Arg(1098) is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity.

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn(2+) metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl(-), but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl(-) dependence. Cl(-) dependence is also lost when the equivalent Arg in the N-domain, Arg(500), is substituted with Gln. The Arg(1098) to Lys substitution reduced Cl(-) binding affinity by approximately 100-fold. In the absence of Cl(-), substrate binding affinity (1/K(m)) of and catalytic efficiency (k(cat)/K(m)) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg(1098) to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl(-)]. The Arg(1098) to Gln substitution also eliminates Cl(-) dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl(-). These findings indicate that: 1) Arg(1098) is a critical residue of the C-domain Cl(-)-binding site and 2) a basic side chain is necessary for Cl(-) dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl(-)-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl(-) are much more important for the hydrolysis of short substrates than for Ang I. Since Cl(-) concentrations are saturating under physiological conditions and Arg(1098) is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl(-)-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.

Angiotensin I-converting enzyme transition state stabilization by HIS1089: evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases.

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His(1089) as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu. At pH 7.5, with Ang I as substrate, k(cat)/K(m) values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k(cat)) with minor effects on ground state substrate binding (K(m)). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His(1089) side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene.

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two-dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.

X-linked myotubular myopathy: refinement of the gene to a 280-kb region with new and highly informative microsatellite markers.

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the off-spring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.

X-linked progressive mixed deafness: a new microdeletion that involves a more proximal region in Xq21.

We report a large two-generation pedigree with seven affected males segregating for an X-linked mixed conductive sensorineural deafness. The patients present with atypical Mondini-like dysplasia, dilated petrous facial canal, dilatation of the internal auditory meatus fully connected with enlarged cochlear canals, and, in one patient, a wide bulbous posterior labyrinth. Obligatory carrier females are mildly affected. Molecular characterization of this family revealed a deletion of locus DXS169, in Xq21.1. Loci DXS72 and DXS26, which, respectively, flank DXS169 proximally and distally, were intact. Since a gene responsible for X-linked progressive mixed deafness with perilymphatic gusher (DFN3) has previously been assigned by deletion mapping to a slightly more distal interval between DXS26 and DXS121, this study indicates either two different deafness genes or the involvement of a very large region in Xq21.

Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies.

Because of the growing importance of the Retinoid X Receptors (RXR alpha, beta and gamma) in the retinoid acid signalling pathway, we have prepared polyclonal and monoclonal antibodies directed against these proteins. For this purpose, either the whole mouse RXR alpha protein expressed in E.Coli, or synthetic peptides corresponding to amino acid sequences common to all RXRs or unique to RXR alpha, beta or gamma, were used as antigens. Antibodies recognizing either all three RXR types (alpha, beta and gamma) or specific for each RXR type were obtained. The antibodies were characterized by immunocytochemistry, immunoblotting, immunoprecipitation and electromobility shift assay (EMSA). These antibodies allowed us to detect the presence of RXR alpha proteins in mouse embryos and in mouse embryonal carcinoma cells (F9 and P19 cell lines) by immunoblotting, immunoprecipitation and EMSA whereas RXR beta could be detected only by EMSA and RXR gamma could not be detected by any of these techniques.

Construction of a high-resolution linkage map for Xp22.1-p22.2 and refinement of the genetic localization of the Coffin-Lowry syndrome gene.

The genes responsible for two X-linked diseases, the Coffin-Lowry syndrome (CLS) and juvenile retinoschisis (RS), have been previously mapped, through linkage studies, to an 8-cM region, in Xp22.1-p22.2, flanked distally by two tightly linked markers, DXS207 and DXS43, and proximally by DXS274. In the present study, five Genethon markers have been assigned to the (DXS207, DXS43)-DXS274 interval using somatic cell hybrids and a meiotic breakpoint panel and ordered together with three markers previously mapped to this region. A genetic map, which includes 13 loci and spans a distance of approximately 13 cM, was derived from linkage analysis using the CEPH families. The most likely locus order and map distances (in centimorgans) are Xpter-DXS16-(3.4)-(DXS207, DXS43, DXS1053)-(2.0)-(DXS999, DXS257)-(1.7)-AFM291 wf5-(1.4) - DXS443 - (2.0) - (DXS1229, DXS365) - (2.1) - (DXS1052, DXS274, DXS41)-Xcen. Analysis of multiply informative crossovers established AFM291 wf5 and DXS1052 as new flanking markers for CLS, which significantly reduces the candidate region for this disease gene to a 4- to 5-cM interval. Three markers, DXS443, DXS1229, and DXS365, mapping within this interval showed complete cosegregation with the disease phenotype, giving a multipoint lod score of 14.2. The present map provides the framework for constructing a YAC contig for the CLS and RS region and should be useful for refining the localization of other disease genes mapping to this region. The panel of somatic cell hybrids characterized for the present study has also allowed us to refine the localization of five genes (CALB3, GRPR, PDHA1, GLRA2, and PHKA2) and two expressed sequence tags (DXS1118E and DXS1006E) previously assigned to the Xp22 region.

Non-specific X-linked mental retardation: linkage analysis in MRX2 and MRX4 families revisited.

We have previously reported linkage analysis in 3 families with non-specific X-linked mental retardation (XLMR). This used RFLPs and was limited by the relatively low informativeness and density of markers available. We have performed a new linkage analysis using microsatellites (including new Genethon markers) in the two most informative families. In the MRX2 family, a lod score of 2.61 at theta = 0.05 had previously been obtained with DXS85 in Xp22.2. We now report a tighter linkage with AFM 135xe7 (DXS989, z = 4.62 at theta = 0.00) and established the order DXS85-DXS207-DXS999 (AFM234 y12)-MRX2, DXS365, DXS1052 (AFM 163yh2), DXS989-DXS1065 (AFM224zf2), DMD 3'. The localization of MRX2 in Xp22.2-p22.1 is thus clearly different from the more distal MRX gene defined by patients with contiguous gene syndromes. In the MRX4 family, a maximum lod score of 2.53 at theta = 0.00 had been obtained with DXS159 in Xq13. Our present study did not show recombination from ALAS2 in Xp11.21 to DXS441 in Xq13.3 (z = 3.38 at theta = 0.00 for the latter marker) and the closest flanking markers are DXS255 in Xp11.22 and DXYS1 in Xq21.3. Reduced recombination around the centromere prevents precise mapping. The localisation of MRX4 overlaps with that of several other MRX families.