Carnosine Dipeptidase Research Articles

The zinc form of carnosine dipeptidase 2 (CN2) has dipeptidase activity but its substrate specificity is different from that of the manganese form

Carnosine dipeptidase II (CN2), a metallopeptidase present in the cytosol of various vertebrate tissues, catalyzes the hydrolysis of carnosine and several other dipeptides in the presence of Mn2+. Although the metal-binding center of mouse CN2 is also able to associate with Zn2+invitro, it was not known whether the zinc form of CN2 has any enzymatic activity. In the present study, we show that Zn2+ has a higher affinity for binding to CN2 than Mn2+, as evidenced by native mass spectrometry. The issue of whether the zinc form of CN2 has enzymatic activity was also examined using various dipeptides as substrates. The findings indicate that the zinc form of CN2 catalyzes the hydrolysis of several different dipeptides including Leu-His, Met-His and Ala-His at a reaction rate comparable to that for its manganese form. On the other hand, the zinc form of CN2 did not catalyze the hydrolysis of carnosine and several other dipeptides that are hydrolyzed by the manganese form of CN2. Substrate specificity was also examined in HEK293T cells expressing CN2, and the findings indicate that Leu-His, Met-His, but not carnosine, were hydrolyzed in the cell culture. These results suggest that the zinc form of CN2 is an active enzyme, but with a different substrate specificity from that of the manganese form.

Serum Carnosine Dipeptidase 1 and Ubiquitin C - Terminal Hydrolase L1 as Markers of Brain Damage in Patients After Carotid Endarterectomy

Introduction: Carotid endarterectomy (CEA) is the recommended surgical procedure in the prevention of ischemic stroke. However, this procedure may lead to vascular neurological complications. The aim of the study was to measure serum carnosine dipeptidase 1 (CNDP1) and ubiquitin C - terminal hydrolase L1 (UCHL1) as markers of brain damage in patients that underwent CEA due to high-grade internal carotid artery stenosis. Material and Methods: This study included 25 patients. Blood samples were taken from the antecubital vein at three different intervals (within a 24 hour period prior to CEA, 12 hours following surgery, and 48 hours after surgery). Serum CNDP1 and UCHL1 levels were measured by a commercially available enzyme-linked immunosorbent assay (ELISA). Results: The study showed that serum CNDP1 and UCHL1 levels were significantly decreased 12 hours after CEA when compared to the level before the surgery. Furthermore, these enzymes levels were normalized 48 hours after CEA. Conclusion: Data from our study showed that CEA significantly affects serum CNDP1 and UCHL1 levels. Moreover, these enzyme levels seems to reflect a brain ischemia resulting from severe internal carotid artery stenosis in patients undergoing CEA.

Association of CTG repeat polymorphism in carnosine dipeptidase 1 (CNDP1) gene with diabetic nephropathy in north Indians.

Background & objectives:CNDP1 gene, present on chromosome 18q22.3-23, encodes carnosinase, the rate-limiting enzyme in hydrolysis of carnosine to β-alanine and L-histidine. Linkage of CTG trinucleotide (leucine) repeat polymorphism in CNDP1 gene with diabetic nephropathy has been observed in several populations. However, this association is conflicting and population-dependent. We investigated this association in type 2 diabetes mellitus (T2DM) patients with and without nephropathy in north India.Methods:A total of 564 individuals [199 T2DM without nephropathy (DM), 185 T2DM with nephropathy (DN) and 180 healthy individuals (HC)] were enrolled. CNDP1 CTG repeat analysis was done by direct sequencing of a 377 base pair fragment in exon 2.Results:The most frequent leucine (L) repeats were 5L-5L, 6L-5L and 6L-6L. 5L-5L genotype frequency was reduced in DN (24.3%) as compared to DM (34.7%, P=0.035) and HC (38.4%, P=0.005). Similarly, 5L allele frequency was lower in DN (46.8%) as compared to DM (57.3%, P=0.004) and HC (60.5%, P<0.001). The genotype and allelic frequencies were similar in DM and HC groups. No gender specific difference was observed in the genotype or allelic frequencies between groups.Interpretation & conclusions:Compared to healthy individuals and those with diabetes but no kidney disease, patients with diabetic nephropathy exhibited lower frequencies of 5L-5L genotype and 5L allele of CNDP1 gene, suggesting that this allele might confer protection against development of kidney disease in this population.

Evidence for an essential role of intradimer interaction in catalytic function of carnosine dipeptidase II using electrospray-ionization mass spectrometry

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β-alanyl-L-histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray-ionization time-of-flight mass spectrometry (ESI-TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI-TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N-terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI-TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time-dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI-TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.

Circulating carnosine dipeptidase 1 associates with weight loss and poor prognosis in gastrointestinal cancer.

BackgroundCancer cachexia (CC) is linked to poor prognosis. Although the mechanisms promoting this condition are not known, several circulating proteins have been proposed to contribute. We analyzed the plasma proteome in cancer subjects in order to identify factors associated with cachexia.Design/SubjectsPlasma was obtained from a screening cohort of 59 patients, newly diagnosed with suspected gastrointestinal cancer, with (n = 32) or without (n = 27) cachexia. Samples were subjected to proteomic profiling using 760 antibodies (targeting 698 individual proteins) from the Human Protein Atlas project. The main findings were validated in a cohort of 93 patients with verified and advanced pancreas cancer.ResultsOnly six proteins displayed differential plasma levels in the screening cohort. Among these, Carnosine Dipeptidase 1 (CNDP1) was confirmed by sandwich immunoassay to be lower in CC (p = 0.008). In both cohorts, low CNDP1 levels were associated with markers of poor prognosis including weight loss, malnutrition, lipid breakdown, low circulating albumin/IGF1 levels and poor quality of life. Eleven of the subjects in the discovery cohort were finally diagnosed with non-malignant disease but omitting these subjects from the analyses did not have any major influence on the results.ConclusionsIn gastrointestinal cancer, reduced plasma levels of CNDP1 associate with signs of catabolism and poor outcome. These results, together with recently published data demonstrating lower circulating CNDP1 in subjects with glioblastoma and metastatic prostate cancer, suggest that CNDP1 may constitute a marker of aggressive cancer and CC.

Analysis of plasma from prostate cancer patients links decreased carnosine dipeptidase 1 levels to lymph node metastasis

• Translation of an antibody array discovery. • Multi-antibody sandwich assay. • Biomarker candidate for lymph node metastasis. There is a need for a better differentiation of aggressive tumors in prostate cancer to design a tailored treatment for each patient, preferably by a minimally invasive analysis of blood samples. In a previous study, we discovered a decrease of plasma levels of carnosine dipeptidase 1 (CNDP1) in association with aggressive prostate cancer. Now this relation has been investigated and characterized further by generating several new antibodies for extended analysis of CNDP1 in plasma. Multi-antibody sandwich assays were developed and applied to 1214 samples from two Swedish cohorts that confirmed decreased levels of CNDP1 in plasma from patients with advanced disease. Therein, data from CNDP1 assays allowed a better differentiation between tumor N stages than clinical tPSA, but did not when classifying T or M stages. Further investigations can now elucidate mechanisms behind decreasing levels of CNDP1 in plasma and primary in regards to lymph node metastasis.

The Influence of a Single Nucleotide Polymorphism within CNDP1 on Susceptibility to Diabetic Nephropathy in Japanese Women with Type 2 Diabetes

BackgroundSeveral linkage analyses have mapped a susceptibility locus for diabetic nephropathy to chromosome 18q22–23, and polymorphisms within the carnosine dipeptidase 1 gene (CNDP1), located on 18q22.3, have been shown to be associated with diabetic nephropathy in European subjects with type 2 diabetes. However, the association of this locus with diabetic nephropathy has not been evaluated in the Japanese population. In this study, we examined the association of polymorphisms within the CNDP1/CNDP 2 locus with diabetic nephropathy in Japanese subjects with type 2 diabetes.Methodology/Principal FindingsWe genotyped a leucine repeat polymorphism (D18S880) that is within CNDP1 along with 29 single nucleotide polymorphisms (SNPs) in the CNDP1/CNDP2 locus for 2,740 Japanese subjects with type 2 diabetes (1,205 nephropathy cases with overt nephropathy or with end-stage renal disease [ESRD], and 1,535 controls with normoalbuminuria). The association of each polymorphism with diabetic nephropathy was analysed by performing logistic regression analysis. We did not observe any association between D18S880 and diabetic nephropathy in Japanese subjects with type 2 diabetes. None of the 29 SNPs within the CNDP1/CNDP2 locus were associated with diabetic nephropathy, but a subsequent sex-stratified analysis revealed that 1 SNP in CNDP1 was nominally associated with diabetic nephropathy in women (rs12604675-A; p = 0.005, odds ratio [OR] = 1.76, 95% confidence interval [CI], 1.19−2.61). Rs12604675 was associated with overt proteinuria (p = 0.002, OR = 2.18, 95% CI, 1.32−3.60), but not with ESRD in Japanese women with type 2 diabetes.Conclusions/SignificanceRs12604675-A in CNDP1 may confer susceptibility to overt proteinuria in Japanese women with type 2 diabetes.

Abstract P3-12-01: Serum biomarkers identification using quantitative proteomics in patients (pts) with untreated brain metastases from HER2-positive breast cancer receiving capecitabine (C) and lapatinib (L) (UNICANCER LANDsCAPE trial)

Abstract Background: The LANDsCAPE phase II study showed that C+L had a significant antitumor activity in previously untreated brain metastases (BM) from HER2-positive breast cancer (BC), with a central nervous system-objective response rate (CNS-ORR) of 67% and a median time to whole-brain radiotherapy (WBRT) of 8.3 months (43 analysed pts). Thus initial C+L combination might be a viable alternative to immediate WBRT in this setting. In this study, serum samples were collected before and during treatment for proteomic-based approaches to identify predictive biomarkers of treatment response. Methods: Baseline (BL) and day 21 (D21) serum samples from highly responsive (R, CNS lesions -volumetric response ≥ 75%, n=6) and non-responsive (NR, CNS-stable or progressive disease, n=6) pts were subjected to isobaric Tag for Relative and Absolute Quantification (iTRAQ)-based proteomics. Samples from each condition were pooled to constitute 4 independent mixes ((BL-R, D21-R, BL-NR and D21-NR). Each of them underwent immuno-depletion of highly abundant proteins, concentration, reduction, alkylation and tryptic digestion. Then, each mix was fractionated and subjected to iTRAQ identification and quantitation using nano-liquid chromatography (LC) and electrospray ionisation (ESI)-orbitrap tandem mass spectrometry (MS/MS) (LTQ-orbitrap, Thermofisher). Differentially expressed proteins were analysed using IPA (Ingenuity® Systems) to highlight biological functions and signalling pathways that were most significantly enriched. Results: iTRAQ-based measurements identified serum proteins with significant (fold-change &amp;gt; 1.5 and p-value &amp;lt; 0.05) differential expression between BL and D21, in R- (n = 38 proteins) and NR- pts (n = 18 proteins). At baseline, 30 proteins were differentially expressed between R-and NR-pts. Among the most differentially expressed proteins, some had been previously described as involved in cancer metastases (between BL and D21: tenascin C, neuropilin-1, Serpin A1, Cathepsin S, prostaglandin D2 synthase, melanoma cell adhesion molecules, procollagen C-endopeptidase enhancer; between R and NR: carnosine dipeptidase 1, endothelial protein C receptor, matrix metallopeptidase 9, cystatin C, lumican, plexin B2, Insulin-like growth factor binding protein acid labile subunit, pro-platelet basic protein, cadherin 5, Protein S). Proteins with differential expression during treatment were involved in various biological processes, including lipid metabolism, small molecule biochemistry, molecular transport, gene expression, cellular growth and proliferation, cellular movement and cancer, as well as various canonical pathways such as acute phase response signalling, LXR/RXR activation and complement system. Interestingly, and as opposed to NR-pts, R-pts specifically showed an overall increase in proteins of acute phase response, with a significant decrease in C-reactive protein (CRP). Conclusion: iTRAQ-based quantitative proteomics identify serum proteins that could predict the therapeutic response to C+L combination in untreated BM from HER2-positive BC. If validated, such biomarkers may help to select the best therapeutic strategy in this setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-12-01.

Purification and identification of two carnosine-cleaving enzymes, carnosine dipeptidase I and Xaa-methyl-His dipeptidase, from Japanese eel (Anguilla japonica)

Three enzymes, carnosine dipeptidase I (EC 3.4.13.20, CNDP1), carnosine dipeptidase II (EC 3.4.13.18, CNDP2), and Xaa-methyl-His dipeptidase (or anserinase: EC 3.4.13.5, ANSN), are known to be capable of catalyzing the hydrolysis of carnosine (β-alanyl-l-histidine), in vertebrates. Here we report the purification and identification of two unidentified carnosine-cleaving enzymes from Japanese eel (Anguilla japonica). Two different dipeptidases were successfully purified to homogeneity from the skeletal muscle; one exhibited a broad substrate specificity, while the other a narrow specificity. N-terminal amino-acid sequencing, deglycosylation analysis, and genetic analysis clearly revealed that the former is a homodimer of glycosylated subunits, encoded by ANSN, and the latter is another homodimer of glycosylated subunits, encoded by CNDP1; that is, Xaa-methyl-His dipeptidase, and carnosine dipeptidase I respectively. This is the first report on the identification of carnosine dipeptidase I from a non-mammal. Database search revealed presence of a CNDP1 ortholog only from salmonid fishes, including Atlantic salmon and rainbow trout, but not from other ray-finned fish species, such as zebrafish, fugu, and medaka whose genomes have been completely sequenced. The mRNAs of CNDP1 and ANSN are strongly expressed in the liver of Japanese eel, compared with other tissues, while that of CNDP2 is widely distributed in all tissues tested.

Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays

There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format.

Examination of association with candidate genes for diabetic nephropathy in a Mexican American population.

Diabetic nephropathy (DN) is a multifactorial complication characterized by persistent proteinuria in susceptible individuals with type 1 and type 2 diabetes. Disease burden in people of Mexican-American descent is particularly high, but there are only a few studies that characterize genes for DN in this ethnic group. Two genes, carnosine dipeptidase 1 (CNDP1) and engulfment and cell motility 1 (ELMO1) previously showed association with DN in other ethnic groups. CNDP1 and ELMO1 were examined along with eight other genes that are less well characterized for DN in a new study of Mexican-Americans. The target sample was patients of Mexican-American ancestry collected from three centers: 455 patients with DN and 437 controls with long-term diabetes but no incident nephropathy. Forty-two, 227, and 401 single nucleotide polymorphisms (SNPs) in CNDP1, ELMO1, and the other eight genes, respectively, were examined. No region in CNDP1 or ELMO1 showed significant P values. Of the other eight candidate genes, an association of DN with a SNP pair, rs2146098 and rs6659783, was found in hemicentin 1 (HMCN1) (unadjusted P = 6.1 x 10(-5)). Association with a rare haplotype in this region was subsequently identified. The associations in CNDP1 or ELMO1 were not replicable; however, an association of DN with HMCN1 was found. Additional work at this and other loci will enable refinement of the genetic hypotheses regarding DN in the Mexican-American population to find therapies for this debilitating disease.

Neuronal pentraxin receptor in cerebrospinal fluid as a potential biomarker for neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by progressive loss of cognitive function, dementia, and problems with movements. In order to find new protein biomarkers of high specificity from cerebrospinal fluid (CSF) of AD and PD patients, one-dimensional gel electrophoresis (1-DE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as well as 2-DE analysis were performed. In 1-DE and LC-MS/MS 371 proteins were identified, among which levels of proteins such as isoform 1 of contactin-1, contactin-2, carnosine dipeptidase 1 (CNDP1), 120 kDa isoform precursor of neural cell adhesion molecule 1 (NCAM-120), alpha-dystroglycan, secreted protein acidic and rich in cysteine-like protein 1 precursor (SPARCL1), isoform 2 of calsyntenin 1 (CLSTN1), and neuronal pentraxin receptor (NPR) showed significant changes in AD or PD CSF compared with normal subjects. In 2-DE analysis approximately 747-915 spots were detected in CSF of AD or PD patients, from which 17-24 proteins with more than a 1.2 fold change were identified by tandem MS. Most proteins identified showed consistent changes in LC-MS/MS and 2-DE analysis. Three proteins that showed significant changes were selected for further validation by Western blot analysis. While NCAM-120 and alpha-dystroglycan exhibited higher levels in both AD and PD CSF compared with normal subjects, the level of NPR was increased only in AD CSF in Western blot analysis. The results were consistent with quantitative analysis of 2-DE spots. A higher level of NPR was also found in AD serum. This study suggests that NCAM-120, alpha-dystroglycan, and NPR are candidate biomarkers in CSF for neurodegenerative diseases, and that the changes in the CSF level of NPR may be specific for AD.