Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1), a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins

Abstract

Background: A partial cDNA clone from dog thyroid presenting a very significant similarity with an uncharacterized mouse EST sequence was isolated fortuitously. We report here the identification of the complete mRNA and of the gene, the product of which was termed "brain cell membrane protein 1" (BCMP1).

Results: The 4 kb-long mRNA sequence exhibited an open-reading frame of only 543 b followed by a 3.2 kb-long 3' untranslated region containing several AUUUA instability motifs. Analysis of the encoded protein sequence identified the presence of four putative transmembrane domains. Similarity searches in protein domain databases identified partial sequence conservations with peripheral myelin protein 22 (PMP22)/ epithelial membrane proteins (EMPs) and Claudins, defining the encoded protein as representative of the existence of a novel subclass in this protein family.Northern-blot analysis of the expression of the corresponding mRNA in adult dog tissues revealed the presence of a huge amount of the 4 kb transcript in the brain. An EGFP-BCMP1 fusion protein expressed in transfected COS-7 cells exhibited a membranous localization as expected. The sequences encoding BCMP1 were assigned to chromosome X in dog, man and rat using radiation hybrid panels and were partly localized in the currently available human genome sequence.

Conclusions: We have identified the existence in several mammalian species of a gene encoding a putative four-transmembrane protein, BCMP1, wich defines a novel subclass in this family of proteins. In dog at least, the corresponding mRNA is highly present in brain cells. The chromosomal localization of the gene in man makes of it a likely candidate gene for X-linked mental retardation.

Figure 1

Nucleotide sequence of dog BCMP1 cDNA. The aminoacid sequence encoded by the ORF appears above the corresponding DNA sequence. The underlined sequence corresponds to the insert of the original clone C60 (see text). ATTTA motifs appear in bold and the polyadenylation signal is highlighted in blue.

Figure 2

Structure of BCMP1 mRNA. Schematic representation of known sequences from mouse and man that are homologous to dog BCMP1 sequence (short EST sequences were not considered). Coordinates are given for each sequence individually. Highly conserved regions are highlighted in red with indication of the percentage of identity relative to the dog sequence. Inverted triangles symbolize ATTTA motifs. They appear in red when the motif is conserved in the dog sequence.

Figure 3

Northern-blot analysis of BCMP1 mRNA expression. PolyA+ mRNA preparations from various dog tissues were probed with the coding region of BCMP1 cDNA. The arrow points to the signal corresponding to the expected 4 kb-long transcript.

Figure 4

Identification of PMP22 and Claudin family signatures in BCMP1 primary structure. Conserved residues are coloured (blue: PMP22 signature, red: claudin signature, violet: overlapping PMP22 and claudin signatures) and conserved spaces between specific residues are over - or underlined. The part of the predicted BCMP1 structure (see fig. 5) to which the primary sequence corresponds is shown (TM1 = first transmembrane domain).

Figure 5

Predicted structure of BCMP1 in the plasma membrane. The structure was drawn on the basis of the predictions obtained from HMMTOP on the Expasy server.

Figure 6

Phvlogenetic tree of four-transmembrane proteins related to PMP22/EMPs and the claudins. See materials and methods section for the description of the tools used for the construction of the tree. Database accession numbers of individual sequences are given in parentheses. Dog BCMP1 and its mouse and human orthologs appear in red.

Figure 7

Subcellular localization of the EGFP-BCMP1 fusion protein. The fusion protein was expressed in COS-7 cells. Parts A and B: observation of EGFP fluorescence in the perinuclear region and at the cell membrane. Parts C and D: cells were permeabilized and the nuclear DNA was stained with ethidium bromide in order to visualize the cell nucleus.