Figure 1. OAP-1 belongs to the tetraspanin family of proteins. (A) Multiple sequence alignment of OAP-1 with murine tetraspanins (mA15, mCD53, mCD63, mCD82), a rat tetraspanin (rTspan-2), and a human tetraspanin (hTspan-3). Boxed residues denote con- servation among TM4SFs. (B) Kyte-Doolittle hydropathy plot of the predicted amino acid sequence of OAP-1, predicting four-transmembrane hydrophobic domains (arrows). Drops of the cell suspension (2 ml) were applied to the center of wells in a 24-well tissue culture dish, which were then placed at 48C for 20 min to al- low the agarose to solidify. The drops were then covered with 50 ml of Sato medium containing 10 mg/ml fibronectin (Sigma-Aldrich) and incu- bated for 30 min at 378C. An additional 350 ml of Sato medium containing platelet-derived growth factor (5 ng/ml) and fibronectin was added to all of the wells with and without antibodies. Cell migration was measured at daily intervals for 1–4 d. At least three experiments were performed and within a single experiment, each condition was tested in four different wells. Statistical significance was assessed by using the Student’s paired t test in which P , 0.05 was considered statistically significant. Results A Novel Protein Interacts with the COOH Terminus of OSP/Claudin-11 A mouse brain cDNA GAL4 activation domain library was screened with three different OSP/claudin-11 GAL4- binding domain baits in pGBT vector: the entire OSP/ claudin-11 ORF, extracellular loop of OSP/claudin-11 be- tween 35 and 84 amino acids, and COOH terminus of OSP/claudin-11 between 122 and 207 amino acids. Only Figure 2. Presence of OAP-1 mRNA and protein in mouse brain and oligodendrocytes. (A) Northern blot analysis of OAP-1 expression. mRNA isolated from normal spinal cord (lane 1), brain (2), testes (3), skeletal muscle (4), heart (5), kidney (6), spleen (7), and lung (8) was probed with a 32P-labeled 1.9-kB cDNA of OAP-1. OAP-1 RNA was abundant in all tissue types. Gel loading was quantified with 18S expression. (B) Northern blot analysis of OAP-1 expression in mouse brain homogenate (BH) and purified oligodendrocytes (OL) from mouse brain. (C) Western blot analysis of OAP-1 protein in oligodendrocyte progeni- tor cells (O2A), purified mouse oligodendrocytes, and mouse brain homogenate. Preincubation of antibody with OAP-1–specific peptide eliminated the 31-kD band confirming antibody specificity (1Pep). transfection with OSP/claudin-11 COOH terminus bait re- sulted in positive colonies. Of 2 3 106 transformants screened, 140 colonies grew on His2 selection medium; two of these were b-galactosidase positive. None of the negative controls grew on His-deficient medium or acti- vated lacZ transcription. OSP/claudin-11 cotransformed with OAP-1 activated the lacZ reporter in a manner simi- lar to our positive controls of pVA3, pTD1 (murine p53 and SV40 large antigen), and pCL1. OAP-1 Is a Member of the Tetraspanin Superfamily OAP-1 cDNA (700 bp) was partially sequenced, and BLAST search revealed that it is a novel clone. The com- plete OAP-1 cDNA was then isolated from a spinal cord cDNA library and composed of 1,679 bp with an ORF of 762 bp (sequence data available from GenBank/EMBL/ DDBJ under accession number AF242591). The predicted protein has 254 amino acid residues and a calculated mo- lecular mass of 28 kD. The original OAP-1 clone isolated from the yeast two-hybrid screen contained nucleotide se- quence 778–1053 of the ORF, corresponding to amino ac- ids 162–254 (COOH termini). Sequence homology and hy- dropathy plot analysis suggested that OAP-1 belongs to the transmembrane 4 superfamily (TM4SF). TM4SFs in- clude $21 different proteins that have four transmem- brane-spanning domains and exhibit high homology in 24 conserved regions that are specific for the TM4SF (Todd et al., 1998). Alignment of the OAP-1 sequence with some murine and human TM4SF members (mA15, mCD53, The Journal of Cell Biology, Volume 153, 2001 298