Antigen processing by endosomal proteases determines which sites of sperm-whale myoglobin are eventually recognized by T cells.

This study reports an identification of the major processing products of an exogenous protein antigen, viz, sperm-whale myoglobin, as obtained after cell-free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by high performance liquid chromatography analysis from those generated after uptake of myoglobin inside live macrophages. The concerted action of the endosomal proteases cathepsin D and cathepsin B can account for nearly all cleavages observed. Cathepsin D appears to be mainly responsible for the initial cleavage of myoglobin, while cathepsin B catalyzes the C-terminal trimming of initially released fragments. The fragments released by cathepsin D contain most, if not all, major epitopes for murine myoglobin-specific helper T cells. Interestingly, each known T cell epitope of myoglobin is located at the very N terminus of a different myoglobin fragment released upon processing. In order to explain this correspondence, noted also in several other protein antigens, a structural relationship is proposed between antigen processing by cathepsin D and antigen recognition by major histocompatibility complex (MHC) class II products. As is demonstrated here, this relationship may be used as a predictive tool for the identification of MHC-binding sequences as well as of T cell epitopes in their naturally occurring form.

Catheptic processing of protein antigens: enzymic and molecular aspects.

Processing of most exogenous protein antigens involves restricted intracellular proteolysis to yield fragments that may be bound specifically by MHC class II glycoproteins. This association is required for functional presentation to antigen-specific T lymphocytes. The proteolytic degradation takes place in antigen-presenting cells, in an acidic, endosomal compartment where antigens arrive after capture and internalization. It is catalyzed by a small number of enzymes, mainly aspartic and thiol proteases, with the cathepsins D and B--in that order--as dominant representatives. Resulting immunogenic protein fragments possess a binding site for mature MHC molecules and a recognition site for T cell receptors; topologically, these sites are interspersed, generally extending over 7-8 consecutive amino acid residues. Binding of peptides by MHC proteins of a particular genotype appears to depend mainly on specific interactions involving a common sequential motif in their primary structure in terms of non-polar and polar/charged amino acid residues and not on a general propensity to adopt a certain regular secondary structure, such as an alpha-helix.

The selectivity of cathepsin D suggests an involvement of the enzyme in the generation of T-cell epitopes.

The selectivity of cathepsin D, a mammalian intracellular aspartyl proteinase involved in the degradation of endocytosed proteins, was studied. For this purpose, several proteins of known primary structure were subjected to mild proteolysis by the enzyme, and the preferentially cleaved peptide bonds were identified. Comparison of the primary structures around these sites indicates that cathepsin D shows a strong preference for peptide bonds within a distinct sequence pattern of amino acids extending over 7 residues. In general, this pattern is most likely to occur within amphipathic alpha-helical structures. These findings and their possible implications are discussed together with additional evidence suggesting an important role for cathepsin D in the processing of protein antigens, an essential step for their recognition by T-cells. Accordingly, it is proposed that the proteolytic activity of cathepsin D is crucial in selecting processing sites and hence the location and structural context of T-cell epitopes for the majority of protein antigens.

Conformational differences between aged and nonaged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin as detected by optical spectroscopy.

Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.

Hydrodynamic characterization of the size and shape of atropinesterase from Pseudomonas putida.

Atropinesterase from Pseudomonas putida has been investigated by means of different ultracentrifugation methods under native and denaturing conditions. The following quantities were determined: sedimentation coefficient, translational diffusion and friction coefficient, partial specific volume and molecular weight. From these data the size, shape and hydration of the enzyme molecule in solution were estimated. The results suggest that atropinesterase is a globular protein which consists of a single polypeptide chain with a molecular weight of about 30,000. In solution under non-denaturing conditions, it occurs mainly as a dimer which hydrodynamically behaves as a rigid impenetrable particle. Calculations based on the spheroid model indicate that this particle resembles a hydrated sphere with a diameter of 6.1 +/- 0.2 nm and a hydration of 0.4 +/- 0.1 g of H2O/g of protein rather than a significantly less hydrated ellipsoid of revolution. Under denaturing conditions dissociation into monomers takes place. The effects of sodium dodecyl sulphate (SDS) on size and shape suggest that dimerization results from side-by-side association of two elongated monomers rather than from end-to-end association. Approximately 57 molecules of SDS are bound per dimer before dissociation occurs concomitant with the additional binding of about 19 molecules of detergent.

P NMR and mass spectrometry of atropinesterase and some serine proteases phosphorylated with a transition-state analogue.

The serine residue in the active center of atropinesterase (AtrE), alpha-chymotrypsin (Chymo), and subtilisin A (Sub) and in alpha-chymotrypsinogen (Chymogen) was labeled with a diisopropylphosphoryl (DP) group. The labeled proteins were studied in buffered aqueous solution under various native and denaturing conditions with 31P NMR before and after being subjected to "ageing", a process leading to conversion of the DP group into a monoisopropylphosphoryl (MP) group. Besides, the model compounds Gly-Ser(DP), Gly-Glu-Ser(DP)-Gly-OEt, and diisopropyl hydrogen phosphate were investigated under similar conditions and in other solvents with different hydrogen-bonding capacity. Mass spectrometry was used to analyze products resulting from ageing in the presence of H2(18)O. The 31P chemical shift of the DP proteins increases according to a simple titration curve upon lowering the pH from 9.0 to 5.0. This is ascribed to protonation of a particular histidine residue in the active center that interacts with a nearby isopropoxy group by hydrogen bonding with the ester oxygen. In DP-AtrE, hydrogen bonding at the phosphoryl oxygen dominates the interaction between substituent and protein; in the other DP proteins, nonbonding interactions become more dominant in the order Chymogen less than Chymo less than Sub. DP-AtrE, DP-Chymo, and DP-Sub age according to first-order kinetics. The pH dependence of the reaction rate constant ka indicates that ageing is catalyzed by the protonated histidine, which is responsible for the increase in chemical shift. The direct interaction between the phosphoryl group and the histidine is lost upon ageing whereas there is an increase in the nonbonding interaction of the remaining isopropyl group with the protein in the order Chymo less than Sub less than AtrE. The maximum value of ka when the histidine is fully protonated (kam) increases in the same order. Ageing of the DP enzymes occurs exclusively by C-O fission, yielding 2-propanol and propene. Since the amount of 2-propanol decreased and that of propene increased in the order Chymo to Sub to AtrE, the increase in kam has been interpreted as a shift in character of ageing from mainly SN2 for Chymo to considerably SN1 for AtrE and Sub. This has been attributed to preferential stabilization of the SN1 transition state by an interplay of hydrogen-bonding and nonbonding interactions between the phosphoryl group and the protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the active sites of atropinesterase and some serine proteases by spin-labeling.

The side chain of the serine residue in the active center of atropinesterase (AtrE), alpha-chymotrypsin (Chymo), and subtilisin A (Sub) was labeled with two paramagnetic reporter groups of different size (label I or II, respectively) by sulfonylation with N-[3-(fluorosulfonyl)phenyl]-1-oxy-2,2,5,5-tetramethyl-pyrroline-3 -carboxamide or N-[6-(fluorosulfonyl)-2-naphthyl]-1-oxy-2,2,5,5-tetramethylpyrroline+ ++-3 -carboxamide. ESR spectra of labeled enzymes in 10 mM phosphate buffer, pH 7.4, were measured at temperatures between 133 and 298 K by using a home-built spectrometer operating in the absorption mode at 10-kHz field modulation. The spectra, in particular those at 276-298 K, were analyzed by computer simulation of the overall line shape according to the methods developed by Freed and co-workers, based on eigenfunction expansion. In the case of AtrE for both labels, the best agreement between experimental and simulated solution spectra was obtained with only one mobility component showing anisotropic, axially symmetric reorientation according to the Egelstaff jump-diffusion model. The axis of preferential reorientation was found to lie in the XZ plane at a polar angle of about 30 degrees with the X axis. The corresponding rotational correlation time (tau parallel) did not show appreciable viscosity/temperature (eta/T) dependence but had a constant value of 4.4 and 2.2 ns for labels I and II, respectively. The rotational correlation time associated with rotation around the axes perpendicular to that of preferential reorientation (tau perpendicular) showed the usual eta/T dependence and had a value of 22.0 ns at 276 K for both labels. The above results strongly suggest that in AtrE both nonpolar reporter groups reside in a pocket near the active serine. Contrary to the situation in AtrE, the overall mobility of the -N-O. fragments in Chymo and Sub was found to result from contributions of at least two distinct motional states, strongly and weakly immobilized. In going from label I to label II, the relative contribution of the latter state increases at the expense of that of the former. This is ascribed to an equilibrium between a relatively free state of the aromatic cores and a firmly bound position in the specificity pocket of these proteases. The apparently more rigid embedding of the spin-labels in the enzyme structure of AtrE suggests that the size of the nonpolar binding pocket in the active center region of this esterase allows a deeper penetration of the aromatic portions of the labels than is possible for the specificity pocket of Chymo or Sub.

Comparsion of the oxygen-enhancement ratio for gamma-ray-induced double-strand breaks in the DNA of bacteriophage T7 as determined by two different methods of analysis.

Bacteriophage T7 was irradiated in a protecting medium under nitrogen and oxygen with 60Co gamma-rays. Double-strand breaks were measured by sucrose-gradient sedimentation and by boundary-sedimentation analysis. Both methods showed that the presence of oxygen during irradiation enhanced the production of double-strand breaks. This is at variance with a recent report which suggests that boundary-sedimentation analysis does not show an effect of oxygen. The discrepancy must be ascribed to differences in interpretation of the sedimentation data.