Daisaku Muno

Both cathepsin B and cathepsin D are necessary for processing of ovalbumin as well as for degradation of class II MHC invariant chain.

The effect of highly selective inhibitors of cathepsins on the processing of ovalbumin (OVA) and the presentation of an OVA-derived antigenic peptide (OVA323-339) by antigen presenting cells (APC) was investigated. Both CA-074 (a specific inhibitor of cathepsin B) and pepstatin A (a specific inhibitor of cathepsin D) showed an inhibitory effect on the IL-2 production from an OVA-specific, I-Ad-restricted helper T (Th) cell clone upon stimulation with OVA presented by the I-Ad-positive APC. In contrast, the presentation of the antigenic epitope, OVA323-339, to the same Th clone was not inhibited by either CA-074 or pepstatin A alone, nor even by the mixture of both inhibitors. When APC were treated with cathepsin inhibitor for 24 h, and then antigen and Th were added to the culture, the presentation of not only OVA but also an OVA-derived antigenic peptide was inhibited by either cathepsin inhibitor alone. In addition, the expression of invariant chain on APC was significantly augmented by the pretreatment of APC with either cathepsin inhibitor. Two main conclusions are drawn from these results. First, not only aspartyl protease, such as cathepsin D, but also thiol protease, such as cathepsin B, is involved in antigen processing by APC. Second, both cathepsin B and cathepsin D are necessary for degradation of the invariant chain (Ii) from the MHC class II alpha beta heterodimer in endosomes in order to express functional MHC class II molecules for binding antigenic peptides.

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins

A selective inhibitor of cathepsin B, a derivative of E‐64 (compound CA‐074), and pepstatin‐asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA‐074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC‐labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.

Generation of both MHC class I‐ and class II‐restricted antigenic peptides from exogenously added ovalbumin in murine phagosomes

The phagosome fraction derived from a murine macrophage cell line (J774.1), which had internalized ovalbumin (OVA)‐coated latex beads, was isolated. The peptides recovered from the phagosome fraction were separated on reverse phase HPLC and each fraction was analyzed for the content of either major histocompatibility complex (MHC) class I‐ or class II‐restricted OVA‐derived peptide. Both peptides were detected in the phagosome fraction after less than 15 min of internalization. It was also indicated that phagosomes degrade OVA protein into both MHC class I‐ and class II‐restricted antigenic peptides by employing the same types of cathepsins. Furthermore, the results suggest that the MHC class I‐restricted peptide rapidly exits from the phagosome to the cytosol. These findings illustrate a potential role for phagosomes not only in MHC class II‐restricted but also in MHC class I‐restricted exogenous antigen presentation pathways. Our results also point to the vital role of phagosomes in non‐cytosolic antigen presentation pathway, in which further degradation of antigens by the proteasome is dispensable.

MHC Class I Presentation of an Exogenous Polypeptide Antigen Encoded by the Murine AIDS Defective Virus

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25‐mer polypeptide (P12–25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H‐2b) CD8+ T cells in vitro. The presentation of P12–25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome‐inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA‐074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12–25 polypeptide. In fact, when P12–25 was digested with purified cathepsin B in vitro, a highly antigenic 11‐mer peptide containing the class I (H‐2Db)‐binding motif was obtained. Our results favor the phagosome/macropinosome‐to‐cytosol‐to‐endoplasmic reticulum (ER)‐to‐cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8+ T cell immunity in vivo.

New fluorogenic, photoactivable, heterobifunctional crosslinking thiol reagents☆

Properties of newly synthesized crosslinking reagents (ACM) and their applications to proteins are studied (ACM is the abbreviation for a series of photoactivable and heterobifunctional crosslinking thiol reagents, each of which has two reactive groups, maleimide and azide). These reagents bind specifically to the sulfhydryl residues of proteins in the first reaction step. Upon photoactivation, the azide group of the coumarin ring reacts with side or main chains of the proteins, and thus intra- or intermolecular crosslinking can be elicited. In addition, the coumarin moiety of the reagents becomes highly fluorescent after photolysis. Therefore, the crosslinking products can be detected by fluorometry with high sensitivity in the pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reaction of ACM with rabbit muscle aldolase led to extensive crosslinking between subunits of the enzyme and maximally 25% of the total subunits were found to be crosslinked to the dimer.

Lysosomal Proteinosis Based on Decreased Degration of a Specific Protein, Mitochondrial ATP Synthase Subunit C

Lysosomal storage diseases are a group of more than 35 genetically determined heterogenous metabolic disorders. Biochemically, they are characterized by deficient activity of specific lysosomal hydrolases and intralysosomal accumulation of one or more substrates in affected cells. They are often categorized according to the chemical nature of the most prominent storage compounds. They include lipid storage diseases, glycogen storage diseases, mucopolysaccharidoses, mucolipidoses and gangliosidoses. However, there are no reports of protein storage diseases (proteinoses) based on deficiency of specific lysosomal proteases. Experimentally, inhibition of lysosomal cysteine proteinases by administration of leupeptin and E-64 causes intralysosomal storage of cytoplasmic proteins in cells (1).

Intracellular functions of lysosomal cysteine proteinases in macrophages.

Macrophages contain high amounts of lysosomal cysteine proteinases that are considered to be important in catabolic process of endogenous and exogenous proteins.Addition of horseradish peroxidase to the cultured macrophages induced an increase in the density of lysosomes in parallel with the accumulation of peroxidase taken up the heavy lysosomes. Degradation of uptaken peroxidase was inhibited by addition of anticathepsin L antibody to the medium, but not by addition of anti-cathepsin B or H antibody. Treatment of cells with E-64 caused intracellular accumulation of endogenous proteins due to inhibition of lysosomal proteolysis, but pepstatin, a potent inhibitor of cathepsin D, showed much less effect.Those results suggest a large contribution of cysteine proteinases, especially cathepsin L to the initiation of lysosomal degradation of both endogenous and exogenous proteins. The levels of mRNAs for cathepsin L and H and cystatin β in macrophages obtained from rats injected with sodium caseinate were several times as high as those in resident macrophages.

Photocross-linking from SH1 of the myosin heavy chain to light chains through the dinitrophenyl group attached to SH1.

Trinitrobenzene selectively dinitrophenylates SH1, a specific thiol in the myosin heavy chain which contains 1 mol of this cysteinyl residue. When the SH1-DNP-myosin thus obtained was irradiated with a mercury lamp, a cross-linked product was formed with a molecular weight of about 220K daltons. It was shown that this product was composed of both heavy and light chains by fluorescence labeling of the heavy chain at SH2, another specific thiol, and immune reaction using an anti-light chain antibody, respectively.