Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site

Abstract

We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40 kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I : a stretch of approx. 70 amino acids with a high cysteine content (approx. 15 %) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca# + -binding site). Expression analysis of

Evaluation of all non‐synonymous single nucleotide polymorphisms (SNPs) in the genes encoding human deoxyribonuclease I and I‐like 3 as a functional SNP potentially implicated in autoimmunity

“…It remains unknown why both DNases disappeared during the evolution of Protostomia from the predicted ancestral species of Eumatazoa, in contrast to Deuterostomia. Next, multiple alignment analysis of the amino acid sequences of DNase I and 1L3 from Animalia organisms available on the genome database, in addition to vertebrate DNases I determined previously [21][22][23][24], was performed; DNase I and 1L3 derived from 40 and 26 species, respectively, were analyzed (Figs S1 and S2). Among the amino acid sequences of animal DNases I, all the organisms fully conserved 27 amino acid residues, which included the four amino acid residues responsible for the active site [25] and two Cys residues that form the disulfide bond responsible for structural stability of the enzyme [26], corresponding to Glu100, His156, Asp234 and His274, and the Cys residues at positions 195 and 231 in human DNase I, respectively.…”

Molecular evolution of shark and other vertebrate DNases I

“…1). Although anion exchange chromatography using resins such as DEAE-Sepharose CL-6B has generally been found useful for the purification of vertebrate DNases I, including the human [11], rat [12], rabbit [13], amphibian [19] and reptile [18] enzymes, shark DNase I was retained on a cation exchange resin but not on an anion exchange one. As shown below, H. japonicus DNase I consisted of 262 amino acid residues; however, it was found to contain more basic amino acids (32 residues) than acidic ones (27 residues), whereas the human enzyme has 24 basic and 31 acidic amino acid residues.…”

“…DNase I activity was assayed using the single radial enzyme diffusion (SRED) method [2,22] or test tube method [11] as described previously, except that 50 mM Hepes/NaOH buffer pH 8.0, containing 20 mM MgCl 2 and 2 mM CaCl 2 was substituted for the reaction buffer. The enzymatic [23], proteochemical [11] and thermal stability [18,19] characteristics of the enzymes were examined as described previously.…”

“…LipofectaminPlus, all oligonucleotide primers, and the 3¢-and 5¢-RACE systems were obtained from Invitrogen; CM-Sepharose CL-6B, Mono S 5/50 GL and Superdex 75 were from Amersham Pharmacia Biotech; the Expanded High Fidelity PCR system was from Boehringer Mannheim GmbH. Antibodies specific to human, hen, Rana catesbeiana (frog), Elaphe quadrivirgata (snake) and Cyprinus carpio (carp) DNases I were prepared using previously described methods [11,[17][18][19]21]. All other chemicals used were of reagent grade and commercially available.…”

“…These, together with other findings suggesting that DNase I or DNase I-like endonucleases may be responsible for internucleosomal DNA degradation during apoptosis [9,10], have focused attention on the potential physiological roles of DNase I. In this context, we have attempted to elucidate the intrinsic intraand extracellular function(s) of DNase I, as well as the phylogenetic origins of the vertebrate DNase I family, by carrying out comprehensive comparisons of the enzymes from lower and higher vertebrates: the biochemical and molecular characterizations of mammalian [11][12][13][14][15][16], avian [17], reptilian [18] and amphibian [19] DNases I have already been reported. Previous studies on piscine DNases I, from Oreochromis mossambica (tilapia) [20] and five different species of the Osteichthye class [21], have demonstrated that these enzymes possess some unique features compared with those of other vertebrates: a relatively high pH for optimum activity and greater structural diversity.…”