A Protease Sensitive Region of Plant and Animal Ribonucleases Belonging to the RNase T2 Family.

Iwama, Masanori; Kusano, Ayumu; Ogawa, Yuko; Ohgi, Kazuko; Irie, Masachika

doi:10.1248/bpb.21.634

Cited by 2 publications

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“…The 26‐kDa form we observed is recognized by two different polyclonal antibodies against RNASET2 and also by an antibody against the C‐terminal HA tag, suggesting that spontaneous proteolysis occurs at the N‐terminus. Indeed, ribonucleases from the Rh/T2/S family have been described to possess protease‐sensitive regions 25 and we previously showed that RNASET2 undergoes physiological proteolysis in mammalian cells, resulting in 31 and 27 kDa isoforms, which are targeted to the lysosome 4. However, further analysis of the human forms showed that they were both generated by a cleavage event occurring in the C‐terminal moiety of the RNASET2 protein.…”

Section: Discussionmentioning

confidence: 89%

Comparison of the baculovirus‐insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

Campomenosi

Cinquetti

Tallarita

et al. 2011

Biotech and App Biochem

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We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.

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Section: Discussionmentioning

confidence: 89%

Comparison of the baculovirus‐insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

Campomenosi

Cinquetti

Tallarita

et al. 2011

Biotech and App Biochem

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Effect of Modification of the Carboxyl Groups of the Sialic Acid Binding Lectin from Bullfrog (Rana catesbeiana) Oocyte on Anti-tumor Activity.

Iwama

Ogawa

Sasaki

et al. 2001

Biological & Pharmaceutical Bulletin

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The sialic acid binding lectin from Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity.2) cSBL is a multifunctional protein which has pyrimidine base specific ribonuclease activity. 3,4) In respect to RNase, cSBL belongs to the RNase A family, and is a very heat stable enzyme. It remained 100% active after heating at 100°C. 3,5) Four RNases belonging to the RNase A family have been isolated from Rana family frogs to date. Their primary structures are shown in Fig. 1. They have anti-tumor activity, except for that from bullfrog liver. 11)They are a simple protein and constituted from 104 to 111 amino acid residues. The anti-tumor activity of one of them, onconase, obtained from Rana pipiens, has been studied in detail, and is on its way to application as an anti-tumor drug, now in phase III clinical trials. 12,13) On the other hand, the anti-tumor activity of cSBL has been reported by Nitta et al.2) Although the mechanism of the anti-tumor activity of cSBL is not fully elucidated to date, the first step of the action was different from that of onconase. That is, the interaction of cSBL with sialoglycoprotein on the surface of tumor cells is thought to be essential for cSBL action. The interaction enhances the internalization of cSBL into the tumor cells. However, in contrast to cSBL, for onconase, the contribution of sialic acid to the action has not been identified to date.Nitta et al. showed that the chemical modification of cSBL with a water-soluble carbodiimide (EDC) in the presence of glycine methylester increased the anti-tumor activity. The The sialic acid binding lectin from bullfrog Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity. In order to investigate the relationship between the net charge of cSBL and its anti-tumor effect, cSBL was modified with a water-soluble carbodiimide (EDC) in the presence of three kinds of nucleophiles, taurine, glycine methylester and ethylenediamine. cSBL having four carboxyl groups was partially modified (ca. 2 residues). The anti-tumor activity of modified cSBLs was in the order of ethylenediamine-modified cSBLϾglycine methylestermodified cSBLϾtaurine modified cSBLмnative cSBL. The results suggested that anti-tumor activity seems to increase with the increase in positive net charge, possibly enhancing the interaction of cSBL with sialoglycoprotein on the surface of tumor cells. The ribonuclease activity of ethylenediamine-modified cSBL decreased with the progress of the reaction, but the number of internalized molecules in the tumor cell increased. Thus, for antitumor activity, a higher incorporation of cSBL with reasonable RNase activity seems to be more important than total RNase activity.

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A Protease Sensitive Region of Plant and Animal Ribonucleases Belonging to the RNase T2 Family.

Cited by 2 publications

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Comparison of the baculovirus‐insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

Comparison of the baculovirus‐insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

Effect of Modification of the Carboxyl Groups of the Sialic Acid Binding Lectin from Bullfrog (Rana catesbeiana) Oocyte on Anti-tumor Activity.

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