|Direct interaction between emerin and lamin A. |
Clements, L; Manilal, S; Love, DR; Morris, GE
Biochemical and biophysical research communications
Emerin is the protein of the inner nuclear membrane that is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. The autosomal dominant form of the disease is caused by mutations in the lamin A/C gene. Several lines of circumstantial evidence have suggested an interaction of emerin with lamins in the nuclear lamina but direct interaction between the two proteins has not yet been demonstrated. We now demonstrate direct interaction between recombinant emerin and lamin A molecules using biomolecular interaction analysis (BIA) and monoclonal antibodies. An emerin-lamin A interaction system may be related in function to the LAP2-lamin B system at the inner nuclear rim.
|Distribution of emerin and lamins in the heart and implications for Emery-Dreifuss muscular dystrophy. |
Manilal, S; Sewry, CA; Pereboev, A; Man, N; Gobbi, P; Hawkes, S; Love, DR; Morris, GE
Human molecular genetics
Emerin is a nuclear membrane protein which is missing or defective in Emery-Dreifuss muscular dystrophy (EDMD). It is one member of a family of lamina-associated proteins which includes LAP1, LAP2 and lamin B receptor (LBR). A panel of 16 monoclonal antibodies (mAbs) has been mapped to six specific sites throughout the emerin molecule using phage-displayed peptide libraries and has been used to localize emerin in human and rabbit heart. Several mAbs against different emerin epitopes did not recognize intercalated discs in the heart, though they recognized cardiomyocyte nuclei strongly, both at the rim and in intranuclear spots or channels. A polyclonal rabbit antiserum against emerin did recognize both nuclear membrane and intercalated discs but, after affinity purification against a pure-emerin band on a western blot, it stained only the nuclear membrane. These results would not be expected if immunostaining at intercalated discs were due to a product of the emerin gene and, therefore, cast some doubt upon the hypothesis that cardiac defects in EDMD are caused by absence of emerin from intercalated discs. Although emerin was abundant in the membranes of cardiomyocyte nuclei, it was absent from many non-myocyte cells in the heart. This distribution of emerin was similar to that of lamin A, a candidate gene for an autosomal form of EDMD. In contrast, lamin B1 was absent from cardiomyocyte nuclei, showing that lamin B1 is not essential for localization of emerin to the nuclear lamina. Lamin B1 is also almost completely absent from skeletal muscle nuclei. In EDMD, the additional absence of lamin B1 from heart and skeletal muscle nuclei which already lack emerin may offer an alternative explanation of why these tissues are particularly affected.
|The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein. |
Manilal, S; Nguyen, TM; Sewry, CA; Morris, GE
Human molecular genetics
A large fragment of emerin cDNA was prepared by PCR and expressed as a recombinant protein in Escherichia coli. Using this as immunogen, we prepared a panel of 12 monoclonal antibodies which recognise at least four different epitopes on emerin in order to ensure that emerin can be distinguished from non-specific cross-reacting proteins. All the mAbs recognised a 34 kDa protein in all tissues tested, though minor emerin-related bands were also detected in some tissues. Immunofluorescence microscopy showed that emerin is located at the nuclear rim in all tissues examined. A muscle biopsy from an Emery-Dreifuss muscular dystrophy (EMDM) patient showed complete absence of emerin by both Western blotting and immunohistochemistry, suggesting a simple diagnostic antibody test for EDMD families. Biochemical fractionation of brain and liver tissues showed that emerin was present in nuclei purified by centrifugation through 65% sucrose and was absent from soluble fractions (post-100,000 g). From these results, together with sequence and structural homologies between emerin, thymopoietins and the nuclear lamina-associated protein, LAP2, we suggest that emerin will prove to be one member of a family of inner nuclear membrane proteins.