Hisao Kakegawa

Participation of cathepsin L on bone resorption

The proteinase responsible for bone collagen degradation in osteo‐resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E‐64 and cystatin A, while no inhibition was observed by CA‐074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E‐64 or cystatin A, but not by CA‐074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer‐graphic modeling based on the stereo‐structure. The common fragment, N‐(L‐trans‐carbamoyloxyrane‐2‐carbonyl)‐phenylalanine‐dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10−7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10−7 M in vitro.

SECRETION AND PROCESSING MECHANISMS OF PROCATHEPSIN L IN BONE RESORPTION

Secretion of procathepsin L into the culture medium from a bone cell mixture was markedly enhanced by addition parathyroid hormone (PTH), 1α,25‐(OH)2D3 or tumor necrosis factor α (TNFα). These stimulators of secretion of procathepsin L enhanced bone pit formation, which was inhibited by E‐64, but not by CA‐074, a specific inhibitor of cathepsin B. Procathepsin L may thus participate in the process of bone collagenolysis during bone resorption. Procathepsin L partially purified from rat long bones under cold conditions was rapidly converted to the mature form under acidic conditions at room temperature. This conversion was inhibited by E‐64, suggesting that the procathepsin L secreted into lacunae is catalytically converted to the mature enzyme by cysteine proteinase(s).

The mechanisms and regulation of procathepsin L secretion from osteoclasts in bone resorption

The secretion mechanisms of cathepsin L from osteoclasts in the process of bone resorption were investigated. The increases in bone pit numbers formed take place by PTH addition in parallel with the increases of cathepsin L and/or L‐like proteinase activities in the culture medium of bone cells, and these were suppressed by the addition ofcalcitonin. The Z‐Phe‐Arg‐MCA hydrolysing activity increased in the medium through the effect of PTH is considered to be a kind of procathepsin L by Western blotting analysis, and was suppressed by calcitonin addition. Furthermore, monensin inhibited not only the PTH‐induced pit formation, but also cysteine proteinase activity in osteoclasts. Therefore, the procathepsin L excreted might be transferred from endothelial reticulum via Golgi and/or via lysosomes.

Linkage between phosphorylated cystatin α and filaggrin by epidermal transglutaminase as a model of cornified envelope and inhibition of cathepsin L activity by cornified envelope and the conjugated cystatin α

Lysine‐rich phosphorylated cystatin α (P‐cystatin α) from newborn rat epidermis is a good substrate for epidermal transglutaminase (TGase) and also one of the component proteins of cornified envelope in the stratum corneum. Since the filaggrin linker segment peptide was efficiently conjugated with P‐cystatin α and was mediated by epidermal TGase in the presence of Ca2+ ions, filaggrin is a candidate for the glutamine‐rich linkage protein to conjugate with lysine‐rich P‐cystatin α. A conjugated protein was formed by epidermal TGase in the activated condition with Ca2+ ions and dithiothreitol. In contrast, the conjugated protein was not formed under chelated conditions with EDTA. The conjugated protein reacted positively with anti‐P‐cystatin α polyclonal antibody (PoAb). The conjugated protein and purified cornified envelope showed an inhibitory effect against papain and cathepsin L, but cathepsin B and H were not inhibited by these P‐cystatin α conjugates. Although the component protein, P‐cystatin α itself, inhibited cathepsin H strongly, these conjugated proteins inhibited specifically the cathepsin L family. The amino acid composition of cornified envelope protein and the conjugated protein of P‐cystatin α and filaggrin linker segment peptide was not completely the same. The conjugated protein of P‐cystatin α and filaggrin linker segment peptide showed the same inhibitory properties against cysteine proteinases as the cornified envelope. These findings suggest that the linkage protein between P‐cystatin α and filaggrin linker segment peptide may be considered a model of cornified envelope, although skin cornified envelope may be conjugated with some additional proteins.

Syntheses and reactions of the diethyl α-alkynylmalonates involving the generation of conjugated allenyl esters as the latent active species: A new approach to the development of cysteine proteinase inhibitors

Abstract Various diethyl α-alkynylmalonates (DAM) having potential as cysteine proteinase inhibitors were synthesized by treatment of diethyl acetyliminomalonate (or ketomalonate) with several lithium acetylides. Hydrolytic decarboxylation of the DAM under the mild basic conditions afforded oxazole derivatives or allenyl esters which caused the Michael type reaction with EtSH.

Immunological significances of invariant chain from the aspect of its structural homology with the cystatin family

The primary structure of p31 of invariant chain (Ii‐chain) shows about 50% homology with those of the cystatin family which are endogenous cysteine protease inhibitors. The binding domains between Ii‐chain and HLA‐DR‐7 were estimated from the structural homology between cystatin and Ii‐chain and also between cathepsins and DR‐7, respectively. The QL64–71 and GS76–88 of Ii‐Chain were estimated to be the binding domains with GG45–51, and VS57–63 of HLA‐DR7, respectively. The purified human Ii‐chain from spleen is capable of forming four molecular forms from monomer to tetramer by redox‐potential dependent disulfide bond formation. The Ii‐chain inhibits cathepsin L and H competitively as a dimer and the Ki value for cathepsin L was 4.1 × 10−8 M, but cathepsin B was not inhibited at all. The Ii‐chain showed mainly a dimer (60 kDa) under the assay condition of cathepsins with cysteine and was not degraded by these cathepsins. The Ii‐chain may play an important role in the regulation of antigenic peptide presentation to MHC class II.

Inhibitory mechanisms of H+‐ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption

The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.The effects of the vacuolar‐type H+‐ATPase inhibitor bafilomycin A1 (baf. A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf. A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.

Novel physiological functions of cathepsins B and L on antigen processing and osteoclastic bone resorption

Lysosomal cathepsin B plays an essential role in the processing of ovalbumin as an exogenous antigen to produce the complex between antigenic-peptide and major histocompatibility-complex class II. Administration of cathepsin B inhibitors, E-64, CA-074 and vitamin B6, caused the strong suppression of the Th-2 type immune responses. We found that pyridoxal phosphate (PAP), a coenzyme form of vitamin B6, inhibits the activities of cathepsin B and L in vitro and vitamin B6 administration induces the inhibition of the lysosomal cathepsin activities in vivo. The production of an antigenic epitope (I323-R339) of ovalbumin by antigen presenting cells was suppressed by cathepsin B specific inhibitors. The ovalbumin dependent production of immunoglobulins (IgE and IgG1) and of the corresponding interleukin (IL-4) was suppressed by cathepsin B inhibitors, while the production of IgG2a and interferon (INF-gamma) was increased. The switch of helper T lymphocyte functions from the type-2 to the type-1 may be induced by the cathepsin B inhibition. The experimental bone pit formation, i.e., osteoclastic bone collagen degradation test, induced by parathyroid hormone was markedly suppressed by the administration of pyridoxal, because of the inhibition of cathepsin L type cysteine proteases in bone.

Chondroitin sulfate proteoglycan is a potent enhancer in the processing of procathepsin L

Abstract The acceleration effect of chondroitin-4-sulfate(CS) proteoglycan on the processing of procathepsin L in vitro was investigated using enzyme purified from the culture medium of MLC cells. Procathepsin L was slightly processed even when it was incubated without CSproteoglycan for 60 min in 50 mM acetate buffer, pH 5.5, and trace amounts of the 31 kDa mature form and 35 38 kDa intermediates of cathepsin L were formed. On the other hand, in the presence of CSproteoglycan, procathepsin L was completely converted to the mature form within the same 60 minute time period. Moreover, ZPheArgMCA hydrolyzing activity was increased significantly by the incubation with CSproteoglycan, while no considerable increase in the activity was observed during the incubation without CSproteoglycan. Since the specific cathepsin L inhibitor, CLIK-195, inhibited the processing of procathepsin L accelerated by CSproteoglycan, the trace amount of cathepsin L activity may participate in the processing. These results suggest that CSproteoglycan may play a role in accelerating the processing of procathepsin L as an endogenous enhancer in the extracellular environment in vivo.