BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D.

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.

Identification of a palmitic acid-modified form of human Sonic hedgehog.

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses.

Accessory cell surface molecules, such as T cell antigen CD2 and its ligand lymphocyte function-associated antigen 3 (LFA-3; CD58), are critical costimulatory pathways for optimal T cell activation in response to antigens. Interaction of CD2 with cell surface LFA-3 not only increases T cell/accessory cell adhesion, but also induces signal transduction events involved in the regulation of T cell responses. In this report, we show that specific interactions of LFA-3 with CD2 can result in T cell unresponsiveness to antigenic or mitogenic stimuli in vitro. By deletion of certain regions of the extracellular domain of LFA-3, we localized the CD2 binding site to the first domain of LFA-3. We then demonstrated that a soluble, purified first domain-LFA-3/IgG1 fusion protein (LFA3TIP) interacts with CD2 and binds to the same CD2 epitope as purified multimeric or cell surface-expressed LFA-3. LFA3TIP inhibits tetanus toxoid, hepatitis B surface antigen, anti-CD3 mAb, Con A, and phytohemagglutinin P-induced T cell proliferation, as well as xenogeneic and allogeneic mixed lymphocyte reactions (MLR). Unlike anti-LFA-3 or anti-CD2 monoclonal antibodies (mAbs) which inhibit T cell responses by blocking LFA-3/CD2 binding, LFA3TIP is capable of rendering T cells unresponsive to antigenic stimuli in situations where T cell activation is independent of CD2/LFA-3 interactions. Furthermore, LFA3TIP, but not blocking anti-CD2 mAbs, is capable of inducing T cell unresponsiveness to secondary stimulation in allogeneic MLR. This inhibition of T cell responses by LFA3TIP occurs through a different mechanism from that of mAbs to LFA-3 or CD2.