Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

Abstract

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA-cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.

Molecular Cloning of the cDNA Encoding Human Deoxyribonuclease II

“…Thus, all tissues with significant levels of DNase II activity (Table I) yielded detectable amplified products specific for DNase II. Therefore, in contrast to DNase I, which shows tissue-specific expression of the gene (23,30), our results indicate that the DNase II gene is ubiquitously expressed in human tissues. It is interesting that, although the biological significance remains to be clarified, high levels of enzyme activity were detected in endocrine tissues such as the pituitary, thyroid, and adrenal glands, probably due to high expression of the DNase II gene.…”

“…The 3Ј-untranslated region of DNase II cDNA had a longer span than that of DNase I cDNA. In humans, this is about 150 bp (16), in rabbits, 147 bp (23), and in the mouse, 163 bp (30). It has been reported (35) that a long 3Ј-untranslated region is a common feature of lysosomal protease mRNAs.…”

“…One unit of DNase II activity was defined as an increase of 1.0 in the absorbance at 260 nm. Levels of DNase II activity in each tissue sample were determined as follows (23). The human tissues were cut into small pieces, washed with cold saline to remove excess blood, homogenized using an Ultra-turrax (IKA-WERK, Staufen, Germany) in 1-2 ml 50 mM Tris (pH 7.5) containing 1.0 mM phenylmethylsulfonyl fluoride (Sigma), then centrifuged at 15,000 ϫ g for 20 min.…”

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

“…. The enzymological and chemical properties of the enzymes, the inhibitory effects of specific antibodies on their activities and their tissue distributions were examined as described previously [4,12,13]. Proteins were determined with a protein assay kit (BioRad, Richmond, CA, U.S.A.) with BSA as a standard.…”

“…Because the additional amino acid residues in amphibian DNases I might be involved in their catalytic activities, this shift to a higher optimal pH might reflect conformational alterations around the active site compared with the other vertebrate enzymes, which have optimal pH values of 6.5-7.0 [10][11][12]24]. Although G-actin is known to be a potent inhibitor of bovine [24], human [10], mouse [13] and rabbit [12] DNase I, the activities of the amphibian enzymes were completely unaffected by G-actin. In addition, Tyr'&, Val'( and Ala""%, which have been assumed to be responsible for actin binding in human DNase I [25], were rarely conserved in the amphibian enzymes.…”