production of monoclonal antibodies against human 6-pyruvoyl tetrahydropterin synthase and...

Vol. 182, No. 2, 1992 January 31, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 810-816

PRODUCTION OF MONOCLONAL ANTlBODlES AGAINST HUMAN 6-PYRUVOYL TETRAHYDROPTERIN SYNTHASE AND IMMUNOCYTOCHEMICAL

LOCALIZATION OF THE ENZYME

Jaime Guzman, Gabriele Schoedon and Nenad Blau

Division of Clinical Chemistry, Department of Pediatrics, University of Zurich, CH-8032 Zurich, Switzerland

Received October 4, 1991

SUMMARY : Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6- pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique. e 1992 Academic Press. Inc.

INTRODUCTION : Tetrahydrobiopterin (BH$ is required as cofactor by the mammalran

aromatic amino acid hydroxylases (1). A lack of BH, leads to hyperphenylalaninemia

and a deficiency of biogenic amine neurotransmitters. 6-Pyruvoyl tetrahydropterin

synthase (PTP synthase) is one of the key enzymes in the biosynthetic pathway of BH4

(2-5). Patients with PTP synthase deficiency, the most common form of BH, deficiency,

lack BH4 almost completely and suffer from severe neurological disorders (6). PTP

synthase deficiency is a heterogeneous genetic disease. Some patients suffer from a

so-called peripheral defect (7). These patients have enough PTP synthase activity to

meet the BH4 requirement for neurotransmitter biosynthesis in the brain, but not

enough to activate the hepatic phenylalanine hydroxylase reaction (8). In some cases it

is difficult to differentiate patients with a partial or peripheral defect from those with a

severe PTP synthase deficiency. Only analysis of urinary pterins combined with

ABBREVIATIONS : BH,, tetrahydrobiopterin; PTP synthase, 6-pyruvoyl tetrahydropterir synthase; mAbs, monoclonal antibodies; FPLC, fast protein liquid chromatography; ELISA enzyme linked immunosorbent assay; IMDM, Iscove’s modified Dulbecco’s Medium PVDF, polyvinylidene difluoride.

0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. 810

Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

measurement of enzyme activity in red blood cells and analysis of neurotransmitter

metabolites in CSF enables differentiation between the variants of PTP synthase

deficiency.

For a better understanding of these heterogenic forms of PTP synthase deficiency

on the protein level, antibodies were thought to be an invaluable tool. Until now only

monoclonal antibodies (mAbs) against salmon liver PTP synthase (9) and an antiserum

to PTP synthase from Drosophila melanogaster (10) have been available. However,

neither of these antibodies crossreacted with the human enzyme. This communication

reports the first successful production of mAbs against human PTP synthase. These

monoclonal antibodies were used for the immunocytochemical localization of PTP

synthase in peripheral blood cells and in human skin fibroblasts.

MATERIALS AND METHODS

Materials Polyvinylidene difluoride (PVDF, Immobilon-P) transfer membranes were from

Millipore (Bedford, MA, USA). All culture reagents and tissue culture water were from Sigma (St. Louis, MO, USA), all the sterile plastics were from Falcon (Oxnard, CA, USA). The tissue culture medium was Iscove’s modified Dulbecco’s Medium (IMDM) from Gibco (Grand Island, New York, USA). Fetal calf serum (FCS) was a tested batch from lnotech (Wohlen, Switzerland). Lymphokine conditioned medium sMLC/sEL-4 was from Bio-Invent (Lund, Sweden). All other chemicals were of analytical grade from commercial sources.

Methods Monoclonal antibodies were produced using a modification of the in vitro

immunization technique described by Borrebaeck and Mdller (11). As antigen, PTP synthase partially purified from human pituitary gland (12), blotted from SDS-PAGE to PVDF membranes, was used (13).

The splenocytes were stimulated for 5 days with the antigen bound to the PVDF membranes at 37 ‘C and 8% CO2 in a humidified atmosphere. The cells were then fused with the nonsecreting myeloma cell line X63/Ag8.653 at a ratio of 2:l in 1 ml of polyethylene glycol 4000 (Merck, Darmstadt, FRG). Fused cells were resuspended in IMDM containing 10% FCS and distributed into seven 96-well tissue culture plates containing peritoneal macrophages. The medium was changed 24 hours later to IMDM- HAT (IMDM complete supplemented with 2%, vol/vol, of 50 x hypoxanthinei aminopterin/thymidine, from Sigma). Seven days after the fusion the supernatants of hybridoma containing wells were screened for specific antibody production by ELISA. Plates were coated with 1 ug/well of partially purified human or 0.3 ug/well of homogeneous salmon liver PTP synthase at 4 ‘C overnight. Hybridomas giving positive results in the first ELISA screening were rescreened in ELISA in duplicate against each antigen and against uncoated wells. Six hybridomas were selected for further subcloning by limited dilution. The lg class and subclass of the monoclonal antibodies were determined using subisotype specific rabbit anti-mouse immuno-globulines (Mouse Typer subisotyping kit, Bio-Rad, Richmond, USA).

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Staining of routine blood smears was carried out using anti-PTP synthase monoclonal antibodies and the alkaline phosphatase/monoclonal anti-alkaline phosphatase labeling technique (14).

Skin fibroblasts were cultured in Nunc SlideFlasks for 4 days. The adherent cells were washed twice with Hanks’Bal. Salt and then air dryed overnight. The slides were fixed for 1 minute in aceton/methanol 1 :l and then processed as for blood smears.

Briefly, slides were incubated with the hybridoma supernatant as first monoclonal antibody over night at 4 ‘C and additionaly at 37 ‘C for 30 minutes in a humidified chamber. The slides were then developed as described previously (15). As negative control, slides were incubated with IMDM complete instead of hybridoma supernatants.

RESULTS AND DISCUSSION

Human pituitary gland PTP synthase has a native molecular weight of 68 kDa. It

consists of 4 subunits of 17 kDa in SDS-PAGE (12). Since it is very difficult to obtain a

homogeneous preparation of the native enzyme, immunization must be carried out with an enzyme preparation obtained from SDS-PAGE. Immunization of mice with the

17 kDa band excised from SDS gel was not successful, as only antibodies reactive with

the denatured enzyme were obtained (9).

PTP synthase is present in all mammals and even in lower organisms. Because of

the low amounts and the weak immunogenicity of PTP synthase, we used an in vitro

immunization system for the production of monoclonal antibodies. This system requires

presentation of the antigen (PTP synthase) in a form compatible with the cell culture

conditions, preserving simmultaneously the antigen immunogenicity. Since partial or

even complete reconstitution of the native structure of antigenic determinants in blotted

proteins is strongly suggested (16), we decided to immunize with blotted PTP synthase.

For this purpose the enzyme fraction after FPLC on Mono Q was blotted to

lmmobilon-P PVDF membrane. Several 17 kDa bands, representing a total amount of about 8 ug of antigen, were cut out of the membrane and directly used for the in vitro

immunization (13).

After 5 days of in vitro immunization with the antigen bound to the PVDF membrane, the fusion and HAT-selection of the hybridomas were performed essentially as

described by Schoedon et al. (17). Initial screening by ELISA against the human PTP

synthase resulted in 125 positive hybridomas. From the clones secreting antibodies that showed the strongest reactivity in ELISA 56 were transferred to 24-well plates. Of these hybridomas 39 were rescreened in ELISA against human pituitary gland and salmon

liver PTP synthase.

In ELISA analysis 21 hybridomas tested showed strong reactivity with the homogeneous PTP synthase from salmon liver. All hybridomas tested against the human pituitary gland enzyme showed strong reactivity in ELISA. There was no

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unspecific reaction of the antibodies with the assay plate (no antigen coated), nor with

the blocking agent BSA.

One of the clones tested, clone 7C6, reacts only with the human enzyme, therefore

we suggest that this clone might detect an antigenic determinant that is not present in

salmon liver PTP synthase. Comparison of the results of amino acid composition

analysis (12) from salmon liver PTP synthase and enzyme derived from human pituitary

gland indicates that the respective sequences are probably very similar. Relative amino

acid contents were identical for all residues except for 3 amino acids. Thus, clone 7C6 might be the most specific one for assaying the human PTP synthase enzyme.

The class and subclass of 6 subcloned antibodies were found to be IgM, with the

exception of two antibodies, Si and 4C6, which are IgGs.

To prove the usefulness of our antibodies for PTP synthase detection in human

blood cells, two pairs of antibodies, 7C6/1G6 and 1 F5/4C8, were selected for

development of a capture-antibody double sandwich ELISA system. In a first attempt,

checkerboard ELISA using erythrocyte lysate as sample and all possible combinations

of capture/detection antibody pairs revealed very sensitive detection of PTP synthase.

The most sensitive detection was achieved with clone 7C6 as capture antibody and an

antigen dilution of 1:3125. Even with higher antigen dilution and lower amounts of

coated capture antibody, detection of PTP synthase in erythrocytes is possible.

Thus, we intend to use the two pairs of antibodies mentioned above for the

development of a capture ELISA for selective screening of PTP synthase deficiency.

The antibody 7C6 might also be valuable for screening of a cDNA library.

The reactivity of the antibodies with human PTP synthase was further investigated

by immunocytochemistry using routine blood smears and skin fibroblasts with the

alkaline phosphatase/ anti-alkaline phosphatase staining technique. A panel of 5

monoclonal antibodies (Sl , lG6, 4C3, 7C6 and 7F8) that were all cross-reactive with

the salmon liver PTP synthase except for clone 7C6, was tested on blood smears fixed

with acetone/methanol (1:l) for 1 minute. A pan-T cell monoclonal antibody (positive

control, Fig. 1C) and myeloma supernatant (negative control, Fig. 1D) served as method

controls and for cell typing. After staining, morphological details were clearly visible and

this enabled us to determine the cell types labeled for PTP synthase and to localize it in

the cells.

As expected, there was staining for PTP synthase in the erythrocytes, but

surprisingly, the staining intensity was very heterogeneous within the erythrocyte population of the same smear (Fig. 1A). Reticulocytes were clearly and intensively

labeled for PTP synthase (data not shown), while most of the erythrocytes showed only weak labeling and some were even unstained. This might explain the earlier finding of Shintaku and Niederwieser (7) who fractionated red blood cells according to their age

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lmmunocytochemical detection of PTP synthase in blood smears. Fig. 1. Labeling with PTP synthase-specific mAb 7F8 in (A) erythrocytes and T-lymphocytes, and (6) granulocytes. (C) Positive control (commercial monoclonal antibody against Tll marker for T-lymphocytes), (D) negative control (IMDM medium and counter staining with hematoxylin). Final magnification x220

lmmunocytochemical detection of PTP synthase in human skin fibroblasts. Fig. 2. Labeling with PTP synthase-specific mAb 7F8 in (A) fibroblasts and (6) negative control (IMDM medium and counter staining with hematoxylin). Final magnification x70

and found the highest PTP synthase activity in the fraction enriched with young cells

, and reticulocytes. They concluded that fully active enzyme might be present only in

these cells and in erythroblasts. Their proposal is strongly supported by our

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immunocytochemical data and we also conclude that old erythrocytes no longer contain

enzymatically or immunologically detectable PTP synthase.

Our finding is also in agreement with the fact that fetal blood cells contain by far the

highest PTP synthase activity, since fetal blood consists mainly of young

erythrocytes (7).

Two other types of cells presented an intense and clear cytoplasmatic staining for PTP synthase, that is, T-lymphocytes and granulocytes (Figs. 1A and 1B). The labeling

in T cells confirms the finding of PTP synthase activity in those cells (18), while the

presence of PTP synthase in granulocytes is demonstrated for the first time. Therefore, there is strong evidence that granulocytes, which contain GTP cyclohydrolase I (17),

the first enzyme on the BH4 biosynthesis pathway, produce BH4.

Since PTP synthase activity was reported in human skin fibroblasts (19, and own

data) we decided to localize the enzyme in those cells by the use of our monoclonal

antibodies. As shown in Fig. 2Athe cytosol of the skin fibroblasts is uniformly stained

for PTP synthase. We also observed a very intensive staining of the cytoplasm of

dividing fibroblasts. This intensive stain might be due to the concentration of the cytosol or to a higher expression of PTP synthase. As for the blood cells, using IMDM complete

as negative control in the fibroblast, no unspecific staining was detectable (Fig. 26). This result is contrary to the previous reported lack of PTP synthase activity in

fibroblasts (7) and enables the use of patients’ fibroblasts and probably amniocytes for

genetic analysis of synthase deficiency.

The monoclonal anti-PTP synthase antibodies provide a powerful tool for further studies of PTP synthase on the protein level. Thus, the variant clinical forms of PTP

synthase deficiency can be classified according to the immunoreactivity of the enzyme.

They are useful also for the development of an ELISA test for selective screening of PTP synthase deficiency.

ACKNOWLEDGMENTS: Cultered fibroblasts were kindly supplied by Prof. B. Steinmann (Metabolic Unit, Dept. of Pediatrics., University of Zurich) and routine human blood smears by Mrs. V. Buob (Laboratory for Clinical Hematology, Dept. of Pediatrics., University of Zurich). We are grateful to Mrs. M. Killen for help in preparing the manuscript. This work was supported by the Swiss National Science Foundation, projects No. 31-26609.89 and 31-28797.90.

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