and deletion of the PMP22 gene is associated with CMT1A and HNPP, respectively, indicating that a gene dosage effect is involved in the pathological mechanism (Patel and Lupski, 1994). Mutations, gene duplication or gene deletion could lead to a nonfunctional protein or interfere with the stoichiometry of protein–protein interactions and ultimately to an unstable myelin structure. PMP22 seems to form heterophilic complexes with P0 in peripheral myelin as well as in ovary carcinoma cells expressing both proteins (D'Urso et al., 1999). Furthermore, PMP22 protein carrying CMT1A-related point mutations accumulates in the ER/Golgi compartments thereby causing a dominant negative effect on the intracellular trafficking of the wild-type PMP22 from the unaffected allele (D'Urso et al., 1998, Naef and Suter, 1999, Naef et al., 1997, Notterpek et al., 1997, Tobler et al., 1999).

It has been speculated that the heterophilic interaction of PMP22 and P0 may be important for a function of these proteins in maintenance and stability of PNS myelin (D'Urso et al., 1999, Scherer and Arroyo, 2002). To evaluate the mechanism of the heterophilic PMP22 and P0 interaction, we have generated a cell adhesion assay with retrovirally transduced HeLa cells expressing PMP22 or P0, respectively, and confirmed the results in GST pull down studies. These data indicate that PMP22 and P0 perform homophilic and heterophilic interactions. Mutations located in distinct regions of the extracellular domain of P0 protein that are associated with CMT1 and DSS disease clearly impair those interactions.

Section snippets

Generation of HeLa cell pools expressing P0, PMP22 and Plasmolipin

To elucidate the interaction of PMP22 and P0 proteins, we transduced HeLa cells with retroviral constructs encoding PMP22, P0 and Plasmolipin (PLA), respectively. Expression of the constructs in G418 resistant cells was confirmed by reverse transcription (RT)-PCR using construct-specific primers (data not shown) and by Western blotting (Fig. 1). To ensure that each protein was ectopically expressed and inserted into the plasma membrane, intact cells were prelabelled by membrane impermeable

Discussion

The molecular mechanisms of membrane assembly and compaction of the multilamellar structure of myelin sheaths is still not well understood. Previous studies have identified P0 as a transmembrane protein with homophilic adhesion capacity (D'Urso et al., 1990, D'Urso et al., 1999, Filbin et al., 1990, Schneider-Schaulies et al., 1990) and, based on crystallography studies it has been suggested that P0 forms interdigitating homotetramers in cis and trans configuration in myelin membranes (Inouye

Antibodies

The following antibodies were used as indicated: Polyclonal anti-PMP22 antibody (1:300; Antibody Service, Dr. Pineda, Berlin, Germany), polyclonal anti-P0 antibody (1:500; Hasse et al., 2002), polyclonal anti-Plasmolipin antibody (1:200; Hamacher et al., 2001) and monoclonal anti-Glutathione-S-Transferase antibody (1:2000; Amersham Pharmacia Biotechnology, Buckinghamshire, UK).

Retroviral overexpression of PMP22, P0 and Plasmolipin in vitro

The open reading frames (ORFs) of rat PMP22 (NM017037), rat P0 (K03242, BF562392) and rat Plasmolipin (PLA, NM022533)

Acknowledgments

This work was supported in part by the Deutsche Forschungsgemeinschaft (Mu630/5-4), the Elterninitiative Kinderkrebsklinik and a Grant from the Medical Faculty of the University of Düsseldorf.

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