Generation of Superoxide and In Vitro Inactivation of Viruses by Aldopentoses

Ren-1 renin is synthesized in the kidney of every mouse. Ren-2 renin has been observed in the submandibular gland (SMG) of male mice carrying two renin genes. However, it is not known if Ren-2 renin is in the kidney and blood of the two-renin gene mice. In this study, a direct ELISA for Ren-2 renin (SMG renin) was established by a sandwich method. This ELISA could measure the Ren-2 active renin in the range from 1 to 100ng and distinguish Ren-2 active renin from not only Ren-1 renin but also Ren-2 prorenin. By a combination of this assay system and conventional methods, the pro-form as well as the active form of Ren-2 renin was found in the kidney and plasma of male AKR mice carrying two-renin genes.     

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.     

DNA insertions distinguish the duplicated renin genes of DBA/2 andM. hortulanus mice

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci,Ren-1 andRen-2, a variantNot I hybridization pattern was observed in the wild mouseM. hortulanus. To determine the basis for this variation, the structure of theM. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in bothMus species. In particular, the sequence at the recombination site between the linkedRen-1 andRen-2 loci was found to be identical in both DBA/2 andM. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences inM. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes in thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 andM. hortulanus mice exhibit different patterns of developmentally regulated renin expression.     

Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family

Mouse submandibular gland prorenin-converting enzyme (PRECE) consists of the two polypeptide chains of 17 and 10 kDa and cleaves mouse Ren-2 prorenin at a dibasic site to yield mature renin. Western blot analysis using an antiserum against this enzyme gave rise to multiple bands in mouse submandibular glands, suggesting that PRECE is a member of a protease family. Partial amino acid sequence analysis of purified PRECE and cloning and sequence analyses of its cDNA indicated that it is identical to the mGK-13 gene product, epidermal growth factor-binding protein type B, which is a member of the glandular kallikrein family and is involved in maturation of epidermal growth factor. Conditioned medium from Chinese hamster ovary cells transfected with an expression plasmid for PRECE had prorenin converting activity. These results indicate that PRECE is involved in the maturation of two bioactive polypeptides expressed in mouse submandibular glands, Ren-2 renin and epidermal growth factor.     

DNA insertions distinguish the duplicated renin genes of DBA/2 and M. hortulanus mice.

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci, Ren-1 and Ren-2, a variant Not I hybridization pattern was observed in the wild mouse M. hortulanus. To determine the basis for this variation, the structure of the M. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in both Mus species. In particular, the sequence at the recombination site between the linked Ren-1 and Ren-2 loci was found to be identical in both DBA/2 and M. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences in M. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes is thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 and M. hortulanus mice exhibit different patterns of developmentally regulated renin expression.     

Close physical linkage of the murine Ren-1 and Ren-2 loci

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.     

Renin enhancer is critical for control of renin gene expression and cardiovascular function

The important cardiovascular regulator renin contains a strong in vitro enhancer 2.7 kb upstream of its gene. Here we tested the in vivo role of the mouse Ren-1c enhancer. In renin-expressing As4.1 cells stably transfected with Ren-1c promoter with or without enhancer, expression of linked beta-geo reporter, stable expression, and colony formation were dependent on the presence of the enhancer. We then generated mice carrying a targeted deletion of the enhancer (REKO mice) and found marked depletion of renin in renal juxtaglomerular and submandibular ductal cells, as well as hyperplasia of macula densa cells. Plasma creatinine was increased, but electrolytes were normal. Male REKO mice implanted with telemetry devices had 9 +/- 1 mm Hg lower mean arterial pressure (p < 0.001), which was partly normalized by a high NaCl diet. Locomotor activity was lower, and baroreflex sensitivity was normal. Markedly reduced mean arterial pressure variability in the midfrequency band indicated a contribution of reduced sympathetic vasomotor tone to the hypotension. In conclusion, the renin enhancer is critical for renin gene expression and physiological sequelae, including response to alteration in salt intake. The REKO mouse may be useful as a low renin expression model.     

Comparison of genetic and endocrine control of renin and kallikrein in mouse submandibular gland

The effects of strain, sex, hypophysectomy and hormone treatment on mouse submandibular gland renin, kallikrein, S2266 hydrolase, and BAEe esterase activities have been examined. Renin activity is determined by the $\text{Rnr}$ locus on mouse Chromosome 1. Female SWR/J mice ($\text{Rnr}$^$\text{s}$/$\text{Rnr}$^$\text{s}$) have 1000-fold higher submandibular gland renin activity than C57BL/6J mice ($\text{Rnr}$^$\text{b}$/$\text{Rnr}$^$\text{b}$). Both strains have similar kallikrein activity. Renin, BAEe esterase, and S2266 hydrolase are substantially higher in male mice compared to females of the same strain whereas kallikrein is not. Dihydrotestosterone and/or thyroxine treatment induces renin, BAEe esterase, and S2266 hydrolase in female mice with little effect on kallikrein. All four enzyme activities are profoundly reduced by hypophysectomy. Dihydrotestosterone and thyroxine are both required to restore renin, BAEe esterase, and S2266 hydrolase to induced levels. Dihydrotestosterone and.or thyroxine restores kallikrein to control levels. We conclude that renin and kallikrein in the mouse submandibular gland are under different genetic and endocrine control. In addition, the synthetic substrate S2266 is not a specific substrate for kallikrein activity in mouse submandibular gland cytosol.     

The crystal structure of the mouse glandular kallikrein-13 (prorenin converting enzyme).

A crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK-13) has been determined at 2.6-A resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin-converting enzyme and cleaves submandibular gland Ren-2 prorenin to yield active renin. The mGK-13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and an active site located in the cleft between two beta-barrel domains. The mGK-13 structure reveals for the first time an ordered kallikrein loop conformation containing a short 3(10) helix. This loop is disordered in the related porcine pancreatic kallikrein and rat submandibular tonin structures. The kallikrein loop is in close spatial proximity to the active site and is also involved in a dimeric arrangement of mGK-13. The catalytic specificity of mGK-13 for Ren-2 prorenin was studied by modeling a prorenin-derived peptide into the active site of mGK-13. This model emphasizes two electronegative substrate specificity pockets on the mGK-13 surface, which could accommodate the dibasic P2 and P1 residues at the site of prorenin cleavage by mGK-13.     

Physical Characterization of Genetic Rearrangements at the Mouse Renin Loci

Many inbred strains of mice have a single locus encoding renin, Ren-1, whereas other inbred strains have two tandemly linked loci, Ren-1 and Ren-2. Each of these renin genes in inbred mice exhibits a unique pattern of tissue-specific expression. As a prerequisite to understanding the structural basis for the expression differences, we have physically characterized the sequence organization of this chromosomal region in both types of strains. Pulsed field gel electrophoresis was initially used to compare the long-range structure of this region in C57BL/6 (Ren-1) and DBA/2 (Ren-1 + Ren-2) mice. The structure in both inbred strains is extremely similar, except for an additional 30 kb containing Ren-2 in DBA/2 mice. The boundaries of the extra 30-kb segment were sequenced and compared to homologous sequences flanking the Ren-1 alleles. This analysis identified the precise recombination site, and also the presence of a large insertion, between the renin loci in DBA/2. The renin gene duplication apparently resulted from recombination between sequences sharing little homology, suggesting that nonhomologous chromosomal breakage and rejoining may have been involved mechanistically in the event.     

The angiotensinogen gene of Swiss mice is closely linked to a retrovirus-like element

Angiotensinogen is cleaved by renin and angiotensin-converting enzyme to liberate the potent vasocontrictor peptide angiotensin II. We have recently identified a cis-acting genetic lesion associated with high levels of angiotensinogen mRNA in the testis and salivary gland of Swiss mice. To determine the molecular basis of this mutation, the Swiss angiotensinogen gene was cloned, and its structure was compared to that from a low-expressing strain (BALB/c). I show that a retrovirus-like element belonging to the intracisternal A-particle gene family has been inserted 9 kb upstream from the cap site of the Swiss angiotensinogen gene. This intracisternal A-particle, named IAP-Agt, segregated concordantly with angiotensinogen expression phenotypes in CXB recombinant inbred mice. However, genomic Southern analysis showed that IAP-Agt was present in some, but not all, inbred laboratory mouse strains displaying high levels of angiotensinogen gene expression. On the basis of this evolutionary evidence, it is unlikely that IAP-Agt is the cause of the angiotensinogen mutation. It is intriguing that Ren-2, the duplicated mouse renin gene, is expressed to high levels in the male salivary gland and also contains a transposed intracisternal A-particle genome.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Kidney and submaxillary gland renins are encoded by two non-allelic genes in Swiss mice.

Two distinct phenotypic groups of inbred strains of mice, with different amounts of submaxillary gland (SMG) renin have been described. We have previously shown that strains with high levels of SMG renin, such as Swiss or AKR mice, have two renin genes, Rn1 and Rn2, per haploid genome, while strains with low levels of SMG, such as BALB/c or C57Bl/6, have only one renin gene. We now report the molecular cloning of cDNA copies of Swiss mouse kidney renin mRNA and present nucleotide sequence data of the recombinant clones. Comparison of these sequences with the sequence of Swiss mouse SMG renin mRNA we have previously reported, demonstrates that Swiss mice express the two non-allelic genes, Rn1 and Rn2.