The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases ofthe kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the chargerelay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and-to a lesser extent-gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the a-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromo- (6) and was tested as a marker for postcoital detection in rape investigations (7). Although the clinical utility of PA has been shown, its biological function and chemical structure are not well characterized (8). We report here the complete amino acid sequence of PA and describe the characteristics of its enzymatic properties. MATERIALS AND METHODSPA was purified from human seminal plasma as described (1). PA was finally purified on a large-pore Vydac (Hesperia, CA) C4 column and eluted either with a linear gradient between 70% (vol/vol) buffer A (buffer A = 0.1% trifluoroacetic acid in H20) and 37% (vol/vol) buffer B (buffer B = 0.1% trifluoroacetic acid in acetonitrile) in 280 min or 10% (vol/vol) buffer B and 80% (vol/vol) buffer B in 60 min.The enzymatic activity of PA was assessed on commercially purified proteins including insulin A, insulin B, gelatin, myoglobin, ovalbumin, fibrinogen (Sigma), and recombinant interleukin 2 (Ala-IL2, Cetus). The substrates (1 mg/ml) were dissolved in either 0.1 M ammonium bicarbonate or 50 mM Tris HCl, pH 7.8, containing PA at 0.1 mg/ml. In some cases, a mass ratio (enzyme:substrate) of 1:20 was used. After an 18-hr digestion at 37°C, the peptides were separated by HPLC using a 60-min linear gradient of 0-80% (vol/vol) buffer B. To determine the peptide bond specificity of PA, hydrazinolysis was performed on each ofthe PA/substrate digestion mixtures, substrate alone, and intact PA as described (9). The free carboxyl-terminal amino acids were lyophilized and analyzed on a Beckman 121MB amino acid analyzer.The kinetics ofPA hydrolytic activity on synthetic substrates were studied by monitoring the absorbance change at room temperature using a Hewlett-Packard 8450A spectrophotometer. The following substrates were used: N...
Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.
Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide–major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR–T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3–specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.
Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.
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