Organization of two genes encoding cytotoxic T lymphocyte-specific serine proteases CCPI and CCPII

The genes encoding two recently described cytotoxic T cell proteases, CCPI and CCPII, have been isolated and sequenced. The organizations of the coding and noncoding portions of the two genes are very similar to each other and also to the gene encoding rat mast cell protease type II. Similarly to other serine protease genes, each of the active-site residues is contained on a separate exon; however, two introns were found in particularly interesting positions. One occurs within the postulated activation dipeptide and the other in a position close to the active-site Asp residue. This latter intron interrupts the amino acid sequence in the invariant core region of the protein. We believe that these genes represent a new subfamily of serine protease genes.     

A serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells

We report here by using stopped-flow fluorometry with three different fluorescent probes that a serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells. When cytotoxic T cells (mouse LC7, H-2b anti H-2d) bound to the specific target cells (mouse mastocytoma P815, H-2d), cytotoxic T cells first increased their membrane fluidity, and calcium then was released from intracellular stores. After that, there was a calcium influx from the external medium into the T cells. All of these steps, however, were blocked by serine protease inhibitors (soybean trypsin inhibitor, N alpha-p-tosyl-L-lysine chloromethyl ketone and tosylphenylalanyl chloromethyl ketone). Bovine pancreatic trypsin and chymotrypsin in the external medium mimicked the signalling events which were triggered by the serine protease on the T cell surfaces. From the reaction time (within 1 s) and its specificity, this serine protease in cytotoxic T cells was considered to be different from a protease which works at the killing stage.     

Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes

Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has provided useful structural information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinases to provide a structurally based sequence alignment: alpha-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat mast cell protease 2 (RMCP2). The root mean square differences in alpha-carbon atom positions among these four structures when compared in a pairwise fashion range from 0.79 to 0.97 A for structurally equivalent residues. The sequences of the two lymphocyte enzymes were then aligned to these proteinases using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. With RMCP2 as a template for CCP1 and the two enzymes RMCP2 and BT as templates for HF, the molecular models were constructed. Intramolecular steric clashes that resulted from the replacement of amino acid side chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angles in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginine residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 position of the substrate. Both CCP1 and HF have a free cysteine in the segment of polypeptide 88 to 93.(ABSTRACT TRUNCATED AT 400 WORDS)     

A serine protease (CCP1) is sequestered in the cytoplasmic granules of cytotoxic T lymphocytes

Based upon the use of a new predictive algorithm, three peptides were synthesized that correspond to likely antigenic sites of the cytotoxic T cell-specific protease cytotoxic cell protein 1 (CCP1). Antibodies raised against these peptides, under reducing conditions, bound to a single protein of m.w. 29,000 found in two actively cytotoxic T cells. This protein was absent from a "cytotoxic" T cell line that had lost its cytolytic properties. By using immunocytologic techniques, the protein was localized within cytoplasmic granules. In addition, granules purified from the cytoplasm of cytotoxic T cells were shown, by Western blot analysis, to contain the protein. The molecule detected by the antibodies behaves, upon reduction, similarly to a diisopropylfluorophosphate-binding protein. Thus, here we provide evidence that CCP1, a protein whose expression correlates with cytotoxicity, is contained within organelles which have been implicated in killing. These findings strongly suggest that CCP1 plays a key role in T cell-mediated target cell lysis.     

Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase

The photorespiratory pathway is comprised of enzymes localized within three distinct cellular compartments: chloroplasts, peroxisomes, and mitochondria. Photorespiratory enzymes are encoded by nuclear genes, translated in the cytosol, and targeted into these distinct subcellular compartments. One likely means by which to regulate the expression of the genes encoding photorespiratory enzymes is coordinated temporal control. We have previously shown in Arabidopsis that a circadian clock regulates the expression of the nuclear genes encoding both chloroplastic (Rubisco small subunit and Rubisco activase) and peroxisomal (catalase) components of the photorespiratory pathway. To determine whether a circadian clock also regulates the expression of genes encoding mitochondrial components of the photorespiratory pathway, we characterized a family of Arabidopsis serine hydroxymethyltransferase (SHM) genes. We examined mRNA accumulation for two of these family members, including one probable photorespiratory gene (SHM1) and a second gene expressed maximally in roots (SHM4), and show that both exhibit circadian oscillations in mRNA abundance that are in phase with those described for other photorespiratory genes. In addition, we show that SHM1 mRNA accumulates in light-grown seedlings, although this response is probably an indirect consequence of the induction of photosynthesis and photorespiration by illumination.