Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies.

TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 antibody T4 disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.

Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173.

BACKGROUND AND AIMS: The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis. METHODS AND RESULTS: Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn2+ and Cu2+ concentrations after dialysis. CONCLUSIONS: Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.

N-acetylated α-linked acidic dipeptidase is identified as an antigen of Histoplasma capsulatum.

Histoplasmosis, one of the most important mycoses, needs to be diagnosed rapidly and accurately. The main method used to diagnose histoplasmosis is serological detection of antibodies to the Histoplasma capsulatum H and M antigens. Several other protein antigens have been reported in H. capsulatum; however, they have not been used for diagnosis. In this study, we explored novel antigens that were detected during H. capsulatum infection. We obtained a protein mixture from H. capsulatum yeast cells after vigorous mixing in a 0.1% Triton X-100 solution. From the resultant pool, we detected nine spots that reacted with sera from patients with histoplasmosis and identified eight seroactive proteins with mass spectrometry. The seroactive proteins were purified, and their antigenicities were tested with an enzyme-linked immunosorbent assay (ELISA). ELISA revealed that the titer of the patients' sera to N-acetylated α-linked acidic dipeptidase was significantly higher than those of healthy volunteers (P < 0.01). These data indicate that N-acetylated α-linked acidic dipeptidase of H. capsulatum is recognized as a major antigen during histoplasmosis.

Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations.

Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1-1.8 vs. 1-50 µg/ml, RYSK- vs. ATLAS-based, P<0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P<0.01 and R: 0.93, P<0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P<0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay × 100% (Spearman-Rang correlation coefficient: -0.6, P<0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies.

Generation of monospecific antibodies based on affinity capture of polyclonal antibodies.

A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

Molecular characterization and expression of dipeptidase 3, a testis-specific membrane-bound dipeptidase: complex formation with TEX101, a germ-cell-specific antigen in the mouse testis.

We previously established an anti-sperm head auto-monoclonal antibody designated Ts4. The immunoreactivity of this antibody was also observed in other reproduction-related cells, such as testicular germ cells and early embryos, suggesting that the Ts4-recognized molecules might play a role in the reproductive process. However, the molecular characteristics and functions of the antigens warrant further clarification. In this study, we primarily attempted identification of the mAb-recognized molecules within the mouse testis. An immunoprecipitation method, together with liquid chromatography-tandem mass spectrometry, revealed that the testicular immunoprecipitants with Ts4 contained dipeptidase 3 (DPEP3), a member of the membrane-bound dipeptidase family. A Western blot analysis using an anti-DPEP3 polyclonal antibody established in this study showed that this molecule was glycosylated and formed a disulfide-linked homodimer within the testis. Expression of DPEP3 protein was observed in the testicular germ cells, but not in the Sertoli or interstitial cells, or in any other major organs. Although Western blot analysis of testicular proteins separated by two-dimensional SDS-PAGE failed to demonstrate binding of Ts4 to DPEP3, we found that DPEP3 forms complexes with Ts4-immunoreactive molecules, such as TEX101, on the surfaces of spermatocytes, spermatids, and testicular spermatozoa. Based on data showing in the present study, further studies concerning DPEP3 on the testicular germ cells may help to clarify the molecular mechanisms of testicular germ-cell development.

Localization and activity of membrane dipeptidase in bovine ciliary epithelium.

PURPOSE: To investigate the localization and the activity of membrane dipeptidase (MDP) in the bovine eye. METHODS: A monoclonal antibody (mAb), 49C mAb, raised against bovine ciliary process was used to examine the localization of MDP. Conversion of leukotriene (LT)D4 to LTE4 was evaluated by enzyme-linked immunosorbent assay for LTE4. Hydrolytic activity (beta-lactamase activity) was evaluated with a fluorometric assay. To clarify the contribution of MDP to conversion of LTD4 and beta-lactamase activity, we separated MDP from other enzymes by 49C mAb-conjugated gel. RESULTS: The antigenic molecule of 49C mAb was shown to be MDP by amino acid sequencing. MDP was immunohistochemically detected in the ciliary pigmented and nonpigmented epithelial cells. Conversion of LTD4 to LTE4 in the ciliary process was much greater than that of the neural retina (NR). beta-Lactamase activity in the ciliary process was apparent, but that in the NR or the retinal pigment epithelium was negligible. Approximately 100% of beta-lactamase activity in the ciliary process was catalyzed by the 49C mAb-bound fraction. Conversion of LTD4 was catalyzed by the 49C mAb-bound fraction (55% of total activity) and by the unbound fraction (45% of total activity). CONCLUSIONS: This study produced the first evidence of the presence of MDP in ciliary epithelial cells. The ciliary epithelium converts LTD4 to LTE4 and shows beta-lactamase activity. Conversion of LTD4 is catalyzed by at least two enzymes, and a major part of the conversion is induced by MDP.

Disruption of gamma-glutamyl leukotrienase results in disruption of leukotriene D(4) synthesis in vivo and attenuation of the acute inflammatory response.

To study the function of gamma-glutamyl leukotrienase (GGL), a newly identified member of the gamma-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGL(tm1)) and in both GGL and GGT (GGL(tm1)-GGT(tm1)) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGL(tm1) show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but approximately 70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C(4) (LTC(4)) to LTD(4), indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD(4). Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC(4) and LTD(4) have distinctly different functions in the inflammatory process.

Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91.

Cattle were vaccinated either with a single recombinant tick antigen, Bm86 or with a combination of two recombinant antigens, Bm86 and Bm91 from the tick Boophilus microplus. In three experiments, the responses of cattle to subsequent challenge with the tick were assessed. The addition of the Bm91 antigen enhanced the efficacy of the vaccination over that with Bm86 alone to a statistically significant degree. Moreover, co-vaccination with two antigens did not impair the response of cattle to the Bm86 antigen. Finally, responses of individual cattle to the two antigens were independent. All of these results may be relevant to the increase in efficacy expected from a dual antigen vaccine.

Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.

This report demonstrates that the investigational prostatic carcinoma marker known as the prostate-specific membrane antigen (PSM) possesses hydrolytic activity with the substrate and pharmacologic properties of the N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase that has been characterized in the mammalian nervous system on the basis of its catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) to yield glutamate and N-acetylaspartate and that has been hypothesized to influence glutamatergic signaling processes. The immunoscreening of a rat brain cDNA expression library with anti-NAALADase antisera identified a 1428-base partial cDNA that shares 86% sequence identity with 1428 bases of the human PSM cDNA [Israeli, R. S., Powell, C. T., Fair, W. R. & Heston, W.D.W. (1993) Cancer Res. 53, 227-230]. A cDNA containing the entire PSM open reading frame was subsequently isolated by reverse transcription-PCR from the PSM-positive prostate carcinoma cell line LNCaP. Transient transfection of this cDNA into two NAALADase-negative cell lines conferred NAAG-hydrolyzing activity that was inhibited by the NAALADase inhibitors quisqualic acid and beta-NAAG. Thus we demonstrate a PSM-encoded function and identify a NAALADase-encoding cDNA. Northern analyses identify at least six transcripts that are variably expressed in NAALADase-positive but not in NAALADase-negative rat tissues and human cell lines; therefore, PSM and/or related molecular species appear to account for NAAG hydrolysis in the nervous system. These results also raise questions about the role of PSM in both normal and pathologic prostate epithelial-cell function.

The immunocytochemical localization of N-acetylaspartyl glutamate, its hydrolysing enzyme NAALADase, and the NMDAR-1 receptor at a vertebrate neuromuscular junction.

Although glutamate is thought to be the neurotransmitter at the invertebrate neuromuscular junction, acetylcholine is accepted as the primary neurotransmitter of the vertebrate motoneurons. N-acetylaspartylglutamate, a dipeptide localized in putative glutamatergic neurons in brain, is also found in high concentrations (> mM) in mammalian motoneurons and the ventral roots of spinal cord. N-acetylaspartylglutamate, which is released from neurons by depolarization in a Ca(2+)-dependent fashion, is implicated in glutamatergic transmission in two ways: it is a partial agonist at NMDA receptors, and it is cleaved to yield extracellular glutamate and N-acetylasparate by the specific peptidase N-acetylated alpha-linked acidic dipeptidase. Given the localization of N-acetylaspartylglutamate in motor neuronal perikarya and axons, we wondered whether N-acetylaspartylglutamate or glutamate cleaved from N-acetylaspartylglutamate by N-acetylated alpha-linked acidic dipeptidase may also play a role in neuromuscular transmission. Here we describe the immunocytochemical detection at the rat neuromuscular junction of N-acetylaspartylglutamate in terminals of motoneurons, of N-acetylated alpha-linked acidic dipeptidase in perisynaptic Schwann cells, and of the NMDAR-1 glutamate receptor subunit on postsynaptic muscle membranes. These results point to a potential role for N-acetylaspartylglutamate at the rat neuromuscular junction. Further, this is the first demonstration of a glutamate receptor protein at vertebrate neuromuscular synapses. Together with other recent findings, our results suggest that glutamate-like molecules are involved in neuromuscular transmission not only in invertebrates but also in veretebrates where they may modulate signaling by acetylcholine.

Structural and immunological studies of GPI-anchored brush border hydrolases.

The complement of brush border hydrolases provides an excellent model system for study of the structure, topology and assembly of plasma membrane proteins. Among the peptidases of the renal brush border are a number of glycosylphosphatidylinositol (GPI)-anchored proteins, especially membrane dipeptidase and aminopeptidase P. Affinity purification protocols have led to the isolation of homogeneous forms of these proteins and membrane dipeptidase has been cloned and expressed in Xenopus oocytes and Cos-1 cells. The core glycan structures of both human and porcine dipeptidase have been determined, allowing direct comparisons of the GPI anchors on the same protein in different species. Aminopeptidase P has been compared in the brush borders of pig kidney and intestine and may well be anchored in distinct ways in the two tissues. Immunological cross-reactivity of polyclonal antibodies to these two proteins has revealed the phospholipase C-cleaved GPI anchor as the common epitope and defined those components of the anchor important for recognition. These antibodies are also proving useful in characterizing GPI-derived mediators that may be involved in cell signalling processes. These abundant ectopeptidases offer a number of advantages for studies of the biochemistry of mammalian GPI anchors.

Human kidney prolidase--purification, preincubation properties and immunological reactivity.

1. Prolidase I (EC 3.4.13.9) was purified from human kidney to SDS-PAGE homogeneity. The molecular weights of native and denatured purified enzyme were estimated to be 115,000 and 55,000, respectively. 2. Agarose electrophoresis revealed migration in the alpha 1 globulin region, and an isoelectric point (pl) of 4.65 was estimated by both isoelectric focusing (IEF) and the titration curve method. 3. Activation by preincubation for 24 hr at 37 degrees C with 1 mM MnCl2 was maintained throughout the purification steps, using gly-pro and phe-pro dipeptides as substrates. 4. Activation in the presence of gly-pro was higher (4.5- to 11-fold) than in the presence of phe-pro (1.3- to 2.3-fold). 5. Lineweaver-Burk plot consisted of one and two lines with gly-pro and phe-pro, respectively. Km, Vmax and the Vmax/Km ratio were increased and the two lines with phe-pro were conserved after prolonged preincubation. 6. A specific polyclonal antibody was raised in rabbits against the purified enzyme and immunoreactivity was investigated between rabbit antiserum and both prolidase I from various tissues and human kidney prolidase II. 7. Prolidase I from liver, erythrocytes and plasma was immunochemically identical to renal prolidase I. The polyclonal antibody did not react with prolidase II. 8. These results indicate that a specific immunoassay might be developed to investigate prolidase I protein in plasma and tissues from patients with prolidase deficiency and hepatic fibrosis.

Localization of a novel pathway for the liberation of GABA in the human CNS.

Serum carnosinase is a dipeptidase, which is synthesized in human brain, where it hydrolyzes homocarnosine to release free GABA. Immunohistochemical procedures were used to demonstrate the presence of this enzyme in several layers of the retina and in certain neuronal tracts of the cerebral cortex, cerebellar cortex, olfactory bulb, hippocampus, and in disseminated tracts presumably from the internal capsule, interspersed among the basal ganglia. The enzyme was also present in the epithelial cells of the choroid plexus and in corpora amylacea, which were seen in many regions of the CNS. Homocarnosine was localized either in the same tracts or in nearby neurons. For example, the Purkinje cells of the cerebellar cortex contained homocarnosine, whereas serum carnosinase was localized in adjacent neuronal projections apparently originating from outside the cerebellar cortex and having probable synaptic contact with the Purkinje cells. These findings suggest that in addition to glutamate decarboxylation, a second metabolic reaction for the formation of free GABA exists in specific neuronal tracts of the human CNS where GABA is released from homocarnosine by the action of serum carnosinase.

Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase.

A polyclonal antiserum raised to the phospholipase C-solubilized form of membrane dipeptidase (EC 3.4.13.11) purified from human kidney was found to cross-react with unrelated trypanosomal and porcine glycosyl-phosphatidylinositol anchored proteins. Those antibodies recognising the cross-reacting determinant (CRD) were isolated by chromatography on a column of immobilized phospholipase C-solubilized porcine aminopeptidase P (EC 3.4.11.9), and the epitopes involved in the recognition were then characterized by immunoelectrophoretic blot analysis and by a competitive ELISA. The phospholipase C-solubilized forms of human and porcine membrane dipeptidase, porcine aminopeptidase P and trypanosome variant surface glycoprotein were recognised by the anti-CRD antiserum, and this recognition was abolished by prior treatment of the proteins with either mild acid or nitrous acid. In contrast, the detergent-solubilized, membrane-forms of human and porcine membrane dipeptidase were not recognised. Of a range of components of the glycosyl-phosphatidylinositol anchor, only inositol 1,2-cyclic monophosphate and the insulin-mimetic disaccharide, glucosaminyl-1,6-inositol 1,2-cyclic monophosphate, inhibited in the micromolar range the binding of the anti-CRD antiserum to immobilized porcine aminopeptidase P. These results indicate that the major epitope recognised by this anti-CRD antiserum is the inositol 1,2-cyclic monophosphate formed on phospholipase C cleavage of the glycosyl-phosphatidylinositol anchor.

Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins.

Two polyclonal antisera were raised in rabbits to the phospholipase C-solubilized forms of pig renal dipeptidase (EC 3.4.13.11) and pig aminopeptidase P (EC 3.4.11.9). These antisera were purified and shown to cross-react with other glycosyl-phosphatidylinositol (G-PI)-anchored proteins isolated from pig, human and trypanosomes. The epitopes involved in this cross-reactivity were characterized by Western-blot analysis after mild acid or nitrous acid treatment of the G-PI-anchored proteins and by a competitive e.l.i.s.a. with other G-PI-anchored proteins and individual components of the anchor structure. These studies revealed that the primary epitope for both antisera is the inositol 1.2-(cyclic)monophosphate that is formed on phospholipase C cleavage of the intact G-PI anchor. Other minor epitopes, such as phosphoethanolamine, probably involve side-chain modifications to the core anchor structure that may be species-specific.

Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.

A single nucleotide change in the prolidase gene in fibroblasts from two patients with polypeptide positive prolidase deficiency. Expression of the mutant enzyme in NIH 3T3 cells.

Prolidase deficiency is an autosomal recessive disorder characterized by mental retardation and various skin lesions. Cultured skin fibroblasts were obtained from two independent patients with abnormal prolidase. Using the polymerase chain reaction, we amplified the entire coding region of human prolidase mRNA derived from patients' fibroblasts. Nucleotide sequence analysis of amplified cDNA products revealed a G to A substitution at position 826 in exon 12, where aspartic acid was replaced by asparagine at the amino acid residue 276, in cells from both patients. An analysis of the DNA showed that the substitution was homozygous. An expression plasmid clone containing a normal human prolidase cDNA (pEPD-W) or mutant prolidase cDNA (pEPD-M) was prepared, transfected, and tested for expression in NIH 3T3 cells. Incorporation of pEPD-W and pEPD-M resulted in the synthesis of an immunological polypeptide that corresponded to human prolidase. Active human enzyme was detected in cells transfected with pEPD-W, but not in those transfected with pEPD-M. These results were compatible with our observation of fibroblasts and confirmed that the substitution was responsible for the enzyme deficiency. As active prolidase was recovered in prolidase-deficient fibroblasts transfected with pEPD-W, this restoration of prolidase activity after transfection means that gene replacement therapy for individuals with this human disorder can be given due consideration.