Yoshimi Yamamoto

Retinal Pigment Epithelium-Derived CTLA-2α Induces TGFβ-Producing T Regulatory Cells

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4+ T cells into Treg cells by producing and secreting CTLA-2α, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2α converted CD4+ T cells into Treg cells in vitro, and CTLA-2α small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2α induced CD4+CD25+Foxp3+ Treg cells that produced TGFβ in vitro. Moreover, CTLA-2α produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4+ T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2α effect on Treg cell induction occurred through TGFβ signaling, because CTLA-2α promoted activation of TGFβ in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.

Yolk proteins from insect eggs: Structure, biosynthesis and programmed degradation during embryogenesis

Abstract Since insect vitellogenesis is a heterosynthetic process, yolk protein follows a complicated fate. This includes: hormonally stimulated gene activity, secretion into the blood, specific recognition and uptake by oocytes, packaging into yolk granules, and proteolysis in developing eggs. During the process, proteins are modified co- or post-translationally. Each of these steps has been intensively studied in insects. This offers a basis for more exciting development not only in the field of insect physiology, but also in more basic or fundamental areas of biochemistry and cellular biology. Bombyx acid cysteine proteinase is a case in point. It is now evident that proteolytic reactions play a key role in the control of many physiological functions. Thus the control mechanisms involved in the proteolytic processes require attention and proteinases have been purified and characterized. In many cases, however, it is difficult to analyze the physiological substrates of them, and difficult to know the regulatory mechanism in vivo . Insect yolk protein systems provide a suitable system for studying regulation of cellular proteinases, since yolk proteins (substrate of proteinases) are purified and characterized. This review summarizes recent progress in understanding the structure, biosynthesis and programmed degradation of yolk proteins in insects.

PURIFICATION AND CHARACTERIZATION OF BOMBYX CYSTEINE PROTEINASE SPECIFIC INHIBITORS FROM THE HEMOLYMPH OF BOMBYX MORI

Protein inhibitors capable of inhibiting BCP (Bombyx cysteine proteinase) were found in the larval-pupal hemolymph of Bombyx mori. Two forms of the inhibitors, named BCPI (BCP inhibitor) alpha and BCPI beta, were purified from the pupal hemolymph by heat treatment and column chromatographies on CM-cellulose, Toyopearl HW-50, Phenyl-Sepharose, and Mono Q. Purified BCPI beta gave a single protein band with a molecular mass of 10,500 daltons on SDS-PAGE. BCPI alpha is mostly composed of the same molecular mass protein as BCPI beta. Both forms were inhibitory towards other cysteine proteinases such as cathepsins L,B and papain but had no effects on trypsin and pepsin. Both forms inhibited the processing of the enzymatically inactive proform of BCP (pro-BCP) to the activated mature BCP. BCPI alpha and BCPI beta shared many other features such as molecular mass determined by gel filtration, antigenicity, and HPLC profiles. NH(2)-terminal amino acid sequencing of the purified inhibitors revealed that three amino acid residues were different in the BCPI alpha and BCPI beta sequences, all others being identical. The hemolymph BCP inhibitor increased activity approximately four- to fivefold at the time of spinning and maintained this level of activity during pupation.

Acquisition of T Regulatory Function in Cathepsin L-Inhibited T Cells by Eye-Derived CTLA-2α during Inflammatory Conditions

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4+ T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2α, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2α’s role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-β, but not IFN-γ inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2α in RPE. Similarly, CTLA-2α via RPE was able to promote TGF-β production by the CD4+ T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-β and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4+ T cells from EAU in CathL knockout mice or rCTLA-2α from EAU animals were found to contain a high population of forkhead box p3+ T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2α, thereby enabling the bystander T cells to be converted into Treg cells via TGF-β promotion.

Acid cysteine proteinase from the eggs of silkmoth, Bombyx mori: Tissue distribution, developmental changes and the sites of synthesis for the enzyme

Abstract Acid cysteine proteinase activities were found in the pupal ovary and hemolymph from the silkmoth, Bombyx mori. The activity in mature eggs was quite high. It increased abruptly 6 days after larval-pupal ecdysis. In the hemolymph, low activity is maintained throughout the pupal stage. These were confirmed using specific antiserum and immunoblotting. Cultured hemocytes from female pupae immediately after larval-pupal ecdysis synthesized and released cysteine proteinase into the medium. Cultured ovary could also synthesize the proteinase, whereas in this case, the protein was not secreted into the medium. Translation of mRNA isolated from hemocytes and ovary directed the synthesis of the enzyme in a reticulocyte lysate cell free system, indicating that there were two major sites of synthesis for cysteine proteinase in the female pupae.

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs : localization of yolk proteins and BCP, and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.

PROREGION OF BOMBYX MORI CYSTEINE PROTEINASE FUNCTIONS AS AN INTRAMOLECULAR CHAPERONE TO PROMOTE PROPER FOLDING OF THE MATURE ENZYME

A cDNA encoding the proform of Bombyx cysteine proteinase (BCP) was expressed at a high level in Escherichia coli using the T7 polymerase expression system. The insoluble recombinant zymogen was solubilized and renatured by modifying a method applied to human pro-cathepsin L. Like the natural BCP precursor, the recombinant proenzyme was spontaneously converted to an active proteinase at pH 3.75. A deletion in the central region of the propeptide resulted in much loss of the activity, suggesting that the propeptide is essential for proper folding during renaturation. In contrast, the renatured mature form of recombinant BCP was not active but regained activity by including the propeptide in the renaturing buffer, suggesting that the propeptide, acting as an intramolecular chaperone, promotes refolding of the associated proteinase domain into an active conformation. The mature form of natural BCP rapidly lost its activity at neutral pH, whereas its proform was stable. The mature enzyme retained some activity in the presence of the propeptide. Arch.

Bombyx acid cysteine proteinase

Summary Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.

Cysteine proteinase from the eggs of the silkmoth, Bombyx mori: Site of synthesis and a suggested role in yolk protein degradation

Abstract A cysteine proteinase with properties similar to mammalian cathepsins has been found in silkmoth eggs. Enzyme activity in the ovary increased abruptly 5–6 days after larval-pupal ecdysis. Activity in the mature ovary is quite high, and high activity is maintained throughout embryonic development. In a previous paper, we reported that the enzyme was synthesized in the ovary. To define the site of synthesis in more detail, follicle cells were isolated from ovaries and cultured in a medium containing 14 C-amino acids. Isolated follicle cells synthesized and secreted cysteine proteinase into the medium. Immunohistochemistry also showed that the major site of synthesis is the follicle cells. Cysteine proteinase is able to degrade vitellin (Vn) and egg specific protein (ESP) in vitro in a profile similar to that found in vivo . The results are discussed in relation to the degradation mechanism of yolk proteins during early embryonic development.