From eBioscience: anti-CD5 (53C7

From eBioscience: anti-CD5 (53C7.3), anti-CD8 (53C6.7), anti-CD19 (eBio1D3), anti-CD93 (AA4.1), anti-B220 (RA3-6B2), anti-IgD (11-26), anti-IgM Gemcabene calcium (II/41), and anti-Ter119 (TER-119). an unstable cytosolic N-terminal 42-aa peptide with NF-BCpromoting activity (Matza et al., 2002b). In contrast, two parallel studies indicated that deficiency of CD74s MHC II chaperone activity was responsible for the failure to accumulate mature B cells normally (Benlagha et al., 2004; Maehr et al., 2004). Hence the role of regulated intramembrane proteolysis in B cell maturation remains unresolved. Regulated intramembrane proteolysis (Wolfe, 2009) is best known from the role of presenilin (-secretase) in Alzheimers disease and in Notch signaling for T cell maturation and leukemia (Selkoe and Kopan, 2003). Transmembrane protein substrates are first cleaved to release their extracellular domain by an ectoprotease, followed by a second intramembrane cleavage mediated by presenilin or signal peptide peptidase (SPP) enzymes that are amenable to pharmacological inhibition (Wolfe and Kopan, 2004; Eder et al., 2007). SPPL2A is an endosome/lysosome-localized member of the SPP-like (SPPL) intramembrane cleaving aspartyl proteases (aspartyl I-CliPs; Behnke et al., 2011). SPPL2 proteases specifically degrade single pass, type II transmembrane proteins, and in vitro studies have identified several potential SPPL2A substrates, such as British precursor protein 2 (BRI2; Martin et al., 2008), membrane FAS ligand (Kirkin et al., 2007), and membrane TNF (Friedmann et al., 2006), but so far a biological role for SPPs remains largely unknown. In this study, we identify an gene (Fig. 1 B). The mutation caused complete skipping of exon 7 in the mRNA, introducing a frameshift and premature stop codon that deleted most of the SPPL2A protein, including seven of the nine transmembrane domains and the protease active site (Fig. 1 B). Retroviral transduction Gemcabene calcium of IL-7Ccultured mutant pre-B cells with wild-type cDNA but not with empty vector restored normal IgD expression on B cells that matured in vitro (Fig. 1 C), confirming the causative role for the truncating mutation in mice. (A) Percentage of CD19+ B cells among blood lymphocytes and representative IgM/IgD flow cytometric plots gated on CD19+ B cells from mice of the indicated genotypes. Numbers in top left corner are geometric mean fluorescence intensity of IgD, and other numbers are percentage of cells in each gate. ****, P < 0.0001. (B) Schematic of splice-donor mutation, the resulting skipping of exon 7, and the effect on the SPPL2A protein. (C) Surface IgD on GFP+IgM+ B cells from IL-7 bone marrow cultures transduced with a retroviral vector encoding wild-type SPPL2A and GFP (thick line) or empty vector encoding GFP alone (thin line), Slc4a1 compared with B cells transduced with empty GFP vector (shaded gray). (D) ELISA analysis of total serum IgM and IgG1 in unimmunized animals. (E) ELISA analysis of serum antibodies to and CGG 2 wk after immunization, against the ABA 4 wk after immunization, and against NP-Ficoll 6 d after booster immunization. Bars represent the mean, and each symbol represents a Gemcabene calcium single mouse. Data are representative of more than five experiments with at least three animals per group in each (A), one experiment (B and C) or two (D), and one experiment with at least five to seven mice per group (E). Homozygous mutant animals had 25-fold less serum IgM and sixfold less IgG1 (Fig. 1 D). Almost no specific antibody was made after immunization with formalin-inactivated and heat-killed and alum-precipitated chicken gamma globulin (CGG) coupled to mutant mice showed normal cellularity (not depicted), normal numbers of pro-B, pre-B, and immature B cells, and normal cell surface IgM and normal onset of IgD expression among the IgMhigh T1 subset (Fig. 2 A). Mature IgD+IgMlow recirculating B cells were selectively decreased in the bone marrow and had very low surface IgD. In the spleen, the T1 subset of recent bone marrow emigrants (CD93+IgMhighCD23?) was present in normal numbers and expressed levels of surface IgM comparable with controls (Fig. 2, B and C). Subsequent maturational stages of T2, T3, and mature CD93?IgMlow B cells were 20-fold reduced, whereas the mature marginal zone B cell subset was decreased 200-fold. Immunofluorescent staining of spleen sections showed that the residual B cells in SPPL2A-deficient mice nevertheless.