青蛙体内Amphinase分子有望治疗脑癌

Enzymatic and Structural Characterisation of Amphinase,  a Novel Cytotoxic Ribonuclease from Rana pipiens Oocytes

Umesh P. Singh1, †, Wojciech Ardelt2, †, Shailendra K. Saxena2, †, Daniel E. Holloway1, †, Eugene Vidunas2, 2, Hung-Suen Lee2, Abha Saxena2, Kuslima Shogen2, , and K. Ravi Acharya1, ,
1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
2Alfacell Corporation, Bloomfield, NJ 07003, USA
Received 21 March 2007;  revised 26 April 2007;  accepted 29 April 2007.  Edited by M. Guss.  Available online 10 May 2007.

Abstract

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1–4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8–99.1% and 38.2–40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of kcat/KM with hypersensitive fluorogenic substrates are 104 and 102-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B1 or B2 subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 Å resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual α2–β1 loop may do so) and the B2 subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.

Keywords: Amphinase; cytotoxic RNase; X-ray crystallography; enzyme efficiency; substrate specificity

Abbreviations: Amph, Amphinase; Amph-1–4, Amphinases-1–4; CpA, cytidylyl-3′,5′-adenosine; DTT, dithiothreitol; 6-FAM-dArC(dA)2-6-TAMRA and rCA, 6-carboxyfluorescein-dArC(dA)2-6-carboxytetrarhodamine; 6-FAM-dArU(dA)2-6-TAMRA and rUA, 6-carboxyfluorescein-dArU(dA)2-6-carboxytetrarhodamine; 6-FAM-dArUdGdA-6-TAMRA and rUG, 6-carboxyfluorescein-dArUdGdA-6-carboxytetrarhodamine; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; ONC, Onconase; PEG, poly(ethylene glycol); PNGase F, peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase from Elizabethkingia (Chryseobacterium/Flavobacterium) meningsepticum; rAmph-2, recombinant Amphinase-2; RC-RNase, Rana catesbeiana ribonuclease; RC-RNase-6, Rana catesbeiana ribonuclease-6; RI, ribonuclease inhibitor protein; RNase A, bovine pancreatic ribonuclease A; rRNA, ribosomal RNA; TFA, trifluoroacetic acid; TPCK, N-p-tosyl-L-phenylalanine chloromethyl ketone; UpA, uridylyl-3′,5′-adenosine

Corresponding authors.
^† U.P.S., W.A., S.K.S. and D.E.H. contributed equally to this work.
² Present address: E. Vidunas, Wyeth Pharmaceuticals, Pearl River, NY 10965, USA.