Lysozyme and bilirubin bind to ACE and regulate its conformation and shedding.

Angiotensin I-converting enzyme (ACE) hydrolyzes numerous peptides and is a critical participant in blood pressure regulation and vascular remodeling. Elevated tissue ACE levels are associated with increased risk for cardiovascular and respiratory disorders. Blood ACE concentrations are determined by proteolytic cleavage of ACE from the endothelial cell surface, a process that remains incompletely understood. In this study, we identified a novel ACE gene mutation (Arg532Trp substitution in the N domain of somatic ACE) that increases blood ACE activity 7-fold and interrogated the mechanism by which this mutation significantly increases blood ACE levels. We hypothesized that this ACE mutation disrupts the binding site for blood components which may stabilize ACE conformation and diminish ACE shedding. We identified the ACE-binding protein in the blood as lysozyme and also a Low Molecular Weight (LMW) ACE effector, bilirubin, which act in concert to regulate ACE conformation and thereby influence ACE shedding. These results provide mechanistic insight into the elevated blood level of ACE observed in patients on ACE inhibitor therapy and elevated blood lysozyme and ACE levels in sarcoidosis patients.

A differential gene expression study: Ptpn6 (SHP-1)-insufficiency leads to neutrophilic dermatosis-like disease (NDLD) in mice.

BACKGROUND: Irradiated syngeneic wild-type mice developed the same neutrophilic dermatosis-like disease (NDLD) after adoptive transfer of bone marrow cells from Ptpn6(meb2/meb2) mutant mice. OBJECTIVE: To analyze differentially expressed genes in the bone marrow of mice with NDLD to gain insight into the role of Ptpn6 in myelopoietic bone marrow pathology, and the mechanisms by which Ptpn6 insufficiency in the hematopoietic cells can lead to the development of skin lesions. METHODS: As Ptpn6 is involved in a myriad of signaling pathways, we used a global approach with microarray technology for the first time to characterize changes in the bone marrow and skin of motheaten-type mice. RESULTS: A total number of 1,511 probe sets in the bone marrow showed at least two-fold changes with FDR <0.05, of which 256 probe sets had over four-fold changes. A group of 63 genes in the bone marrow of NDLD mice had more than a 4-fold change with FDR <0.0001. From 503 genes encoding proteins with ITIM motif that binds to Ptpn6, 109 were up-regulated and 83 were down-regulated. We found that genes encoding hematopoietic receptors, neutrophil chemoattractants, Toll-like receptors (Tlr1, Tlr2 and Tlr4) and C-type lectin innate immunity receptors (Clec4e, Clec4d, Clec4n, Clec4a2 and Clec4a3) were significantly up-regulated in both NDLD bone marrow and skin. The Il1b gene was also significantly overexpressed in skin samples, confirming the importance of the IL-1/TLR pathway in the development of early skin inflammation in NDLD mice. CONCLUSION: Our results suggest that innate immunity genes play a major role in development of neutrophilic dermatosis-like disease in mice.

A novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP.

BACKGROUND: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. CONCLUSIONS AND SIGNIFICANCE: A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.

A novel splice-site mutation in angiotensin I-converting enzyme (ACE) gene, c.3691+1G>A (IVS25+1G>A), causes a dramatic increase in circulating ACE through deletion of the transmembrane anchor.

BACKGROUND: Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. METHODS AND RESULTS: Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. CONCLUSIONS: We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences.

An angiotensin I-converting enzyme mutation (Y465D) causes a dramatic increase in blood ACE via accelerated ACE shedding.

BACKGROUND: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. METHODOLOGY/PRINCIPAL FINDINGS: HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE--between Arg1203 and Ser1204--was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. CONCLUSIONS/SIGNIFICANCE: The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).

Alteration in the gene encoding protein tyrosine phosphatase nonreceptor type 6 (PTPN6/SHP1) may contribute to neutrophilic dermatoses.

We have found a B2 repeat insertion in the gene encoding protein tyrosine phosphatase nonreceptor type 6 (PTPN6) in a mouse that developed a skin disorder with clinical and histopathological features resembling those seen in human neutrophilic dermatoses. Neutrophilic dermatoses are a group of complex heterogeneous autoinflammatory diseases that all demonstrate excessive neutrophil infiltration of the skin. Therefore, we tested the cDNA and genomic DNA sequences of PTPN6 from patients with Sweet's syndrome (SW) and pyoderma gangrenosum and found numerous novel splice variants in different combinations. Isoforms resulting from deletions of exons 2, 5, 11, and 15 and retention of intron 1 or 5 were the most common in a patients with a familial case of SW, who had a neonatal onset of an inflammatory disorder with skin lesions and a biopsy specimen consistent with SW. These isoforms were associated with a heterozygous E441G mutation and a heterozygous 1.7-kbp deletion in the promoter region of the PTPN6 gene. Although full-length PTPN6 was detected in all other patients with either pyoderma gangrenosum or SW, it was always associated with splice variants: a partial deletion of exon 4 with the complete deletion of exon 5, alterations that were not detected in healthy controls. The defect in transcriptional regulation of the hematopoietic PTPN6 appears to be involved in the pathogenesis of certain subsets of the heterogeneous group of neutrophilic dermatoses.

Spontaneous insertion of a b2 element in the ptpn6 gene drives a systemic autoinflammatory disease in mice resembling neutrophilic dermatosis in humans.

We found a spontaneous autosomal mutation in a mouse leading to neutrophil infiltration with ulceration in the upper dermis of homozygous offspring. These animals had increased neutrophil numbers, associated with normal lymphocyte count, in peripheral blood and bone marrow, suggesting a myeloproliferative disorder; however, granulocyte precursor proliferation in bone marrow was actually reduced (because circulating neutrophils were less susceptible to apoptosis). Neutrophil infiltration of the skin and other organs and high serum levels of immunoglobulins and autoantibodies, cytokines, and acute-phase proteins were additional abnormalities, all of which could be reduced by high-dose corticosteroid treatment or neutrophil depletion by antibodies. Use of genome-wide screening localized the mutation within an 0.4-Mbp region on mouse chromosome 6. We identified insertion of a B2 element in exon 6 of the Ptpn6 gene (protein tyrosine phosphatase, non-receptor type 6; also known as Shp-1). This insertion involves amino acid substitutions that significantly reduced the enzyme activity in mice homozygous for the mutation. Disease onset was delayed, and the clinical phenotype was milder than the phenotypes of other Ptpn6-mutants described in motheaten (me, mev) mice; we designated this new genotype as Ptpn6(meB2/meB2) and the phenotype as meB2. This new phenotype encompasses an autoinflammatory disease showing similarities to many aspects of the so-called neutrophilic dermatoses, a heterogeneous group of skin diseases with unknown etiology in humans.

Cloning and characterization of a single-chain fragment of monoclonal antibody to ACE suitable for lung endothelial targeting.

Monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) demonstrates selective accumulation in lung tissue of the rat, hamster, cat, monkey and human after systemic injection. It has also been demonstrated that mAb 9B9 is the useful tool for targeting therapeutic agents or genes to lung endothelium. In this study, we describe the generation and characterization of a single-chain derivative (scFv) of mAb9B9 (scFv 9B9). In vitro, scFv9B9 retains the ability of the parental antibody to recognize human and rat ACE when expressed both on the surface of phage and as a soluble protein in prokaryotic and eukaryotic expression systems. The ability of scFv 9B9 presented by phage or the soluble protein labeled with I(125) to recognize ACE in the pulmonary circulation was also confirmed in an in vivo rat model. Sequence analysis revealed a putative glycosylation site in close proximity to the complementarity determining region 2 (CDR2) of the scFv 9B9 heavy chain. Mutation of Asn68 to Gln in the heavy chain of scFv 9B9 eliminated the glycosylation site and significantly improved the binding affinity of scFv 9B9 to human ACE as determined by cell ELISA and Western Blot. Moreover, Asn68Gln scFv 9B9 showed a greater rate of secretion at 30°C than wild type scFv 9B9, but had a decreased thermal stability at 37°C. The development of a stable and functional single-chain format of mAb 9B9 which specifically recognizes human and rat ACE represents a novel antibody-based reagent suitable for targeted delivery of drugs/genes to the pulmonary circulation.

Angiotensin I-converting enzyme Gln1069Arg mutation impairs trafficking to the cell surface resulting in selective denaturation of the C-domain.

BACKGROUND: Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death. METHODOLOGY/PRINCIPAL FINDINGS: Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10-20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold. CONCLUSIONS/SIGNIFICANCE: Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.

Angiotensin I-converting enzyme mutation (Trp1197Stop) causes a dramatic increase in blood ACE.

BACKGROUND: Angiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots. METHODOLOGY/PRINCIPAL FINDINGS: We have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension. CONCLUSIONS/SIGNIFICANCE: We have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease.

Two major interacting chromosome loci control disease susceptibility in murine model of spondyloarthropathy.

Autoimmune spondylitis was induced in BALB/c mice and their MHC-matched (BALB/c x DBA/2)F1 and F2 hybrids by systemic immunization with cartilage/intervertebral disk proteoglycan (PG). As in human ankylosing spondylitis, the MHC was the major permissive genetic locus in murine PG-induced spondylitis (PGIS). Two major non-MHC chromosome loci with highly significant linkage were found on chromosomes 2 (Pgis2) and 18 (Pgis1) accounting for 40% of the entire F2 trait variance. The dominant spondylitis-susceptibility allele for Pgis2 locus is derived from the BALB/c strain, whereas the Pgis1 recessive allele was present in the disease-resistant DBA/2 strain. The Pgis1 locus significantly affected the disease-controlling Pgis2 locus, inducing as high incidence of spondylitis in F2 hybrids as was found in the spondylitis-susceptible parent BALB/c strain. Additional disease-controlling loci with suggestive linkage were mapped to the chromosomes 12, 15, and 19. Severity of spondylitis in F2 mice positively correlated with serum levels of amyloid A, IL-6, and Pg-specific Abs, and showed negative correlation with Ag-induced T cell proliferation, IFN-gamma, IL-4, and TNF-alpha production. A major locus controlling serum IL-6 was found on chromosome 14 near osteoclast differentiation factor Tnfsf11. Locus on chromosome 11 near the Stat3 and Stat5 genes controlled serum level of the Ig IgG2a isotype. The two major genetic loci Pgis1 and Pgis2 of murine spondylitis were homologous to chromosome regions in human genome, which control ankylosing spondylitis in human patients. Thus, this animal model of experimentally induced spondylitis might facilitate the identification of spondylitis-susceptibility genes in humans.

Induction of arthritis in SCID mice by T cells specific for the "shared epitope" sequence in the G3 domain of human cartilage proteoglycan.

OBJECTIVE: To study the immunologic function and determine the fine epitope structure of a synthetic peptide p135H ((2373)TTYKRRLQKRSSRHP) of the G3 domain of human cartilage proteoglycan (aggrecan), which contains a highly homologous sequence motif of the shared epitope (QKRAA), the most common sequence motif in HLA-DR4 alleles, which predispose humans to the development of rheumatoid arthritis (RA). METHODS: Synthetic p135 peptides with altered sequences were used for (hyper)immunization of arthritis-susceptible BALB/c mice and then challenged with a single dose of cartilage proteoglycan. Human p135 (p135H) and mouse p135 (p135M) synthetic peptides of the G3 domain of aggrecan were used to prime lymphocytes, which were then used for adoptive transfer of arthritis into "presensitized" SCID mice, determining cross-reactivity among p135 peptides and their analogous sequences, and generating T cell hybridomas. T cell hybridomas were also used for arthritis transfer into SCID mice and for characterizing the fine epitope structure of T cell receptor (TCR) and major histo-compatibility complex (MHC) binding sites of the immunogenic/arthritogenic p135H sequence. RESULTS: While p135H peptide-(hyper)immunized mice became sensitized, they developed arthritis only after injection of a single dose of cartilage proteoglycan aggrecan. An altered peptide sequence (p135H-AA) carrying the shared epitope motif (QKRAA) was as effective as the natural peptide p135H sequence for inducing arthritis. Mouse p135M-specific lymphocytes induced arthritis with a lower incidence, but synthetic peptides to Escherichia coli heat-shock protein (DnaJ) or HLA-DR4 allele (both having the shared epitope sequence with different flanking regions) were also positive. Fine epitope sequence recognition of an arthritogenic T cell hybridoma derived from p135H-primed lymphocyte population was determined. Interestingly, in the most central position, a basic amino acid triplet of p135H peptide was found to be the MHC-binding motif, whereas the flanking amino acids bound to the TCR. CONCLUSION: Peptide p135H, corresponding to the peptide sequence in the G3 domain of human cartilage proteoglycan aggrecan, is immunogenic/arthritogenic in BALB/c mice. Peptide p135H includes a highly homologous motif of the shared epitope, a sequence that is overrepresented in bacterial heat-shock proteins, envelope protein of human JC polyomavirus, and numerous HLA-DR4 alleles. Since the G3 domain of cartilage proteoglycan aggrecan with the p135 sequence is "lost" during the normal metabolic turnover of cartilage proteoglycan or in pathologic conditions, an antigenoriented T cell migration into joints of presensitized (susceptible) individuals may contribute to the organ-specificity of RA.

Sex effect on clinical and immunologic quantitative trait loci in a murine model of rheumatoid arthritis.

OBJECTIVE: To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F(2) hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome. METHODS: (BALB/c x DBA/2)F(2) hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor alpha, interleukin-1 [IL-1], IL-6, interferon-gamma, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F(2) hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed. RESULTS: Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction. CONCLUSION: Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.