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The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understanding both the synthesis and the catabolism of CD23 are, thus, important issues. Membrane CD23 is cleaved by a disintegrin and metalloproteinase 10 (ADAM10) and this cleavage influences the ability of CD23 to regulate IgE. In contrast to the belief that cleavage is a cell surface event, endosomal neutralization with NH 4 Cl was found to dramatically reduce CD23 cleavage, suggesting that the majority of CD23 cleavage occurred subsequent to internalization in the endosomal pathway and not at the cell surface. In line with this, full-length CD23 was shown to be sorted in an ADAM10-dependent manner into exosomes. Greatly increased ADAM10-mediated CD23 cleavage was seen at endosomal pH. Additionally, the stalk region of CD23 was found to interact with ADAM10 and ADAM10 binding of CD23 was found to be protease independent. SPR analysis of the interaction indicated about a 10-fold increase in the R max at endosomal pH (pH 5.8) compared with pH 7.4, whereas the affinity of the interaction was not significantly changed. The R max change, combined with the increased cleavage at endosomal pH, indicates greater accessibility of the CD23 stalk region for ADAM10 at the lower pH. These results indicate a model where CD23 internalization results in ADAM10-dependent incorporation into exosomes, followed by partial cleavage of CD23 by ADAM10 prior to being released from the cell. The increased cleavage at endosomal pH also has implications for other ADAM10 substrates.The cleavage of plasma membrane-bound proteins and their subsequent release from the cell is described as ectodomain shedding. Historically this was believed to be a cell surface process. However, recently a second mechanism was discovered, involving the release of proteins after their internalization and subsequent trafficking through the endosomal pathway to multivesicular bodies (MVB) 2 (1). In the MVB, the proteins can interact with their sheddase and be cleaved and released or be sorted into exosomes and released as full-length proteins. Exosomes are small, 30 -100 nm, membrane containing vesicles that are released by a wide variety of cells. As they have high expression of major histocompatibility complexes (MHC) molecules they have been investigated as cell-free transporters of cancer antigens in cancer immunotherapy treatments (2). Exosome released from B cells have also been extensively studied. B cell-derived exosomes express integrins (3), which allow them to bind, and potentially present antigen to T cells. They also transport antigens, including peptides derived from allergen between antigen presenting cells (reviewed in Ref. 4). B cell exosome secretion is also increased upon activation (5) and interaction with T cells (6). However, the secretion of exosomes by cells as well as the mechanism that controls the sorting of proteins into exosomes are still poorly understood. The low-affinity receptor of immunoglobulin E (CD23) is an important regulator of IgE pro...

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ABSTRACT:Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissuespecific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.

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