Complete amino acid sequence of PO protein in bovine peripheral nerve myelin.

Abstract. The extracellular domain of the myelin Po protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that Po earl behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., E S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of Po must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of Po proteins truncated at the COOHterminal to the full-length Po protein. Po cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated Po proteins at the surface as did a cell line expressing the full-length Po. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the fuU-length Po had formed large aggregates, while the cells expressing the truncated Po molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length Po was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of Po lacking 52 amino acids and none of Po lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of Po to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.

Section: Expression Of Truncated Po Proteins In Cho Cellssupporting

confidence: 51%

The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain.

Wong

Filbin

1994

“…PLP contains four highly hydrophobic regions and has been proposed to have up to four membrane-spanning regions (Popot et al, 1991) . P0, on the other hand, has a relatively hydrophobic, but glycosylated, extracellular domain, a single transmembrane domain, and a very basic cytoplasmic domain (Sakamoto et al, 1987;Lemke and Axel, 1985) . As shown here, PMP-22 also displays a reciprocal pattern of expression compared to PLP and, like PLP, has four hydrophobic domains which may serve as membrane-spanning regions.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13)

Snipes

Suter

Welcher

et al. 1992

383

229

“…2, lanes i and j). Sugar residues, which represent approximately 6% by weight of the molecule, are attached to Po protein via a single N-linkage at asparagine 93 (Everly et al, 1973;Kitamura et al, 1976;Lemke and Axel, 1985;Sakamoto et al, 1987). The Po proteins expressed by Po/TPo52 and Po/TPo59 cell lines are of the same approximate molecular weights as when they are expressed individually in CHO cells, indicating that they are all glycosylated (Fig.…”

Section: Coexpression Of Full-length Po and Truncated Po Proteins In Cho Cellsmentioning

confidence: 99%

Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain.

Wong

Filbin

1996

Abstract. The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin. 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the fulllength Po, we coexpressed both molecules in CHO ceils. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.