The rat mast cell antigen AD1 (homologue to human CD63 or melanoma antigen ME491) is expressed in other cells in culture.
Previously we reported that the mAb AD1 recognized a heavily glycosylated 50- to 60-kDa protein (AD1 Ag) sterically close to the high-affinity IgE receptor on rat basophilic leukemia (RBL-2H3) cells. The N-terminal amino acid sequence of the AD1 Ag was nearly identical to that of human CD63 (melanoma-associated Ag ME491). In this study we cloned the cDNA of AD1 Ag from a rat basophilic leukemia 2H3 cDNA library. An open reading frame of 238 amino acids was identified that contained the N-terminal 43 amino acid sequence. No evidence of a signal peptide was found. However, four predominantly hydrophobic stretches of sequence were predicted to form membrane-spanning helices, and three putative N-glycosylation sites were identified. The AD1 Ag and CD63 were highly conserved between rat and human, suggesting that the sequence of this protein is important for its function. By immunostaining various rat tissues, the AD1 Ag was found localized to mast cells. However, it was located to lysosomes, secretory granules and the plasma membrane of RBL-2H3 cells and to lysosomes and plasma membrane of many other cultured cell lines. The AD1 Ag could be induced by placing cells in culture. Fibroblasts and hepatocytes freshly isolated from rat embryos stained very weakly for AD1 Ag; however, after 24 to 48 h in culture they were strongly positive. This increase in the expression of the AD1 Ag was accompanied by an increase in detectable RNA message. Therefore, AD1/ME491/CD63 Ag is a mast cell marker in tissue, but is also associated with other cells in culture.
Nishikata
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Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
Oliver
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C
Mergenhagen
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SE
Siraganian
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RP
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007942:Leukemia, Experimental; D008407:Mast Cells / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D060149:Tetraspanin 30; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D000954:Antigens, Surface; C554586:CD63 protein, human; C554588:Cd63 protein, rat; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D060149:Tetraspanin 30; D004247:DNA