Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.

The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the Kd-restricted nonamer influenza nucleoprotein residues 147-155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O-permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.

The activities of thiol proteases in the rat visceral yolk sac increase during late gestation.

While the rat YVS has been shown to possess an active lysosomal proteolytic system, there are no published reports on the identity of these proteases nor on their changes in activity during the latter half of gestation. We have used specific synthetic substrates to show that cathepsins B, L and H are present in this organ from days 12.5 to 20.5 of gestation. Cathepsins B and L exhibit a marked increase in activity beginning on day 15.5 of gestation. By days 19.5-20.5, cathepsin B activity is increased tenfold over that observed on day 12.5. The activity of cathepsin L may be elevated on day 12.5, decreases more than half by day 14.5 and then increases fourfold by day 20.5. The activity of cathepsin H does not change throughout this period nor do the cathepsins exhibit marked changes in activity in the placenta during this same period or in the PYS from days 12.5 to 14.5 of gestation. These results indicate a specific increase in VYS cathepsin B and L activities late in gestation. These enzymes may be involved in meeting the nutritional needs of the embryo and/or in the degenerative changes which may occur in the VYS and PYS prior to parturition. Studies on the degradation of rat serum albumin by extracts of day 19.5 VYS indicate that cathepsin L may be the quantitatively most important protease in late gestation.

The amino acid sequence of toxin RpIII from the sea anemone, Radianthus paumotensis.

The amino acid sequence of the sodium channel toxin RpIII from the sea anemone Radianthus paumotensis has been determined. The protein is homologous with five analogous toxins from three anemone species, and is most similar to a less toxic protein, RpII, from the same organism. Twelve residues are conserved in all six toxins, one of which is an arginine residue thought to be essential for toxicity. The others (Cys, Gly, Pro and Trp) tend to be conserved in other sets of homologous proteins to maintain functional folds. Comparisons of the sequences suggest the existence of two separate but related classes of toxins cumon the three species of anemone.

NMR analysis and sequence of toxin II from the sea anemone Radianthus paumotensis.

Toxin II from Radianthus paumotensis (RpII) has been investigated by high-resolution NMR and chemical sequencing methods. Resonance assignments have been obtained for this protein by the sequential approach. NMR assignments could not be made consistent with the previously reported primary sequence for this protein, and chemical methods have been used to determine a sequence with which the NMR data are consistent. Analysis of the 2D NOE spectra shows that the protein secondary structure is comprised of two sequences of beta-sheet, probably joined into a distorted continuous sheet, connected by turns and extended loops, without any regular alpha-helical segments. The residues previously implicated in activity in this class of proteins, D8 and R13, occur in a loop region.

Variation of DNA polymerase activities of rat giant trophoblast cells in mid-gestation.

The activities of the DNA polymerases alpha, beta, and gamma were determined in rat giant trophoblast cells during mid-gestation. Trophoblasts at this period of time have ceased to divide but continue to carry out DNA endoreduplication resulting in polyploidy. DNA polymerase-alpha activity in extracts was found to drop sharply from a high level at Day 11 to 1/10 of that level at Day 12 and to continue at a constant level thereafter. A similar pattern of activity was observed for polymerase-gamma, however, in this case the drop represented 50% of the activity at 11 days. Polymerase-beta showed no significant change in activity during this period of development. Endoreduplication (polyploidy) continued during this period as measured by a linear increase in chromosomal DNA content. The sharp drop in alpha-polymerase activity from Day 11 to Day 12 appears to result from the loss of a protein(s) which specifically stimulate(s) alpha-polymerase.

Chromatography of dipeptidyl aminopeptidase I on inhibitor-Sepharose columns.

A number of affinity materials for the purification of dipeptidyl amino-peptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1) have been prepared and tested. These material include peptide and amino acid inhibitors bound to agarose and reversible sulfhydryl adsorbents. Several of these materials are effective affinity adsorbents. The most useful material to be employed in combination with earlier purification methods is acetoxy-anilinomercuri-Sepharose. This removes proteins which are contaminants of some preparations and yields consistently high specific activities. Results with affinity and hydrophobic columns indicate that the primary interactions of the enzyme with amino acid and peptide derivative inhibitors are ionic in nature. This results is in agreement with the conclusions reached in studies of the interactions of these inhibitors with dipeptidyl aminopeptidase in solution.

The mechanism of action of dipeptidyl aminopeptidase. Inhibition by amino acid derivatives and amines; activation by aromatic compounds.

A variety of amino acid and peptide amides have been shown to be inhibitors of dipeptidyl aminopeptidase. Among these compounds derivatives of strongly hydrophobic amino acids are the strongest inhibitors (Phe-NH2, Ki = 1.0 +/- 0.2 mM), while amides of basic amino acids were somewhat less effective (Lys-NH2, Ki = 36 +/- 3 mM). Short chain amino acid amides are notably weaker inhibitors (Gly-NH2, Ki = 293 +/- 50 mM). The interaction of the side chains of compounds with the enzyme appears to be at a site other than that at which the side chain of the amino-penultimate residue of the substrate interacts since the specificity of binding is different. Primary amines have been shown to inhibit, e.g., butylamine, Ki = 340 +/- 40 mM, and aromatic compounds have been shown to stimulate activity toward Gly-Gly-NH2 and Gly-Gly-OEt (phenol, 35% stimulation of activity at a 1:1 molar ratio with the substrate). The data suggest that inhibition involves binding at the site occupied by the free alpha-amino group and the N-terminal amino acid.

Chromatographic evidence for the existence of multiple forms of cathepsin B1.

Preparations of ox spleen cathepsin B1 have been found to give multiple peaks of activity upon chromatography on DEAE-cellulose in NaCl gradients or by equilibrium chromatography in 0.05m-NaCl. CM-cellulose gradient chromatography also shows several cathepsin B1 peaks. This evidence indicates that ox spleen cathepsin B1 can exist in at least three to five forms. All forms have the same molecular weight, are thiol-activated and are inhibited by typical thiol inhibitors. The possible sources of this multiplicity of activity are discussed and a possible physiological role for cathepsin B2 is suggested.