CD23 shedding: requirements for substrate recognition and inhibition by dipeptide hydroxamic acids.

CD23 (low affinity IgE receptor, FcepsilonRII) is expressed as a Type II extracellular protein on a variety of cells such as B cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in regulation of IgE synthesis. CD23 is released from the cell surface by a metalloprotease, analogous to the cleavage of other cell surface molecules such as TNF-alpha. This activity has been extensively studied with respect to biochemical characterization and ability to cleave specific mutants of CD23. Both local sequence and distal domains have been shown to affect cleavage of CD23. Selective dipeptide hydroxamic acid inhibitors of CD23 processing have been identified and demonstrated to very potently and selectively inhibit CD23 processing.

Blockade of CD28-B7, but not CD40-CD154, prevents costimulation of allogeneic porcine and xenogeneic human anti-porcine T cell responses.

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.

CD23 (FcepsilonRII) release from cell membranes is mediated by a membrane-bound metalloprotease.

CD23 (low-affinity IgE receptor, FcepsilonRII) is expressed as a Type II extracellular protein on a variety of cells such as B-cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in the regulation of IgE synthesis. Here we report that the release of CD23 from the cell surface is mediated by a metalloprotease. An assay utilizing purified CD23 and an neo-epitope antibody specific for one of the known cleavage products is described and used to demonstrate unambiguously the cleavage of CD23 by a distinct protease. Characterization of the mechanism of CD23 processing shows that the protease exists as an integral membrane protein with a functional molecular mass of approx. 63 kDa as determined by gel-filtration chromatography. The CD23-cleaving activity found in enriched plasma membranes from RPMI 8866 cells is inhibited by the metalloprotease inhibitors 1, 10-phenanthroline and imidazole and by the matrix metalloprotease inhibitor batimastat, but not by inhibitors of cysteine proteases, serine proteases or acid proteases. The same or a similar activity that cleaves CD23 to the known 33 kDa fragment and is inhibited by batimastat is present in diverse cell types such as unstimulated fibroblasts and monocytic cell lines not expressing CD23, as well as in the Epstein-Barr virus-transformed B-cell line, RPMI 8866, which constitutively expresses CD23.

10-Formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): a potent inhibitor of glycinamide ribonucleotide transformylase.

The synthesis of 10-formyl-5,8,10-trideazafolic acid (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) is reported. The target compound was prepared by a convergent synthesis utilizing the alkylation of hydrazone 5 with benzylic bromide 6 to construct the core heterocycle 7. The aldehyde 3 and related agents were evaluated as inhibitors of purN GAR Tfase and avian AICAR Tfase. Compound 3 exhibited potent inhibition of GAR Tfase with a Ki of 0.26 +/- 0.05 microM. In contrast, 3 exhibited more moderate inhibition of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase), with Ki of 7.6 +/- 1.5 microM.

Abenzyl 10-formyl-trideazafolic acid (abenzyl 10-formyl-TDAF): an effective inhibitor of glycinamide ribonucleotide transformylase.

The synthesis of N-[7-(2-amino-3,4-dihydro-4-oxo-quinazolin-6-yl) -6-formyl-1-oxo-heptyl]-L-glutamic acid (2, abenzyl 10-formyl-5,8,10-trideazafolic acid) as a potential enzyme-assembled tight binding inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. The inhibitor was prepared by a convergent synthesis utilizing the sequential alkylations of acetaldehyde dimethylhydrazone with 6 and 8. The agent exhibited effective inhibition of GAR Tfase (Ki = 4.5 +/- 0.3 microM) and more modest inhibition of AICAR Tfase (Ki = 42 +/- 11 microM).

Formyl phosphate: a proposed intermediate in the reaction catalyzed by Escherichia coli PurT GAR transformylase.

The Escherichia coli purT encoded glycinamide ribonucleotide transformylase (GAR transformylase) serves as an alternate enzyme in the production of formyl GAR for use in de novo purine biosynthesis. This enzyme differs from the previously known purN encoded enzyme in size, sequence, and substrates; ATP and formate are required as opposed to formyl tetrahydrofolate. Kinetic studies of the wild-type PurT enzyme described here demonstrate that formyl phosphate behaves as a chemically and kinetically competent intermediate. The requirement for ATP and GAR in these reactions is consistent with previous steady-state kinetic results, which demonstrated that all substrates must be bound before catalysis. Kinetic characterization of a mutant, which releases formyl phosphate into solution, and positional isotope exchange studies also support the assignment of formyl phosphate as a plausible intermediate.

A rapid screen of active site mutants in glycinamide ribonucleotide transformylase.

Specific and saturation site-directed mutageneses have been used to alter each polar residue within 6 A of the catalytic center of glycinamide ribonucleotide transformylase (EC 2.1.2.2). These mutants were rapidly screened for catalytic activity using functional complementation of auxotrophic cells. This screen allows a rapid qualitative estimate of enzyme activity for each of these mutants. These results have shown that none of the polar residues close to the catalytic center of the enzyme are irreplaceable, although several are important for full catalytic activity, namely, Asn106, His108, Ser135, and Asp144. A mechanism is proposed in which a fixed water molecule mediates the required proton transfers between substrate and cofactor, while the formyl group is transferred from 10-formyltetrahydrofolate by direct nucleophilic attack by the amine of glycinamide ribonucleotide. The active site polar residues may act to alter the pKa values of the attacking and leaving amino groups within a putative tetrahedral intermediate in order to facilitate the transfer of the formyl group.