Peptidase D Research Articles

The Clinical Landscape with Biochemical and Immunological Indicators of Prolidase Deficiency

This article examines Prolidase Deficiency (PD), an autosomal recessive disorder marked by diverse clinical symptoms and significant biochemical and immunological abnormalities. Originating from mutations in the peptidase D (PEPD) gene, PD disrupts the metabolism of proline-rich proteins, leading to a range of manifestations. Clinically, PD presents early with growth delays, recurrent infections, and autoimmune disorders, with neurological impacts including developmental delays and intellectual disabilities. Skin issues like chronic ulcers and eczema are common, and respiratory complications add to the disease's complexity. Gastrointestinal features and hematological conditions such as anemia and thrombocytopenia further complicate PD. Immunologically, PD is associated with hypergammaglobulinemia and systemic lupus erythematosus (SLE), highlighting the immune system's involvement. Biochemically, imidopeptiduria serves as a critical diagnostic marker. Diagnostics range from high-performance liquid chromatography to MALDI-TOF MS, while treatment strategies are diverse, reflecting the challenge of managing PD. This study emphasizes the need for a comprehensive approach to understand and manage the multifaceted nature of PD.

A Case report of rare disease Prolidase deficiency in a 15-year-old Pakistan boy

Case presentation Prolidase enzyme plays a crucial role in proline-rich proteins metabolism and physiological processes such as inflammation, cell proliferation, wound healing, angiogenesis, and carcinogenesis. Due to mutations in the peptidase D (PEPD) gene, the catalytic activity of prolidase loss results in prolidase deficiency. Deficiency of prolidase enzyme is an autosomal inborn metabolic rare genetic disorder that has neither any proper treatment nor consensus for treatment. With approximately 100 cases recorded worldwide, the submitted manuscript describes the 2nd recorded case of prolidase deficiency, an extremely uncommon autosomal recessive disorder associated with collagen metabolism, in a 15-year-old Pakistan boy. The disorder typically becomes apparent during infancy. Affected individuals may have enlargement of the spleen (splenomegaly); in some cases, both the spleen and liver are enlarged (hepatosplenomegaly). Diarrhea, vomiting, and dehydration may also occur. People with prolidase deficiency often develop skin lesions, especially on their hands, feet, lower legs, and face. The severity of the skin involvement, which usually begins during childhood, may range from a mild rash to severe skin ulcers. The severity of symptoms in prolidase deficiency varies greatly among affected individuals. Here we present the report of a 15-year-old boy who has all the clinical manifestations of deficiency of prolidase. This is the 2nd case in Pakistan's 229,488,994 million population.

Dysregulation of macrophage PEPD in obesity determines adipose tissue fibro-inflammation and insulin resistance.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes.

Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

Tumor suppressor p53, a critical regulator of cell fate, is frequently mutated in cancer. Mutation of p53 abolishes its tumor-suppressing functions or endows oncogenic functions. We recently found that p53 binds via its proline-rich domain to peptidase D (PEPD) and is activated when the binding is disrupted. The proline-rich domain in p53 is rarely mutated. Here, we show that oncogenic p53 mutants closely resemble p53 in PEPD binding but are transformed into tumor suppressors, rather than activated as oncoproteins, when their binding to PEPD is disrupted by PEPD knockdown. Once freed from PEPD, p53 mutants undergo multiple posttranslational modifications, especially lysine 373 acetylation, which cause them to refold and regain tumor suppressor activities that are typically displayed by p53. The reactivated p53 mutants strongly inhibit cancer cell growth in vitro and in vivo. Our study identifies a cellular mechanism for reactivation of the tumor suppressor functions of oncogenic p53 mutants.

Abstract 2487: disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities

Abstract Background & Goals: The p53 tumor suppressor is one of the most important determinants of cell fate and is critical for tumor suppression. It functions by regulating the expression of a plethora of genes via sequence-specific DNA binding and also by transcription-independent, mitochondria-mediated mechanisms. Not surprisingly, the TP53 gene, which encodes p53, is frequently mutated in human cancer. For example, TP53 is mutated in about 75% of HER2-positive breast cancer and about 85% of triple negative breast cancer. The majority of TP53 mutations are missense mutations, changing one amino acid per mutant, most of which occur in its DNA-binding domain. p53 mutants show loss of tumor suppressor functions or gain of oncogenic functions. We recently found that peptidase D (PEPD), also known as prolidase, binds via its C-terminal sequence to the proline-rich domain (PRD) of wild type p53 and that disrupting the binding activates p53. The enzymatic activity of PEPD is not required for this function. Our objective in the present study is to determine whether PEPD also regulates p53 mutants. We focused on some of the most common oncogenic p53 mutants, including R175H, R248Q, R273H and R280K. Results: Our study shows that 1) each p53 mutant binds directly to PEPD and binds to PEPD in cells in a significant quantity, 2) disrupting their binding in cells by PEPD knockdown reactivates each p53 mutant, unleashing both transcription-dependent and -independent tumor suppressing activities, 3) after leaving PEPD, p53 mutants undergo acetylation, mono-ubiquitination and phosphorylation, which drive their refolding and reactivation, 4) Lysine 373 acetylation is essential for their reactivation, and 5) p53 mutants and p53WT show almost no difference in PEPD-binding and response to PEPD KD. Conclusions & Therapeutic Relevance: In conclusion, we have identified a cellular mechanism by which the tumor suppressor functions of p53 mutants are strongly reactivated, which is neither cell-specific nor mutant-specific. Mainly, p53 mutants bind to PEPD in cancer cells, and disrupting the complex causes posttranslational modifications of mutants freed from PEPD, which drive their refolding and unleash their tumor suppressor activities. This discovery advances our understanding of the function and regulation of p53 mutants and breaks new ground for developing novel cancer treatment strategies. Citation Format: Lu Yang. disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2487.

The Role of Kruppel‐like Factor 6 in Prolidase Regulation

Prolidase, also known as peptidase D (PEPD), is a hydrolase that cleaves dipeptides containing C-terminal proline or hydroxyproline. These dipeptides are generated largely during collagen turnover, therefore prolidase catalysis is an essential rate-limiting step during collagen biosynthesis. Consequently, prolidase plays a significant role in collagen metabolism, matrix remodeling, and wound healing. Defective wound healing is one of the hallmarks of Prolidase Deficiency, a rare autosomal recessive disorder characterized by the excretion of prolidase substrates in the urine. Although prolidase is implicated in wound healing, its molecular and cellular regulation remains understudied. Our initial in silico analysis of the PEPD promoter (PEPDpro) identified key regulatory elements upstream of the transcription start site. We identified Kruppel-like factor 6 (KLF6), a zinc-finger transcription factor associated with vascular injury, wound healing, and collagen metabolism. To study the interaction between KLF6 and prolidase, we amplified the promoter from the human genome and inserted it into a luciferase reporter construct. Our preliminary data demonstrate that KLF6 enhances PEPD promoter activity in a dose-dependent manner. Additionally, KLF6 is regulated by transforming growth factor β1 (TGFβ1), and our data illustrate that TGFβ1 drives PEPD promoter activity. These results will generate new knowledge on the molecular regulation of prolidase and aid in developing therapeutic approaches to regulate prolidase expression in various physiological and pathological conditions such as wound healing.

Abstract 336: A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer

Abstract Background & Goals: HER2 is an oncogenic receptor tyrosine kinase (RTK) implicated in several types of human cancer. It is strongly expressed in about 20% of breast cancer (BC), known as HER2-positive BC (HER2-BC), due to gene amplification. HER2 amplification or overexpression is a strong predictor of poor disease prognosis. Several HER2-targeting drugs are available for treating HER2-BC, including monoclonal antibodies trastuzumab (Ttzm) and pertuzumab, T-DM1 (Ttzm coupled to a microtubule inhibitor), and tyrosine kinase inhibitors (TKIs) lapatinib and neratinib. While these agents have greatly improved disease outcomes, primary and acquired drug resistance, including cross-resistance, is common. Overcoming drug resistance remains a major unmet medical need in HER2-BC. Notably, HER2 remains overexpressed in drug-resistant HER2-BC cells. We recently found that recombinant human peptidase D (PEPD), also known as prolidase, strongly downregulates HER2 and EGFR in cancer cells in vitro and in vivo via its binding to HER2 and EGFR. The enzymatic activity of PEPD plays no role in its modulation of HER2 and EGFR, and we subsequently focused on recombinant PEPDG278D, an enzymatically inactive mutant (point mutation at codon 278). Our objective in the present study is to determine whether PEPDG278D is active in preclinical models of HER2-BC resistant to clinically available HER2 inhibitors and if so, its mechanism of action. Results: PEPDG278D was exceedingly effective against HER2-BC cells that are resistant to Ttzm and other HER2 inhibitors. The cells carried activating PIK3CA mutations, low expression of PTEN, and/or cyclin E overexpression. All the cell lines also overexpressed MUC4 which shields HER2 from Ttzm and stabilizes HER2. However, PEPDG278D rapidly bound to and freed HER2 from MUC4, and rapidly disrupted the interaction of HER2 with other RTKs, including MET and IGF1R. PEPDG278D subsequently induced persistent downregulation of HER2 via internalization and lysosomal degradation. PEPDG278D also downregulated EGFR. Thus, PEPDG278D suppresses multiple RTKs either directly or indirectly in HER2-BC cells. In contrast, Ttzm cannot perform any of these functions of PEPDG278D. PEPDG278D was highly active in mouse tumor models of HER2-BC resistant to Ttzm and also enhanced the therapeutic efficacy of paclitaxel. However, adverse effects of PEPDG278D were not detected in the animal studies. Conclusions & Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that HER2 remains a critical target in drug-resistant HER2-BC. The strong therapeutic activity of PEPDG278D in preclinical models of drug-resistant HER2-BC, which carry clinically relevant molecular changes that confer resistance to current HER2 inhibitors provides the scientific premise for potential evaluation of this recombinant human protein in patients with drug-resistant HER2-BC. Citation Format: Lu Yang, yun Li, Arup Bhattacharya, yuesheng Zhang. A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 336.

PEPD is a pivotal regulator of p53 tumor suppressor

p53 tumor suppressor responds to various cellular stresses and regulates cell fate. Here, we show that peptidase D (PEPD) binds and suppresses over half of nuclear and cytoplasmic p53 under normal conditions, independent of its enzymatic activity. Eliminating PEPD causes cell death and tumor regression due to p53 activation. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin and H2O2 each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for the stress response, but this also renders cells dependent on PEPD for survival, as it suppresses p53. This finding provides further understanding of p53 regulation and may have significant implications for the treatment of cancer and other diseases.

Interaction between genes and macronutrient intake on the risk of developing type 2 diabetes: systematic review and findings from European Prospective Investigation into Cancer (EPIC)-InterAct

Background: Gene-diet interactions have been reported to contribute to the development of type 2 diabetes (T2D). However, to our knowledge, few examples have been consistently replicated to date.Objective: We aimed to identify existing evidence for gene-macronutrient interactions and T2D and to examine the reported interactions in a large-scale study.Design: We systematically reviewed studies reporting gene-macronutrient interactions and T2D. We searched the MEDLINE, Human Genome Epidemiology Network, and WHO International Clinical Trials Registry Platform electronic databases to identify studies published up to October 2015. Eligibility criteria included assessment of macronutrient quantity (e.g., total carbohydrate) or indicators of quality (e.g., dietary fiber) by use of self-report or objective biomarkers of intake. Interactions identified in the review were subsequently examined in the EPIC (European Prospective Investigation into Cancer)-InterAct case-cohort study (n = 21,148, with 9403 T2D cases; 8 European countries). Prentice-weighted Cox regression was used to estimate country-specific HRs, 95% CIs, and P-interaction values, which were then pooled by random-effects meta-analysis. A primary model was fitted by using the same covariates as reported in the published studies, and a second model adjusted for additional covariates and estimated the effects of isocaloric macronutrient substitution.Results: Thirteen observational studies met the eligibility criteria (n < 1700 cases). Eight unique interactions were reported to be significant between macronutrients [carbohydrate, fat, saturated fat, dietary fiber, and glycemic load derived from self-report of dietary intake and circulating n–3 (ω-3) polyunsaturated fatty acids] and genetic variants in or near transcription factor 7–like 2 (TCF7L2), gastric inhibitory polypeptide receptor (GIPR), caveolin 2 (CAV2), and peptidase D (PEPD) (P-interaction < 0.05). We found no evidence of interaction when we tried to replicate previously reported interactions. In addition, no interactions were detected in models with additional covariates.Conclusions: Eight gene-macronutrient interactions were identified for the risk of T2D from the literature. These interactions were not replicated in the EPIC-InterAct study, which mirrored the analyses undertaken in the original reports. Our findings highlight the importance of independent replication of reported interactions.

Abstract P6-07-04: Targeting ErbB2 with human PEPD

Abstract ErbB2, also known as Her2 or Neu, belongs to the ErbB family of plasma membrane-bound receptor tyrosine kinases, which also include ErbB1, ErbB3 and ErbB4. ErbB2 is best known for its involvement in human breast cancer. ErbB2 gene amplification occurs in ∼20% of breast cancer, and ErbB2 amplification or overexpression is a strong predictor of poor disease prognosis. ErbB2-targeted therapies, particularly humanized monoclonal antibody trastuzumab (Ttzm) in combination with chemotherapy, have shown considerable clinical efficacy. However, primary and secondary resistance remains a clinical challenge, and Ttzm, produced in mammalian cells, is very expensive. We have found that human prolidase, also known as peptidase D (PEPD) among several other names, binds to ErbB2 with high affinity (Kd = ∼7 nM) and binds as a homodimer (493 amino acids per subunit) to subdomain 3 in the extracellular domain of ErbB2. Each monomer of PEPD binds to one copy of ErbB2. However, PEPD is a weak ErbB1 binder (Kd = ∼5 μM) and does not bind to ErbB3 or ErbB4. PEPD is the first-ever natural ligand of ErbB2, and unlike the other ligands of ErbB receptors, it is devoid of an EGF motif. PEPD has been long known to hydrolyze dipeptides with proline or hydroxylproline at the carboxy terminus, but the dipeptidase activity of PEPD is not involved in ErbB2/ErbB1 modulation. In cells overexpressing ErbB2, where both activated dimers and inactive monomers of ErbB2 exist, as ErbB2 overexpression causes spontaneous dimerization, auto-tyrosine phosphorylation and recruitment and activation of downstream signals, PEPD rapidly binds to ErbB2 homodimers (&amp;lt;∼10 min) and silences the existing ErbB2-SRC signaling, a key oncogenic pathway of ErbB2, by disrupting SRC association with ErbB2. In contrast, PEPD binds to ErbB2 monomers relatively slowly (&amp;gt;∼30 min), but this binding causes ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. PEPD binding to ErbB2 subsequently causes pronounced ErbB2 depletion, resulting from its internalization and degradation. PEPD also strongly inhibits the DNA synthesis, anchorage-independent growth and invasion of cells that overexpress ErbB2, but has no effect on cells without overexpression of ErbB2. In fact, cells become sensitized to inhibition by PEPD upon achieving stable ErbB2 overexpression. Thus, the overall impact of PEPD on ErbB2 is inhibitory, and PEPD targets cells addicted to ErbB2. In ErbB2-overexpressing cells, at equimolar concentrations, PEPD was more effective than Ttzm in driving ErbB2 depletion, but is weaker than Ttzm in stimulating ErbB2 phosphorylation. In mouse tumor models, PEPD administered by intraperitoneal injection (Monday, Wednesday, Friday) at 0.2-2 mg/kg body weight strongly inhibited the growth of ErbB2-overexpressing tumors, but had no impact on tumors without ErbB2 overexpression, and the PEPD-treated mice showed no adverse effects. Given that the findings described above were made using human PEPD generated in bacteria, there is a distinct possibility that recombinant human PEPD may be a low cost alternative to Ttzm. Further investigation of the antitumor activity of PEPD and its modulation of ErbB2 signaling is warranted. Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. Targeting ErbB2 with human PEPD [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-04.

Modulation of the Association between the PEPD Variant and the Risk of Type 2 Diabetes by n-3 Fatty Acids in Chinese Hans

Background/Aims: Type 2 diabetes (T2D) is modulated by the interactions between genetic and dietary factors. This study sought to examine whether the associations of genome-wide association study (GWAS)-identified genetic variants with T2D risk were modulated by n-3 fatty acids in Chinese Hans. Methods: Six hundred and twenty-two T2D patients and 293 healthy controls were recruited. Erythrocyte phospholipid fatty acids were measured by standard methods. Nine GWAS-identified T2D-related single-nucleotide polymorphisms (SNPs) were genotyped. These SNPs were all identified in GWAS of Asian populations with a high minor allele frequency (>0.2). Results: Among the 9 SNPs, only rs3786897 at PEPD (peptidase D) showed a significant interaction with n-3 fatty acids (p_interaction after Bonferroni correction = 0.027). The rs3786897 A allele was associated with a higher risk of T2D [GA+AA vs. GG: odds ratio (OR) = 2.16, 95% confidence interval (CI) 1.32-3.55] when n-3 fatty acids were lower than the population median, but no significant association (GA+AA vs. GG: OR = 0.63, 95% CI 0.35-1.12) was observed when n-3 fatty acids were higher than the median. Conclusions: The association between the PEPD genetic variant and the risk of T2D was modulated by n-3 fatty acids. Higher n-3 fatty acids may abolish the adverse effect of the risk allele at PEPD for T2D.

Abstract 4274: Prolidase directly binds and activates epidermal growth factor receptor and stimulates downstream signaling.

Abstract Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxy terminus. Unexpectedly, we have found that recombinant human PEPD directly binds and activates epidermal growth factor receptor (EGFR), and that EGFR activation by PEPD in turn leads to activation of multiple signaling molecules downstream of EGFR, including but may not be limited to AKT, ERK and STAT3. EGFR is a cell surface receptor that plays a major role in a variety of cellular responses, and EGFR activation is known to result in increase in DNA synthesis, cell growth, cell proliferation and cell migration, which perhaps are most clearly demonstrated in cancer cells. The ability of PEPD to activate EGFR signaling is neither cell-specific nor dependent on its enzymatic activity; in the latter case, an enzymatically inactive PEPD mutant was similarly effective in activating EGFR, AKT, ERK and STAT3. PEPD is also a novel inducer of cyclooxygenase-2 (Cox-2), known to play an important role in cancer, inflammation and other diseases, but Cox-2 induction by PEPD also depends on EGFR activation. In line with the pro-survival and pro-proliferation activities of EGFR and Cox-2, PEPD stimulates cell proliferation and DNA synthesis in cultured cells. However, PEPD could activate EGFR only when it was present in the extracellular space, as forced expression of human PEPD via transient gene transfection failed to activate EGFR and the downstream signaling proteins. This result is nonetheless consistent with EGFR being a cell surface receptor. Although PEPD is normally a cytosolic protein, it is apparently released from injured cells and tissues in vivo, and such release appears to result in EGFR activation and activation of its downstream signals. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor. Indeed, there are several notable differences between PEPD and EGF with regard to binding and activation of EGFR and activation of downstream signaling molecules. In conclusion, PEPD is a new ligand of EGFR and presents a novel mechanism of EGFR activation. Citation Format: Lu Yang. Prolidase directly binds and activates epidermal growth factor receptor and stimulates downstream signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4274. doi:10.1158/1538-7445.AM2013-4274

Prolidase Directly Binds and Activates Epidermal Growth Factor Receptor and Stimulates Downstream Signaling

Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation.

Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

BackgroundAbdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database.MethodsNine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies.ResultsSeveral SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples.ConclusionsAssociation testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression.

A nonsense mutation of PEPD in four Amish children with prolidase deficiency

Encoded by the peptidase D (PEPD) gene located at 19q12-q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C-terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma-like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR-amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 T > C in exon 11, resulting in a premature stop-codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation.

A novel nonsense mutation of the PEPD gene in a Japanese patient with prolidase deficiency.

A nonsense mutation at amino acid residue 184 in the human peptidase D (PEPD) gene caused the production of a truncated polypeptide. Characterizing molecular defects in patients provides clues to elucidate the relationship between the phenotype and the genotype.

Genetic variation in some populations of the golden-striped salamander, Chioglossa lusitanica (Amphibia: Urodela), in Portugal.

Genetic variation in the golden-striped salamander (Chioglossa lusitanica) was assessed in 231 individuals from four Portuguese populations by means of horizontal starch gel electrophoresis and isoelectric focusing. Three of 19 enzyme systems, representing 21 presumptive loci, were found to be polymorphic: phosphoglucomutase 1 (PGM1), peptidase B (PEPB), and peptidase D (PEPD). The observed average heterozygosity in Chioglossa lusitanica (0.027) is significantly lower than that observed for other amphibians, either urodeles or salamandrids. Differences in allele frequencies and the presence of private alleles are indicative of a high degree of population differentiation. PEPD, in particular, seems to be a diagnostic locus separating the southernmost population studied from the others.

Genetic mapping in Xenopus laevis: eight linkage groups established.

Inheritance of alleles at 29 electrophoretically detected protein loci and one pigment locus (albinism) was analyzed in Xenopus laevis by backcrossing multiply heterozygous individuals generated by intersubspecies hybridization. Pairwise linkage tests revealed eight classical linkage groups. These groups have been provisionally numbered from 1 to 8 in an arbitrarily chosen order. Linkage group 1 includes ALB-2 (albumin), ADH-1 (alcohol dehydrogenase), NP (nucleoside phosphorylase), and ap (periodic albinism). Linkage group 2 contains ALB-1 and ADH-2, and probably is homeologous to group 1. Linkage group 3 comprises PEP-B (peptidase B), MPI-1 (mannosephosphate isomerase), SORD (sorbitol dehydrogenase), and mIDH-2 (mitochondrial isocitrate dehydrogenase). Linkage group 4 contains GPI-1 (glucosephosphate isomerase) and EST-4 (esterase 4). Linkage group 5 contains GPI-2 and PEP-D (peptidase D). Linkage group 6 comprises ACP-3 (acid phosphatase), sME (cytosolic malic enzyme), and GLO-2 (glyoxalase). Linkage group 7 consists of sSOD-1 (cytosolic superoxide dismutase), GPD-2 (glycerol-3-phosphate dehydrogenase), mME (mitochondrial malic enzyme), and the sex determining locus. Linkage group 8 includes FH (fumarate hydratase) and TRF (transferrin). Recombination frequencies between linked loci showed differences related to the genomic constitution (parental subspecies) and to the sex of the heterozygous parent. Independent assortment was observed between the duplicate ALB loci. This is true for the duplicate ADH, GLO, and MPI loci as well, supporting the view that these genes have been duplicated as part of a genome duplication that occurred in the evolutionary history of X. laevis. Comparative analysis of genetic maps reveals a possible conservation of several linkages from the Xenopus genome to the human genome.

Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1, -C2, and -E and LDLR) and the genes C3, PEPD, and GPI.

We report the regional mapping of human chromosome 19 genes for three apolipoproteins and a lipoprotein receptor as well as genes for three other markers. The regional mapping was made possible by the use of a reciprocal whole-arm translocation between the long arm of chromosome 19 and the short arm of chromosome 1. Examination of three separate somatic cell hybrids containing the long arm but not the short arm of chromosome 19 indicated that the genes for apolipoproteins CI, CII, and E (APOC1, APOC2, and APOE, respectively) and glucose-6-phosphate isomerase (GPI) reside on the long arm, whereas genes for the low density lipoprotein receptor (LDLR), complement component 3 (C3), and peptidase D (PEPD) reside on the short arm. When taken together with previous studies, our results suggest the following physical gene map: pter-LDLR-C3-p13.2-PEPD-centromere-(APOE, APOC1, APOC2, GPI)-qter. In addition, we have isolated a single lambda phage carrying both APOC1 and part of APOE. These genes are tandemly oriented and are separated by about 6 kilobases of genomic DNA. Since previous family studies indicate tight linkage of APOE and APOC2, the apolipoprotein genes APOC1, APOC2, and APOE form a tight complex on the long arm of chromosome 19, suggesting the possibility of coordinate regulation.

Further regional localization of the genes for human acid alpha glucosidase (GAA), peptidase D (PEPD), and alpha mannosidase B (MANB) by somatic cell hybridization.

We have further regionally localized the gene for human acid alpha glucosidase (GAA) to 17q21----q23 by examination of hybrid clones derived from a fusion between human fibroblasts carrying a 17/19 balanced translocation (17pter----17q23::19p13.3----19pter; 19qter----p13.3::17q23----17qter) and a mouse line deficient in thymidine kinase. These hybrids were constantly maintained in HAT selective media in order to select for the presence of the human thymidine kinase gene on the intact chromosome 17 (17q21-q22) or the 17/19 (17pter----17q23::19p13.3----19pter) translocation chromosome. We detected human GAA by rocket immunoelectrophoresis, using a human specific heterologous antibody raised against human acid alpha glucosidase (GAA) (Honig et al. 1984). Three secondary clones, which contained the 17/19 translocation and no intact chromosome 17 or 19, were still positive for GAA. Two of these secondary clones contained the distal portion of the 17/19 translocation chromosome, with a break in the band 17q21 (probably at 17q21.2), attached to a mouse chromosome. Combined with earlier results (Weil et al. 1979; Nickel et al. 1982; Honig et al. 1984), the gene for GAA can be assigned to 17q21.2----17q23. Additionally, these clones were negative for human peptidase D (PEPD), alpha mannosidase B (MANB), and phosphohexose isomerase (PHI). Combined with previous results (Ingram et al. 1977; Bruns et al. 1979), these results exclude the genes for PEPD and MANB from 19pter----19p13.3 and confirm the exclusion of the gene for PHI from this segment of chromosome 19 (Wilson et al. 1984; Ingram et al. 1977).