Osamu Hosomi

MOUSE DEOXYRIBONUCLEASE I (DNASE I) : BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION, CDNA STRUCTURE AND TISSUE DISTRIBUTION

Mouse urinary deoxyribonuclease I (DNase I) resembles rat and human DNase Is in terms of its proteochemical and enzymological properties. Furthermore, mouse DNase I was demonstrated to be immunologically closer to the rat than to the human enzyme. A 1176 bp full length cDNA encoding mouse DNase I was constructed from RNA obtained from the kidney and parotid glands. The amino acid sequence up to the 45th residue from the N‐terminal of the mature enzyme was identical to that deduced from the cDNA sequence. This DNase I was distributed most densely in the parotid glands from the standpoint of both enzyme activity and gene transcript levels.

Identification of the three non‐identical subunits constituting human deoxyribonuclease II

We purified DNase II from human liver to apparent homogeneity. The N‐terminal amino acid sequences of each of three components constituting the purified mature enzyme were then separately determined by automatic Edman degradation. A combination of this chemical information and the previously reported nucleotide sequence of the cDNA encoding human DNase II [Yasuda et al. (1998) J. Biol. Chem. 273, 2610–2626] allowed detailed elucidation of the enzymes subunit structure: human DNase II was composed of three non‐identical subunits, a propeptide, proprotein and mature protein, following a signal peptide. Expression analysis of a series of deletion mutants derived from the cDNA of DNase II in COS‐7 cells suggested that although a single large precursor protein may not be necessary for proteolytic maturation, the propeptide region L17–Q46 may play an essential role in generating the active form of the enzyme.

Two novel screening methods for selecting monoclonal antibodies which specifically inhibit DNase I enzyme activity

Two novel screening methods, single radial enzyme diffusion and the DNA-cast polyacrylamide gel electrophoresis, for selecting monoclonal antibodies which detect human deoxyribonuclease I (DNase I) enzyme activity are described. The former was adopted for initial screening to select potential objective antibodies from numerous hybridoma culture supernatants, because it was easy to perform and a powerful mass-screening tool. The latter was utilized for the subsequent precise selection of the antibodies in the supernatants selected after preliminary screening by the former, because it was clearly more accurate and sensitive, although the procedure was slightly more complicated. The consecutive use of these two methods resulted in the isolation of 25 anti-human DNase I antibodies, all of which specifically inhibited the activity of human DNase I.

Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I.

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.

Isolation and Characterization of Anti-H Antibody from Egg Yolk of Immunized Hens

An anti-H antibody was demonstrated to be produced in egg yolk as well as in serum of hens which were immunized with human type O red blood cells. The antibody in egg yolk was isolated with polyethylene glycol and ethanol and was purified by affinity chromatography using immunoadsorbant beads immobilized with H type 2 hapten (Fuca1----2Gal beta 1----4GlcNAc beta). Hemagglutination reaction of the antibody from egg yolk was inhibited by human saliva samples from secretor types irrespective of their ABO blood types, and by immunoadsorbant beads which contain Fuca1----2Gal beta structures.

Structure and organization of the human deoxyribonuclease II (DNase II) gene

The structure of the human gene for deoxyribonuclease II (DNase II; EC 3.1.22.1) was determined using several specific primers based on the human DNase II cDNA sequence [Yasuda et al. (1998). J. Biol. Chem.273, 2610–2616] in a polymerase chain reaction‐based strategy. The gene spanned about 6 kb and consisted of 6 exons. No canonical TATA or CAAT boxes could be identified within the 1341 nucleotides upstream of the putative transcription start site, although the 5′‐flanking region contained a CpG island and several putative binding motifs for transcription factors Sp1 and ETF. These properties indicate that the DNase II gene is a housekeeping gene and this is compatible with its ubiquitous expression in human tissues. Three different cleavage/polyadenylation sites were identified in the 3′‐flanking region, leading to the production of multiple DNase II mRNA species. However, a comparison of the entire translated sequences of the gene from a pair of subjects with homozygous DNase II phenotypes H and L revealed no differences in the nucleotide sequences.

Xenopus laevis Pancreatic DNase I: Purification and Immunological Characterization

Deoxyribonuclease I (DNase I) was purified from Xenopus laevis pancreas to apparent electrophoretic homogeneity using a series of column chromatographies. The purified enzyme showed a molecular mass of about 36 kDa and maximum activity at pH 7.0–8.0, required divalent cations, Ca2+ and Mg2+, for its activity, and was inhibited by EDTA, EGTA and an antibody specific to the enzyme, but not by G-actin. The N-terminal amino acid sequence of the enzyme up to the 37th residue shared 38–44% homology with that of mammalian DNases I derived from bovine, ovine, porcine, rat, mouse, rabbit and human. A systematic survey of DNase I activity distribution in 20 different kinds of frog tissues showed that the pancreas and rectum produced higher amounts than other tissues. This is the first report concerning the purification and chemical and immunological characterization of frog pancreatic DNase I.

HISTO-BLOOD GROUP LEWIS GENOTYPING FROM HUMAN HAIRS AND BLOOD

SummaryThe expression of histo-blood group Lewis antigens is determined by the Lewis-type α1→3/4fucosyltransferase (Le enzyme) encoded by Fuc-TIII gene (Le gene). The genotyping of Le genes by the PCR-RFLP methods established recently and partly modified in this study was found to be useful not only for determining the genuine Lewis blood types of samples such as human hairs and blood stains but also for distinguishing non-genuine Lewis-negative phenotypes frequently observed in pregnant women from genuine ones. The availability of the present PCR-RFLP methods for the paternity tests was also discussed.

Molecular Cloning of cDNA Encoding Xenopus laevis Deoxyribonuclease I

A 1200-bp cDNA encoding Xenopus laevis deoxyribonuclease I (X. laevis DNase I) was constructed from the total RNA of a X. laevis pancreas using a rapid amplification of cDNA ends method. When the cDNA was transiently transfected into COS-7 cells, the recom-binant polypeptide exhibited similar enzymological properties to those of the native pancreatic DNase I. The recombinant enzyme was considerably more labile than most other vertebrate DNase I enzymes. The X. laevis DNase I polypeptide was larger than any other known vertebrate DNase I, containing a unique Cys-rich stretch of 68 or 70 amino acid residues at the carboxyl terminus, and it had less well conserved binding sites for the Ca2+, G-actin and DNA, and two DNase I signature motifs. These alterations might account for its heat instability.

Polymerization of a vitelline envelope (VE)-like structure produced by using fractionated VE extracts from carp eggs.

Fractionation of vitelline envelope (VE) extracts from carp eggs made possible the efficient polymerization of a VE-like structure. The structure corresponded to the fourth layer of the VE or fertilization envelope (FE), and its organization was achieved by reassembly in vitro after solubilization of the sheets composed of filamentous substances or network-like aggregates which were induced by a cortical alveolus sialoglycoprotein or thrombin. The sialoglycoprotein was a serine proteinase and immunolocalized only in the structure at the periphery of cortical alveoli, not in the VE and yolk granules. Ultrastructural features of the VE-like structure suggested that reassembly in vitro occurred via several intermediates in the process of polymerization. A polyclonal antibody produced against one of the assembled VE components, a 64 kDa protein, more intensely immunostained the outer periphery of the VEs than other areas, and immunoelectron microscopy showed that immunogold particles specifically labeled reassembled VE-like structures and major skeletons of the networks or network-like sheets. The protein with a molecular weight of 64 kDa was found to be a DNase. Thus, these results suggest a new approach to investigating not only the FE assembly process in vitro but also the organizing relationship between the major skeleton of the VE or FE and other additional constituents.