Tracing myelin protein zero (P0) in vivo by construction of P0-GFP fusion proteins

Abstract

Background: Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein.

Results: In contrast to the membrane-localized wild type P0-GFP the Ala221fs P0-GFP protein was detectable almost only in the cytoplasm of the cells, and a complete loss of adhesion function was observed.

Conclusions: The present study provides evidence that GFP is a versatile tool to trace in vivo effects of P0 and its mutations. Not only a loss of adhesion function as a result of the loss of the RSTK domain, but also altered intracellular trafficking indicated by a loss of membrane insertion are possible consequences of the Ala221fs mutation.

Figure 1

Light microscopy of S2 cells. In (A) the P0 transfected S2 cells are not induced to express protein and show only limited aggregation tendencies. In (B) the transfectants were induced with copperions and cell aggregates are clearly visible already 1-2 hours after induction. Pictures were taken at 10 x magnification.

Figure 2

Fluorescence microscopy of a S2 cell transfected with the Ala221fs mutation in fusion with GFP (vector pEXII). None of the different layers analysed by fluorescence microscopy showed a membrane insertion of P0-GFP. Fluorescent cytoplasmic vesicles (arrows) are clearly visible. The nucleus is free of fluorescence (see fig. 4).

Figure 3

Aggregated insect cells after transfection with wild type P0/GFP fusion in vector pEXII. The fluorescent cell surface indicates the membrane incorporation of the wild type P0-GFP fusion protein which initiates the enhanced cell-cell agglutination.

Figure 4

S2 cells transfected with GFP inserted in the pEXII expression vector. The overall staining of the cells expressing GFP only is clearly visible. Also the nucleus shows green fluorescence in contrast to the cells expressing wild type P0-GFP fusion protein (see fig. 2,5).

Figure 5

Fluorescence microscopy of S2 cells expressing wild type P0/GFP in three different layers. In all layers, green vesicles are clearly visible in the cytoplasm. In (B) and (C) the equator of the cells is matched and the green fluorescence of the membrane is visible indicating the functional incorporation of wild type P0-GFP fusion proteins. Arrows indicate wild type P0-GFP in cytosolic vesicles and at the cell surface. The nuclei are free of fluorescence in contrast to the isolated GFP fluorescence (see fig. 4).

Figure 6

Semiquantitative determination of RNA in non transfected and transfected S2 and High5 insect cells by RT-PCR. The respective RT-PCR products derived from the different transfectants revealed the expected sizes as indicated by the positive controls: wild type P0, GFP, P0-GFP and GAPDH. The GAPDH products indicate similar RNA amounts for each experimental value.

Figure 7

Western blot analysis of P0 protein levels with antibodies directed against the V5-epitope. The V5 epitope fused in-frame with P0 reveals expected molecular weights for GFP and GFP fused with P0 proteins. Lane 1: purified recombinant GFP. Lane 2: crude protein extract of sciatic nerve. Lane 3: protein extract of GFP-V5 transfected insect cells. Lane 4: protein extract of wild type P0-GFP-V5 transfected cells. Lane 5: moelcular weight standard.

Figure 8

Adhesion assay with S2 cells transfected with wild type P0, mutated P0 (Ala221fs), wild type P0-GFP and mutated P0-GFP. Adhesion is seen with wild type P0 (indicated in the figure as P0wt) with and without GFP fusion. The negative controls, GFP alone, empty vector and non-transfected cells, as well as the Ala221fs mutation (indicated in the figure as P0GC) do not show adhesion.

Figure 9

Adhesion assay with High5 cells transfected with wild type P0, mutated P0 (Ala221fs), wild type P0-GFP and mutated P0-GFP. Adhesion is seen with wild type P0 (indicated in the figure as P0wt) with and without GFP fusion. The negative controls, GFP alone, empty vector and non-transfected cells, as well as the Ala221fs mutation (indicated in the figure as P0GC) do not show adhesion.