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

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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 6pyruvoyl 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

aromatic amino acid hydroxylases and a deficiency

of biogenic

(BH$ is required as cofactor by the mammalran

(1). A lack of BH, leads to hyperphenylalaninemia

amine

neurotransmitters.

6-Pyruvoyl

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

tetrahydropterin 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

synthase deficiency

is a heterogeneous

so-called peripheral

defect (7). These patients have enough

meet the BH4 requirement

severe

PTP synthase

(6). PTP

genetic disease. Some patients suffer from a

for neurotransmitter

enough to activate the hepatic phenylalanine is difficult to differentiate

disorders

biosynthesis

hydroxylase

PTP synthase

activity to

in the brain, but not

reaction (8). In some cases it

patients with a partial or peripheral defect from those with a 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 Copyright All rights

$1.50

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

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activity in red blood cells and analysis of neurotransmitter

in CSF enables

differentiation

between

the variants

of PTP synthase

deficiency. For a better understanding on the protein level, antibodies monoclonal

antibodies

of these heterogenic

were thought to be an invaluable

neither of these antibodies

melanogaster

crossreacted

reports the first successful production antibodies

deficiency

tool. Until now only

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

to PTP synthase from Drosophila

monoclonal

forms of PTP synthase

(10) have been available.

However,

with the human enzyme. This communication of mAbs against human

were used for the immunocytochemical

PTP synthase.

These

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 IMDMHAT (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). 811

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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. the low amounts and the weak immunogenicity

Because of

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

conditions,

preserving

simmultaneously

even complete reconstitution

with the cell culture

the antigen immunogenicity.

Since partial or

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 812

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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 salmon liver PTP synthase.

Comparison

that is not present in

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

blood cells, two pairs of antibodies, development checkerboard

of a capture-antibody

for PTP synthase detection

7C6/1G6

and 1 F5/4C8,

in human

were selected

for

double sandwich ELISA system. In a first attempt,

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 development

mentioned

above for the

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 alkaline

phosphatase/

monoclonal

using routine blood smears and skin fibroblasts anti-alkaline

phosphatase

staining technique.

with the

A panel of 5

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

control, Fig. 1C) and myeloma supernatant

antibody (positive

(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 813

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Fig. 1. lmmunocytochemical detection of PTP synthase in blood smears. 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 Fig. 2. lmmunocytochemical detection of PTP synthase in human skin fibroblasts. 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. these

cells

and

They concluded in erythroblasts.

that fully active enzyme might be present only in Their 814

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is strongly

supported

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data and we also conclude that old erythrocytes

synthase

no longer contain

detectable PTP synthase.

Our finding is also in agreement highest

AND

activity,

with the fact that fetal blood cells contain by far the since

fetal

blood

consists

mainly

of young

(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 They are useful also for the development PTP synthase deficiency.

of the enzyme.

of an ELISA test for selective screening of

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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