Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IAs class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP139–151) and used it to analyze myelin PLP139–151-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IAs tetramers stimulated and stained the PLP139–151-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP139–151, but not a control T cell line specific for PLP178–191. We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH (∼8.0) and neuraminidase treatment enhances the staining capacity of PLP139–151 tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP139–151-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP139–151-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0 -11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (k lim ) greater than 1000 s ؊1 at pH above 8.5, 10°C. The kinetic parameter k lim /K d for the fast phase exhibits a bell-shaped pH dependence, with two pK a values of 9.3 ؎ 0.1 and 10.0 ؎ 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for k lim gives a single pK a value of 7.1 ؎ 0.2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7)1 is an iron-sulfur containing flavoprotein isolated from the bacterium Methylophilus methylotrophus W 3 A 1 that catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde (presumably through an imine intermediate that spontaneously hydrolyzes once dissociated from the enzyme), (CH 3 The enzyme is a homodimeric protein having a subunit molecular mass of 83 kDa, with each subunit containing a covalently linked 6-S-cysteinyl FMN cofactor and a bacterial ferredoxin type 4Fe/4S center; each subunit also possesses 1 equivalent tightly bound ADP of unknown function (1-7). The physiological electron acceptor for TMADH is an electron transferring flavoprotein (ETF), an ␣␤ dimer of molecular mass 62 kDa. ETF contains 1 equivalent of FAD, which cycles between oxidized and (anionic) semiquinone oxidation states (8), and 1 equivalent AMP, whose function remains unclear (9). Full reduction of TMADH requires three electrons per subunit, two...

CSF-1, the major regulator of macrophage (Mφ) development, has three biologically active isoforms: a membrane-spanning, cell surface glycoprotein, a secreted glycoprotein, and a secreted proteoglycan. We hypothesized that there are shared and unique roles of individual CSF-1 isoforms during renal inflammation. To test this, we evaluated transgenic mice only expressing the cell surface or precursors of the secreted CSF-1 isoforms for Mφ accumulation, activation, and Mφ-mediated tubular epithelial cell (TEC) apoptosis during unilateral ureteral obstruction. The only difference between secreted proteoglycan and secreted glycoprotein CSF-1 isoforms is the presence (proteoglycan) or absence (glycoprotein) of an 18-kDa chondroitin sulfate glycosaminoglycan. We report that 1) cell surface CSF-1 isoform is sufficient to restore Mφ accumulation, activation, and TEC apoptosis to wild-type levels and is substantially more effective than the secreted CSF-1 isoforms; 2) the chondroitin sulfate glycosaminoglycan facilitates Mφ accumulation, activation, and TEC apoptosis; 3) increasing the level of secreted proteoglycan CSF-1 in serum amplifies renal inflammation; and 4) cell-cell contact is required for Mφ to up-regulate CSF-1-dependent expression of IFN-γ. Taken together, we have identified central roles for the cell surface CSF-1 and the chondroitin sulfate chain on secreted proteoglycan CSF-1 during renal inflammation.

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected in k lim /K d ), and in the breakdown of the E ox ⅐S complex (as reflected in k lim ). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pK a ϳ9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), 1 an iron-sulfur containing flavoprotein from the bacterium Methylophilus methylotrophus (sp. W 3 A 1 ), catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde. The enzyme is a homodimer, and each subunit contains an unusual covalently linked 6-S-cysteinyl FMN cofactor and a bacterial ferredoxin-type 4Fe/4S center, as well as 1 equivalent of tightly bound ADP of unknown function (1-6). The physiological electron acceptor of TMADH is an electrontransferring flavoprotein, a 62-kDa heterodimer containing 1 equivalent each of FAD (7) and AMP (8). Electron-transferring flavoprotein is thought to oxidize reduced TMADH in two successive one-electron steps, cycling between the quinone and (anionic) semiquinone oxidation states. The availability of a high resolution structure for TMADH (6) and the cloned and overexpressed gene for the enzyme (10, 11) has made it possible to examine many aspects of the reaction mechanism by conventional site-directed mutagenesis. These have included studies of the role of (i) the 6-S-cysteinyl FMN in catalysis (11-13), (ii) cation-bonding in substrate recognition (14), and (iii) residues on the surface of TMADH involved in electron transfer to electron-transferring flavoprotein (15).The reaction of TMADH with trimethylamine exhibits three sequential kinetic phases (16 -18): a fast phase that represents bleaching of the 6-S-cysteinyl FMN, an intermediate phase that reflects intramolecular electron transfer from dihydroflavin to the 4Fe/4S center to generate the flavin semiquinone and reduced 4Fe/4S center, and a slow phase that involves formation of an unusual spin-interacting state of the enzyme in which the unpaired magnetic moments of t...