Biochemical Basis of Prolidase Deficiency

Biochemical Basis of Prolidase Deficiency Polypeptide and RNA Phenotypes and the Relation to Clinical Phenotypes Fumio Endo,* Akito Tanoue,* Akito Kitano,* Jiro Arata,t David M. Danks,9 Charles M. Lapiere,1 Yoshihiro Sei,1 Sybe K. Wadman,** and Ichiro Matsuda* *Department ofPediatrics, Kumamoto University Medical School, Kumamoto, Japan; tDepartment ofDermatology, Okayama University Medical School, Okayama, Japan; §The Murdoch Institute, Royal Children's Hospital, Victoria, Australia; IlDepartment of Dermatology, University ofLiege, Liege, Belgium; 'Department ofDermatology, Kanazawa Medical University, Uchinada, Japan; and ** Wilhelmina Children's Hospital, Utrecht, The Netherlands Abstract sis of di- and tripeptides with carboxy-terminal proline and Cultured skin fibroblasts or lymphoblastoid cells from eight seems to play an important role in the recycling of proline. As patients with clinical symptoms of prolidase deficiency were a consequence of the deficiency of prolidase, massive imido- analyzed in terms of enzyme activity, presence of material peptiduria and relatively lowered levels of plasma proline are antibodies, biosynthesis of the poly- present in the patients (1). The precise mechanisms involved crossreacting with specific mental retardation, and peptide, and mRNA corresponding to the enzyme. There are at in the development of skin lesions, least two enzymes that hydrolyze imidodipeptides in these cells other symptoms including splenomegaly, osteoporosis, and and these two enzymes could be separated by an immunochem- frequent infections have not been completely elucidated. ical procedure. The specific assay for prolidase showed that More than 20 cases of prolidase deficiency have been re- the enzyme activity was virtually absent in six cell strains and ported, and some of the patients had no clinical symptoms of was markedly reduced in two (< 3% of controls). The activities the disease (2, 3). The symptoms do not seem to be related to of the labile enzyme that did not immunoprecipitate with the severities produced by the enzyme defect, as deduced from anti-prolidase antibody were decreased in the cells (30-60% of individual case reports. Clinical symptoms appear gradually; controls). hence, an assessment of the symptoms at the time of the diag- Cell strains with residual activities of prolidase had immu- nosis or during the relatively short period of the investigations nological polypeptides crossreacting with a Mr 56,000, similar is difficult. The hydrolytic activities toward imidodipeptides in to findings in the normal enzyme. The polypeptide biosynthesis crude extracts of tissues and cells may not solely represent the activity of a single enzyme (4) and activities of prolidase in in these cells and the controls was similar. Northern blot anal- of yses revealed the presence of mRNA in the polypeptide-posi- cases of a deficiency may not reflect residual activities pro- tive cells, yet it was absent in the polypeptide-negative cells. lidase itself, which is genetically defective. in the We purified human prolidase to apparent homogeneity The substrate specificities analyzed partially purified monoclonal and anti- enzymes from the polypeptide-positive cell strains differed, and prepared anti-prolidase polyclonal due to different mutations. Thus, there seems to be bodies for use in the immunochemical analysis of prolidase presumably et al. most a a molecular heterogeneity in prolidase deficiency. deficiency (5). Boright (6) recently developed between the clinical polyclonal antibody directed against prolidase and used it to There was no apparent relation symp- We used the and the that mental re- analyze cells from prolidase-deficient patients. toms biochemical phenotypes, except a cDNA clone to the was in the The antibodies to identify corresponding tardation present polypeptide-negative patients. mRNA (7). Based on the partial amino acid activities of the labile enzyme may not be a major factor in human prolidase Clin. Invest. 1990. sequence of the purified enzyme and the nucleotide sequence modifying the clinical symptoms. (J. structure of human * of the cDNA clones, the primary prolidase 85:162-169.) biosynthesis * crossreacting materials defi- has been deduced (8). ciency * mRNA - peptidase - prolidase We have now examined the biochemical basis of prolidase deficiency using cultured skin fibroblasts or cultured lympho- Introduction blastoid cells obtained from subjects from families with proli- Prolidase deficiency is an autosomal recessive disorder charac- dase deficiency. The availability of the specific antibodies and terized by mental retardation, various skin lesions, and abnor- the cloned cDNA facilitated a definition of the polypeptide malities of collagenous tissues (1). Prolidase catalyzes hydroly- and RNA phenotypes of these patients. The biochemical and clinical phenotypes were compared. Part of this work was presented at the Annual Meeting of the Japanese Methods Society for Inborn Errors of Metabolism, Tokyo, Japan, October 1988. Please address reprint requests to Dr. Fumio Endo, Department of Materials. IgG-EP2, EP1O, and EP38 mouse MAbs that bind to Pediatrics, Kumamoto University Medical School, Honjo 1-1-1, Ku- human prolidase, and IgG-NR, a control mouse MAb directed against mamoto 860, Japan. an irrelevant antigen, were prepared by the hybridoma technique de- Received for publication 12 June 1989 and in revised form 28 Au- scribed elsewhere (5). Human prolidase was then purified from periph- gust 1989. eral erythrocytes using an immunoaffinity gel (9). Polyclonal anti- serum was developed in rabbits by injecting purified human prolidase. J. Clin. Invest. Prolidase-Sepharose 4B was prepared using cyanogen bromide-acti- © The American Society for Clinical Investigation, Inc. vated Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the 002 1-9738/90/01/0162/08 $2.00 manufacturer's description. The gel preparation containing 2 mg of Volume 85, January 1990, 162-169 purified prolidase per ml of Sepharose 4B was used to isolate the 162 Endo et al. specific IgG that binds to prolidase from the anti-prolidase rabbit electrophoresis in formamide-containing agarose gels (1%) as described serum. Immobilized mouse IgGs on agarose beads were prepared using (15). Conditions for hybridization and washing were as described (8). purified IgGs and cyanogen bromide-activated Sepharose 4B (1 mg of Enzyme assay. Imidodipeptide-hydrolyzing activities were mea- purified IgG per ml). sured using glycyl-L-proline, L-alanyl-L-proline, and L-leucyl-L-proline The cDNA insert (PL-2 1) corresponding to human prolidase as substrates. The activities in the crude cell extracts were measured at mRNA was cloned and characterized (8). It was found to cover nearly 370C in a total vol of 250 ul containing 0.1 M Tris-HCl, pH 8.0, 10 the full length of the coding region and 3' noncoding region of the mM MnCl2, and 10 mM of a substrate peptide after 15 min of prein- mRNA and was used as a probe for RNA blotting analysis. The DNA cubation unless otherwise stated. The proline released from the pep- probe was radiolabeled as described (10). [a-32P]CTP (3,000 Ci/mmol) tides was assayed by the method of Chinard ( 16) as described ( 17). and [35S]methionine (1,100 Ci/mmol) were purchased from ICN ra- For the immune titration of prolidase activity, various amounts of diochemicals (Irvine, CA). Nitrocellulose membranes were obtained mouse or rabbit IgG were incubated with the crude cell extracts for 1 h from Schleicher and Schuell (Dassel, FRG). at room temperature and a 50-id suspension of protein A agarose (1: 1) Preparation of cell extracts. Confluently cultured monolayers of was added. The enzyme activity in the supernatant was then measured. skin fibroblasts cultured as described (1 1) were collected by a rubber The enzyme activity that bound to mouse IgG EP2 was measured as policeman and washed three times with PBS. The cell pellets were follows: the samples were prepared by incubating the crude cell ex- suspended in ice-cold PBS and then disrupted by sonication (Handy tracts and EP2-agarose at room temperature for 1 h. The gel was Sonic UR-20P; Tomy Seiko Co. Ltd., Tokyo, Japan). The EBV-trans- washed three times with 50 mM Tris-HCl, pH 7.6, containing 10 mM formed lymphoblastoid cells were cultured as described (12), washed MnC12. The gel suspension was preincubated for 15 min at 370C and three times with PBS, and sonicated. The disrupted cells were centri- the substrate peptides were added. After an appropriate incubation fuged at 12,000 g for 3 min and the supernatants were used for further time at 37°C, with vigorous shaking, the supernatant was collected and analyses. used for the determination of proline. Mouse IgG EP2 did not inhibit Immunoblot analyses. The extracts (100 Ag protein) were mixed the enzyme activity of prolidase. Alternatively, the prolidase was with agarose gel (50 ul of 1:1 suspension) that contained covalently eluted from the gel with 0.1 M Na2CO3 for enzyme assay (9). bound mouse monoclonal IgGs to immunoprecipitate the prolidase. Patients. Cultured skin fibroblasts or cultured lymphoblastoid cells The suspensions were incubated for 1 h at room temperature on an were obtained from eight patients with prolidase deficiency. Clinical end-to-end rotor, centrifuged, and the pellets washed three times with 1 features are summarized in Table I. Two patients died (cases 2 and 4) ml of ice-cold 50 mM Tris-HCl buffer, pH 7.0. Finally, the pellets were ;and skin lesions were present in all the patients. In severe cases there mixed with an equal volume of 125 mM Tris-HCI, pH 6.8, 4% SDS were deep skin ulcers or lymphedema. Mild skin lesions included (wt/vol), 10% (vol/vol) glycerol, and 2% (vol/vol) mercaptoethanol. eruption, teleangiectagia, and pigmentation. Two ofthe first cousins of The suspensions were placed in a boiling water bath for 1 min, cooled the case 5 patient had a prolidase deficiency with similar skin lesions at room temperature, and centrifuged. A sample of the supernatant and other clinical features. Skin lesions of the case 7 patient were was subjected to SDS-PAGE.

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