Clinica Chimica Ada, 191 (1990) 185-200 Elsevier
185
CCA 04818
Comparative biochemical and immunological studies on gamma-glutamyltransferases from human kidney and renal cell carcinoma applying monoclonal antibodies Peter Fischer, Jiirgen E. Scherberich and Wilhelm Schoeppe Center of Internal Medicine, Department of Nephrology, Hospital of the J. W. Goethe- University, Frankfurt (FRG) (Received 15 January 1990; revision received 2 July 1990; accepted 5 July 1990) Key words: gamma-Glutamyltransferase (EC 2.3.2.2); Monoclonal antibody; Renal cell carcinoma; Human kidney; Isoenzyme
Summary We have purified gamma-glutamyltransferases (GGT) from human kidneys and renal cell carcinomas, and fractionated them according to different lectin-binding properties of the isoenzymes. Native polyacrylamide gel electrophoresis and isoelectric focusing revealed different GGT-bands (even after desialylation) not only among kidney and renal carcinoma, but also among Con A-affine tumor fractions separated by ion-exchange chromatography. i%4,of native GGTs were between 106 to 161 kDa, the pl ranged from pH 3 to 4 (pH 5 to 6 after desialylation). Monoclonal antibodies to GGT were produced. One of these, of IgG, class and designed 138Hl1, recognizes human kidney GGT and, in addition, GGT from renal cell carcinomas and liver carcinomas. The specificity of mAb 138Hll for GGT was confirmed by Western blotting, by immunohistochemistry and by immunoprecipitation. The potential usefulness of mAb 138Hll in monitoring renal cancer patients and in identification of renal cancer metastases is currently being studied.
Introduction gamma-Glutamyltransferase (GGT, EC 2.3.2.2) of human kidney is a membrane-bound enzyme [ l] that transfers the gamma-glutamyl moiety of gamma-
Correspondence to: Dr. J.E. Scherberich, Center of Internal Medicine, Dept. of Nephrology, University Hospital, Theodor-Stem-Kai 7, D-6000 Frankfurt 70, FRG. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
186
glutamyl compounds such as glutathione to an acceptor which may be an amino acid or a di- or tripeptide or water (for review see [2] and [3]). The principal functions of GGT may be hydrolysis and metabolism of glutathione [4,5]. GGT is of great clinical interest as a marker for several diseases (rev. in [6,7]), for prenatal diagnosis of congenital abnormalities [8], and as a tumor cell marker [9]. Raised activities or novel forms of GGT in several tumors have been described [lo-171. GGT-positive tumor cells have growth advantages and may be resistant towards cytostatic attack due to detoxification properties of GGT together with glutathione [18-201. Furthermore, GGT is used as a marker for the development of the blood-brain barrier [21-231. Our aim was to further investigate GGT as a potential tumor and cell development marker. In the present paper we compare biochemical and immunological properties of GGT isoenzymes purified from human kidney and renal cell carcinoma (RCC). We describe the production and some attributes of novel monoclonal antibodies (mAb) against these human GGT isoenzymes [24,25]. One of these mAbs, designed 138Hl1, was found not only to react with kidney GGT but also to have high affinity towards GGT from renal cell carcinoma. Materials and methods Reagents
Cacodylic acid, bromelaine, gamma-Glu-MNA, Fast Blue B, and 2,2’azinobis(3-ethyl-benzthiazoline-6-sulfonic acid) .2 NH,-salt (ABTS) were obtained from SERVA (Heidelberg), aquacide I, glycylglycin, and polyethylene glycol (PEG) 4000 (for gas chromatography grade) from Merck (Darmstadt), 3-amino-9-ethylcarbazole (AEC) from Ortho (Neckargemtind), GGT-neu Monotest-a from Boehringer (Mannheim), Aerosil (= diatomaceous earth, SiO,) from Degussa (Frankfurt), Antibody-Multiplier ABM-N/S from Linaris (Bettingen), and Ewing Sarcoma Growthfactor (ESG) from Costar (Badhoevedorp, The Netherlands). Rabbit-antimouse Ig (RaM) was obtained from Nordic (Tilburg), rabbit-anti-mouse Ig HRPconjugated from Dakopatts (Hamburg), and biotin/streptavidin system were obtained from Amersham (Braunschweig). Electrophoresis equipment and column material were obtained from Pharmacia/LKB (Freiburg), cell culture and ELISA plates from NUNC (Wiesbaden). All cellculture media were from Biochrom (Berlin). Hybridoma-medium contained 80% RPMI, 20% medium 199, glycine, gentamicin, and various amounts of fetal calf serum (FCS) or Ultroser Y (Gibco, Eggenstein). Phosphate buffered saline (PBS), pH 7.4, contained 0.35 g NaH?PO, .l H,O, 2.68 g Na,HPO, +12 H,O, and 8.48 g NaCl in 1 liter distilled water. 25J-Protein A was a gift from MPI for Biophysics, Frankfurt. Other reagents were of analytical grade. Isolation of GGT from human kidney and RCC
The isolation procedure was based on the method of Szewczuk and Baranowski [26]. Freshly frozen (-75” C) normal human kidney (that could not be used for transplantation) or RCC were thawed, and homogenized in ice-cold PBS with an omnimixer (SORVALL). The homogenate was mixed with bromeiaine (10 g/400 ml
187
for kidney, 10 g/2 1 for RCC), and incubated on a shaker for 3 h at 37’ C. After centrifugation at 40000 X g (30 rnin, 4” C), the supernatant was filtered. The supernatants from kidney and RCC were then further processed in slightly different ways: Kidney The kidney supematant was mixed with aerosil and centrifuged again (40 000 X g, 30 mm, 4’ C) to remove lipid-containing material. The supernatant was then extracted with a half vol of n-butanol. The filtered water-phase was precipitated with 1.5 vol ice-cold acetone. The precipitate was resuspended in a small vol of PBS, and dialyzed four times against water at 4” C. After cent~fugation (40 000 x g, 30 mm, 4“C), the clear, brown supernatant was placed onto a wheat germ-agglutinin (WGA)-Sepharose column. Bound fractions (WGA+) were eluted with 100 g/l N-acetyl glucosamine [27]. The eluates (and the unbound fractions = WGA-) were further separated through gel filtration (Sephacryl S-200). The fractions cont~~ng GGT-activity were pooled, concentrated in a dialysis-tube coated with aquacide, and fractionated by DEAE ion exchange c~omato~aphy 1261. All purified GGT fractions were concentrated with aquacide, steril filtered, and stored at -2OOC. Renal ceil carcinoma RCC supematant was directly extracted with n-butanol without prior aeros~-treatment and precipitated with acetone as described above. Then the RCC-supematant was fractionated with 52% and 70% ~o~umsulfate at 4’C. The 70%pellet was resuspended and dialyzed against water, and centrifuged at 40000 x g for 30 mm at 4’ C. The resulting RCC-supematant was also fractionated using lectin-affinity chromatography, but with concanavalin A (Con A)-Sepharose. Bound glycoproteins (Con A+) were eluted with 100 g/l alphamethyl-~ucopyranosid f27j. The eluates (and unbound fractions = Con A-) were further separated by ion exchange chromatography 1261. Pooled GGT-containing fractions were further purified by gel filtration (Sephacryl S-200). A portion of the resulting (Con A-affine) RCC-GGT was further purified using FPLC-hydrophobic interactivity chromatography (phenyl superose HR 5/5-column). The sample was applicated in a high-salt buffer (1.5 mol/l (NH4@04 and 20 mm01 Tris/HCl, pH 7.5) and bound RCC-GGT was eluted using a linear gradient with decreasing salt concentration of the buffer, Protein and enzyme assays
Total protein was determined according to the method of Smith et al. using bicin~ho~~~ acid 1281.GGT-activity was determined at 37 OC using a standardized enzyme kit with L-g~a-~ut~yl-3-~arboxy-4-~troa~~d as substrate. Electrophoresis
Electrophoreses were performed in the PhastSystem using precast gels. The handling of the gels, silver staining, and electrophoretic transfer of proteins onto ~tr~ell~ose have been described in detail previously 1291.The following modifications were made: native protein fractions (1 pi/slot) and the highMW marker kit
188
were used for native gradient PAGE (8-25%). For isoelectric focusing, IEF-gels of pH 3-9, without buffer strips, and the IEF-calibration kit pH 3-10 were used according to the producer’s manual. For studies with desialylated GGT, 100 ~1 of each GGT fraction were treated with 400 mU neuraminidase for 16 h at 37 * C. Gels were stained for GGT-activity 30-60 rnin at 37°C in 50 ml cacodylate buffer pH 7.2 containing 10 mg gala-L-glutamyl-4-methoxy-2-naphthyla~d~ 10 mg glycylglycin, and 50 mg Fast Blue B salt. The reaction was stopped by rinsing the gels with water. Production ojpoly- and monoclonal antibodies
Female BALB,/c-mice were primed with 0.5-0.8 ml of purified GGT emulsified in 0.5 ml ABM-S adjuvant, followed by repeated booster injections after 14, 28, 38, and 49 days with 0.2 ml GGT containing ABM-N adjuvant and, finally, three days before cell-fusion without adjuvant. Before splenectomy, the mice were anaesthetised with ether and bled. Somatic cell hybridization was based on methods described earlier [30-321. Mouse X63Ag8.653 myeloma-cells were harvested and suspended (8 min, 90 X g) together with twice the number of spleen-cells. The pellet was carefully mixed with 1.5 ml PEG 4000 (10 g/10 ml hyb~doma-medium, without FCS) and after 1 min stepwise diluted with 1, 3, and 16 ml hybridomamedium. After centrifugation (8 min, 90 X g) the cells were resuspended in hybridoma-medium containing 20% FCS and seeded in nine 96-well cellculture-plates (50 pi/well) containing feeder-cells (mouse peritoneal macrophages). After 24 h and during the following 2 wk, hybridoma cells were fed and selected with medium containing hypoxanthine/ a~opte~ne/ thymidine. Positive hybridomas were cloned by picking and by limiting dilution (in medium supplemented with hypoxanthine/ thymidine and ESG without feeder-cells). ELISA
Growing clones were screened for secreting antibodies in a specific ELISA. 96-nop-plates (TSP-system) were coated with 250 &‘well GGT diluted in sodiumbicarbonate-buffer (1.2 ml/100 ml, pH 9.6) at 4°C overnight. Free binding sites were blocked with BSA 100 g/l in PBS, washed with BSA 1 g/l, Tween 20 0.05% in PBS and the plates then incubated in the hybridoma supematants. After washing, the plates were incubated with 125 ~l/lOO ml rabbit-anti-mouse Ig serum, biotin conjugated, washed again and incubated with 200 ,ul/lOO ml biotin-strepta~dinHRP conjugate. For visualization of the peroxidase reaction, ABTS in acetate buffer, pH 4.6, with 50 $/lo0 ml H,Oz (30%) was used. Absorption was measured in an ELISA-reader at 405 nm. ~ete~~natio~
of Ig s~c~~ses
For determination of the antibody subclasses we used an ELISA similar to the one described above. Instead of GGT, Immunoplates were coated with rabbit-antimouse Ig ,antibodies diluted 1: 2 000. Furthermore, subclass-specific anti-mouse-biotin conjugates were used in the detection system.
189
Western blotting
Electrophoretic protein-transfer was monitored by staining the proteins with amido black [29] or with india ink [33]. For specific staining with antibodies, the NC-strips with the blotted proteins were incubated over night with PBS at 37 o C, and blocked for 2 h with PBS containing 10% FCS and 0.5% Tween 20. The strips were incubated overnight at 4O C with the specific antibodies diluted in ‘blocking buffer’ (mAb 1: 2, pAb 1: 250). The strips were then washed twice in PBS containing 0.5% Tween 20 and twice in ‘blocking buffer’, followed by incubation (1 h 37 o C) in rabbit-anti-mouse IgG/IgMserum diluted 1: 500 with 10% FCS/PBS. After washing, the NC strips were incubated with ‘25J-Protein A (lo6 cpm/lane) and the specifically labelled bands were visualized on a X-ray film. Immunoprecipitation
Purified RCC-GGT was incubated over night together with mAb 138Hl1, pAb against GGT or X63-supernatant (control). The suspension was separated in native PAGE without prior centrifugation. Parallel gels were stained for GGT or with silver. Modified double and triple immunodiffusion assay
1% agarose gels were moulded onto glass plates and 3-mm wells were punched into the gels. GGT and mAb were mixed and incubated over night. In the double-immunodiffusion assay, this complex was run against serial dilutions of anti-mouse Ig serum. In the triple-diffusion assay GGT, mAb and anti-mouse Ig serum were applied simultanously to the gel (20 pi/well). After incubation, the gels were rinsed in water for 2 h, dried and stained for GGT as described above. Immunohistochemistry
Cryosections of various human or animal organs (7-10 pm) were incubated 1 h at 25 o C with 50-100 ~1 hybridoma-supematant or diluted pAb (1: 1000 in PBS). Slices were washed in PBS, incubated with rabbit-anti-mouse HRP-conjugated serum, developed with AEC substrate and counterstained with hematoxyline. Supernatants of X63.Ag8 cells and other mAbs from our laboratory [34] served as controls. Staining for GGT-activity was done in the same manner as with native-gels. Kidney sections were incubated l-10 min, RCC-sections up to 30 min. Results Purification of GGT The results of the purification
steps are summarized in Table I. However, a small portion of RCC-GGT was further purified using analytical-scale FPLC-hydrophobic interactivity chromatography, resulting in total recovery of GGT-activity and separation from remaining impurities (native PAGE, silver-stained, not shown) in one single step. Electrophoresis of different GGT fractions
Significant differences among the relative molecular mass (M,) and the isoelectric points (~1) of the GGT fractions of human kidney and RCC became visible in
190 TABLE
1
Purification of GGT from normal human kidney and from renal cell carcinomas Total protein
Total act
Total vol
Spec act
Yield
(mg)
(U)
(ml)
(U/m&
(%)
Total purif. (-fold)
16,850 4,628 3,250 2,402 984 61 27
8,424 3,493 2,534 2,614 2,399 1,525 1,403
500 605 650 88 125 22 90
0.5 0.75 0.78 1.1 2.4 25 52
100 41 30 31 28 18 17
1 1.5 1.6 2.2 4.8 50 104
DEAE, corm.
11
1,091
14
cont.
120
349
21
62,210 33,490 10,115 8,365 4,655 1,854 316 132
4,161 3,716 3,638 3,638 2,662 2,287 1,086 577
1,730 1,300 350 350 350 33 35 17
1 Normal kidney: Bromeiaine sup. Aerosil treated Butanol extract Acetone precip. Diatysed WGAt, cone. Gel filtration
WGA-,
5 Renal cell carcinomw Bromelaine sup. Butanol extract Acetone precip. Dialysed 52% amm.sulf.sup. 70% amm.sulf.sed. Con A+, wnc. All DEAE, wnc. Gel filtration, cone.
47
45h7
24
Con A- (purified)
36
863
97
a
b
,,,,.
.-.(. .___, “‘_.,__f_I.j_ ) ‘.‘
99 2.9
0.07 0.11 0.36 0.43 0.57 1.2 3.4 4.4 9.7 24
13
198
4
5.8
100 89 87 87 64 55 26 14
1 1.6 5.1 6.1 8.1 17 49 63
11
139
21
343
kDa 669 440
232 140
67
.- ..~ 1
2345
,W.’
67
Fig. 1. Native PAGE of GGT &enzymes with Phast-System. Gels were stained for GGT-activity, the standard (S) was stained with silver. a. Untreated GGT-fractions. b. N~~~~ treated GGT-fractions. Equal numbers in Figs. 1 and 2 belong to equal fractions in all four gels: 1, diluted Con A-affine RCC-GGT, 2, Con A+ RCC-GGT not DEAE binding; 3, Con A+ RCC-GGT early DEAE-fraction; 4. Con A+ RCC-GGT, late DEABfraction; 5, RCC-GGT not Con A-affine; 6, normal kidney GGT binding to WGA; 7, normal kidney GGT, WGA-.
191
b
a
PH 9
5
3 S Fig. 2. Isoelectric focussing (IEF) of gamma-glutamyltransferase (GGT’) isoenzymes in polyacrylamide gels with Phast-System. Gels were stained for GGT-activity, the standard (S) was stained with silver. a. Untreated GGT-fractions. b. Neuraminidase treated GGT. (Fractions see Fig. 1.)
native gradient PAGE and IEF, also after desialylation of GGT with neuraminidase. Details are described in the figure legends (Figs. 1 and 2). An overview of the various M, of native GGTs is given in Fig. 3. Immunisation protocol Immunisation with the new adjuvants ABM-S and ABM-N resulted in polyclonal antibodies (pAb) of high titers. PAb against GGT could be diluted at least 1 : 1000 to 1: 10000 in immunohistochemistry or ELISA.
range
of bands norm.GGT, WGK RCC ConA+.DEAE (weak band)
161 kDa early
RCC GGT ConA-; norm.GGT WGA+ (mean value) 135kDa
RCC RCC
ConA+.DEAE,$;;$;,DEAE
RCC
ConA+.DEAE
(main
Fig. 3. Comparison
band)
of the M, of the GGT-isoenzymes
Imta
early 106kDa
separated
in native PAGE.
192
Cell fusion efficiency
From 750 wells, 158 showed growth of hybridomas which were tested in GGTantigen specific ELISA. Absorbance greater than 1.7 X blank was called positive, and the resulting 5 supematants were further screened by immunohistochemistry. Three clones (138Hl1, 146B4, and 146B6) producing mAbs with strong staining of proximal tubules were selected and subcloned. Subclasses of the mAbs
The subclasses of the antibodies as determined 138Hll and IgM for mAbs 146B4 and 146B6.
by ELISA were IgG, for mAb
Antibody specificity
In contrast to the mAbs 146B4 and 146B6, the specificity of mAb 138Hll GGT could be verified in several tests:
for
Western blotting
Purified fractions of human kidney and RCC-GGT were separated in native and IEF gels and electrophoretically transfered to nitrocellulose (NC). Blotted GGTbands were specifically stained by enzyme activity or by mAb 138Hll (autoradiography) in parallel NC-strips (Fig. 4). In case of kidney GGT, the bands of bound mAb 138Hll corresponded to the enzyme-stained GGT bands in native PAGE and IEF respectively (Fig. 4, lanes 1, 3, 5, 7). Tumor GGT was also detected by mAb 138Hl1, but not aII enzymatic active bands were stained after IEF (Fig. 4, lanes 2, 4, 6, 8).
+ 1
2
3
4
56
78
Fig. 4. Immunoblotting with mAb 138Hll; the bands detected by the antibody in native PAGE (lanes 3 and 4) and in IEF (lanes 7 and 8) corresponded to the bands that were stained for GGT activity (lanes 1 and 2 and lanes 5 and 6, respectively) in a parallel run. Lanes 1,3, 5, and 7 contained kidney GGT, lanes 2,4,6,8 RCC-GGT.
193
=GGT
2345678 1 Fig. 5. Native PAGE stained for GGT-activity. Purified RCC-GGT mixed with mAb 138Hll (lane S), GGT antisera (lanes 3 and 4), control antibodies (lanes 1 and Z), X63-supematant (6), or diluted with PBS (7), and GGT alone (8) were incubated overnight and separated in the gel. Normal migration (cf. Fig. 1) was seen for the controls (1, 2, 6-8), total inactivation or precipitation (at the origin, top) in case of the GGT antisera (3 and 4). In contrast, in case of mAb 138Hll (lane 5), the GGT band migrated slower and was stained at a M, of - 322 kDa belonging to a complex of IgG, and GG’I”(arrow).
Suspensions of RCC-GGT mixed with mAb 138Hl1, pAb or controls (after over night-incubation, without centrifugation) were separated in native PAGE and stained for GGT (Fig. 5). Normal electrophoretic migration of the GGT band was seen for the controls (lanes 1, 2, 6-8), whereas the pAb against GGT absolutely precipitated or inactivated the enzyme (only little staining at the origin of start (top), lanes 3, 4). In contrast, in the case of mAb 138Hll the GGT band was stained at a M, of - 322 kDa belonging to a complex of IgG, and RCC-GGT (lane 5, arrow). Modified double and triple immunodiffusion
Because mAbs usually do not precipitate antigens, we used a modified Ouchterlony assay with a second precipitating antiserum. In the double-effusion assay, the preformed complex of mAb 138Hll and GGT (in the inner well) was run against serial dilutions of the anti-mouse serum, resulting in a ring of precipitated but active GGT (Fig. 6a). In the triple-diffusion assay, GGT, mAb 138Hl1, and anti-mouse Ig-serum were applied to the gel at the same time. A pr~ipitation line of active GGT complexed to antibodies was formed between the antibody-wells (Fig. 6b, c).
194
b
Fig. 6. Modified Ouchterlony assay with double and triple immuno-diffusion in an agarose gel stained for GGT activity. Enzymatic active GGT-mAb-complexes could be precipitated by this method: (a) 1, preincubated complex of normal GGT and mAb 138Hll (see Fig. 5); 2-7, 1: 1 to 1: 32 dilutions of rabbit-anti-mouse Ig (RaM) serum; (b) 8, RCC-GGT; 9, RaM serum; 10, mAb 138Hll; (c) 11, normal GGT; 12, RaM serum diluted 1: 8; 13, mAb 138Hll; 14, RaM serum.
Immunohistochemistry Various cryosections of human kidneys, RCC and other GGT-containing human organs and tumors were stained by mAb 138Hll (Table II). Quantitative differences between mAb 138Hll and the polyclonal anti-GGT antibodies were demonstrated for kidneys: different segments of the proximal tubules were stained with decreasing intensity from Sl to S3 by the mAb (Fig. 7) corresponding to enzymatic GGT-staining. No difference in intensity of staining of Sl to S3 was seen with the polyclonal antibodies (not shown).
195 TABLE II Immunohistochemistry Oman
with mAb 138Hll on human organs No. nos./tested
Intensity
Remarks
14/14 3/3 O/l l/l I/I 3/3 I/I O/I O/l O/5 O/l I/I o/3
+++++
proximal tubules proximal tubules -
++t
epithelium -
14/15 I/l 2/2 2/2 o/12 o/2
++/+++++ +++++ +/+++ ++++
tumor cells tumor cells tumor cells crypts _ -
N#tWd
Kidney Fetal kidney Colon Colon polyp Stomach Liver Testicle Arteria Muscle Squamous epithel. Thyroid gland Thyroid struma Lymphatic ganglion TWi&: Renal cell Ca a Hepatocell. Ca Liver metastases Stomach Ca Squamous ceil Ca Pancreas adeno Ca
-I++-!-/+++ f ++++ ++++ -
some epithelium crypts gall canaliculi spermatides _
-
a Ca. carcinoma
Fig. 7. Normal human kidney. Strong staining with mAb 138Hll of the luminal brushborder membrane of the proximal tubule (PT); glomeruli (Glo) and distal tubules (DT) are negative. Intensity of staining varies between the different segments of FT (X 100, counterstained with hematoxyline).
196
Fig. 8. Renal cell carcinoma. A GGT-typical, trabecular staining profile is observed. Whereas stroma-cells (S) are GGT free, the tumor cells (arrows) express high GGT levels. Notice that the tumor cells are stained on all sides, in contrast to the polarized normal PT-cells in Fig. 7 ( x 100.hematoxyline).
Each but one of the tested RCCs was stained by mAb 138Hll. In well differentiated clear cell RCCs, a typical staining of tumor cells resembling a trabecular structure was achieved with mAb 138Hll (Fig. 8) similar to enzymatic GGT-staining and to the pAbs. Tumor stroma was not stained (Fig. 8). In human liver, the gall canaliculi were stained by 138Hl1, in colon segments the lumina of the crypts, and in fetal kidneys (20th week of gestation) the forming proximal tubules (Table II) were stained. No reaction of mAb 138Hll was seen on rat, porcine or bovine kidney-sections. Strong staining of proximal tubules in kidneys became evident, but only little staining of some tumor cells in RCC was observed with the mAbs 146B4 and 146B6 (not shown). Discussion Purification of
GGT
The method described here for the purification of GGT appears easier and faster to perform than our method described earlier using plasma membrane-fractions isolated from kidney cortex through differential centrifugation. Butanol extraction and acetone precipitation allowed us to handle great sample volumes also in the case of renal tumors containing low GGT. In contrast, precipitation with aerosil for elimination of lipids and lipoproteins appeared unsuitable for this purpose. Whereas aerosil precipitation functioned well in small test volumes (data not shown), using
197
this technique for all of the 500 ml sample resulted in nearly 60% loss of GGT-activity, due to unspecific protein binding capacity of aerosil [35]. WGA was used to further purify kidney GGT due to its affinity for the major portion of renal GGT (WGA also binds RCC-GGT). Because normal kidney does not bind to Con A ( < 2%), this lectin was used to separate a RCC-specific GGT isoform (- 50% binding of total RCC-GGT). However, hydrophobic interactivity chromatography, yet only tested with a small portion of RCC-GGT, will also be helpful for large-scale purification of GGT. Immunisation procedure
Using ABM-N and ABM-S as an adjuvant for challenging mice resulted in serum antibody-titers comparable to that after immunisation with Freund’s adjuvant (unpublished results). However, the liquid ABM solutions were easier to handle and produced no visible side effects, such as abscesses, in the animals. GGT isoforms
Various human GGT isoforms have been found. Whilst kidney GGT gave a single peak in DEAE chromatography, RCC-GGT was eluted in several fractions according to various M, (Fig. la, 3) and multiple isoelectric points (Fig. 2a) as verified in native PAGE and IEF. Differences in 44, were still left after treatment with neuraminidase (Fig. lb), but the pattern of the fractions in IEF appeared to be identical (despite intensity of staining, Fig. 2b). However, normal kidney GGT revealed an exceptional band in the range of pH 5 (Fig. 2b, lane 6). The GGT fractions with various affinity to the lectins Con A and WGA also disclosed various 44, as confirmed by electrophoresis. Whilst Con A- RCC-GGT seems to be identical in M, to normal GGT (Fig. 3), the major Con A+ RCC-GGT fraction (eluted early from DEAE) showed an exclusive M, of 106 kDa, and an extreme p1 of - pH 3. The highest M, of 161 kDa was found for WGA-negative kidney GGT. This GGT-isoform was also demonstrated (as a weak band) for the Con A+ (early DEAE) RCC-GGT fraction. Whereas the appearance of a novel Con A-affine GGT-isoform in RCCs has been described by Hada et al. [15], they did not observe multiple forms of Con A+ GGT in ion-exchange chromatography. However, they described molecular masses of 90 kDa and 130 kDa for normal kidney and RCC-GGT respectively, but they reported faster migration of RCC-GGT in native, nongradient electrophoresis compared to normal GGT, even after neuraminidase treatment. Whilst their latter results correspond to our data, the differences in molecular masses may be explained by the different methods used. The appearance of different elution patterns of tumor and normal GGT-fractions during ion-exchange chromatography have been described by Yamaguchi et al. [36]. In case of normal pancreas GGT, they found two peaks in Mono-Q ion-exchange chromatography compared to a single peak for carcinomatous pancreas GGT. In case of normal kidney GGT they found one single elution peak comparable to our results with DEAE ion-exchange chromatography or Mono-Q chromatography (not shown). The research group did not examine RCC-GGT. Whether these differences among the various observed GGT-isoforms are only
198
due to qualitative as well as quantitative differences in glycosilation, or whether there is another gene of the GGT multigene family in the human genome [37] expressed in RCC (real isoenzyme!) still remains to be answered [38]. Monoclonal
antibodies
to GGT
Recently, the production of monoclonal antibodies to GGT from various organisms has been described. Green et al. [39] have generated mAbs to rat kidney GGT, which, when tested on mesenchymal and epithelial kidney tumors, only reacted with some remnant proximal tubules but failed to bind to tumor cells; fetal kidneys were negative, too. Komatsu et al. [40] have described mAbs to rabbit kidney GGT. These mAbs showed no common epitopes with GGT from human kidney. Yasuda, Shiozawa et al. [41] produced mAbs against rat kidney GGT which cross-reacted with human kidney GGT, and, while this paper was in preparation, a mAb against human kidney GGT was produced [42]. Taniguchi et al. [43] produced an IgM-type mAb against GGT of human hepatocellular carcinoma that cross-reacted with human kidney GGT but not with GGT of normal liver. A recent publication from Donati et al. [44] described several mAbs to human kidney GGT. Nevertheless, so far no mAb capable of recognizing GGT from renal cell carcinomas has been described. In contrast, mAb 138Hl1, as selected here, revealed strong reaction with human RCC and some other carcinomas. No cross-reactivity with other species tested so far has been seen-even porcine kidney showed no reaction-but reaction of rnAb 138Hll with differently glycosylated GGT isoenzymes of all tested human organs known to contain GGT was observed. In contrast to the low specific enzymatic activity of RCC-GGT, an intensive staining of the tumor cells from renal clear cell carcinomas and papillomas was obtained with mAb 138Hll. In well differentiated RCC, a regular, trabecular staining pattern of tumor cell membranes was demonstrated. These results present evidence that the 138Hll epitope is strongly expressed on plasma membranes of RCC. It appears possible that in patients with RCC, GGT of tumor origin is shed into urine or into the blood circulation. Different levels of immunoreactive and enzymatic active GGT forms have been obtained for hepatomas [43] and were demonstrated in a pilot study with a competitive ELISA using mAb 138Hll and urine of patients with RCC (unpublished results). This may allow monitoring patients suffering from RCC before and after removal of the tumor in an ELISA using mAb 138Hll. Since no basal membrane barrier is built by RCC in contrast to tubule epithelia, mAb 138Hll is of potential use for identifying tumor cell metastases settled in regional or peripheral lymphnodes or other organs. Work is now in progress for in vitro binding-studies of mAb 138Hll after tumor nephrectomy, similar as reported previously [45]. Acknowledgements
The authors thank Prof. Dr. D. Baron (Boehringer, Penzberg) and Dr. G. Wolf (presently at the University of Philadelphia) for valuable practical advice, Prof. Dr. Dr. H. Fasold (Dept. of Biochemistry, University of Frankfurt) for his continued
199
discussion and support of this work, Drs. Koepsell, Seibecke, and Ms. Ollig (MPI of Biophysics, Frankfurt) for their support concerning autoradiography of the blots, Dipl.-Biol. Schindler (Pharmacia, Freiburg) for his support with the FPLC, and Ms. M. Haimerl for excellent technical assistance. This work was supported in part by a grant of the Paul and Ursula Klein Foundation, Frankfurt (Main). References 1 Scherberich JE, Gauhl C, Mondorf W. Biochemical, immunological and ultrastructural studies on brush-border membranes of human kidney. Curr Probl Clin Biochem 1978;8:85-95. 2 Tate SS, Meister A. gamma-Glutamyl transpeptidase from kidney. Methods Enzymol 1985;113:400419. 3 Allison RD. gamma-Glutamyl transpeptidase: kinetics and mechanism. Methods Enzymol 1985;113:419-437. 4 Curthoys NP, Hughhey RP. Processing of the propeptide form of rat renal gamma-glutamyl transpeptidase. Enzyme 1979;24:383-403. 5 Hanigan MN, Pitot HC. gamma-Glutamyl transpeptidase its role in hepatocarcinogenesis. Carcinogenesis 1985;6:165-172. gamma-Glutamyl transpeptidase. Adv Clin Chem 1975;53-107. 6 RosalkiSB. Pathobiochemie und differentialdiagnostische WertigI Kottgen E. Die gamma-Glutamyltransferase. keit. Internist 1980;21:231-235. activity in the amniotic fluid 8 Macek M, Annem G, Gustavson KH, et al. gamma-Glutamyltransferase of fetuses with chromosomal aberrations and inborn errors of metabolism. Clin Genet 1987;32:403408. M, Phares W. gamma-Glutamyl transpeptidase: a tumor cell marker with a pharmaco9 Vanderlaan logical function. Histochem J 1981;13:865-877. T, Nakagen M, Hattori N. Purification of gamma-glutamyl 10 Toya D, Sawabu N, Ozaki K, Wakabayashi transpeptidase (g-GTP) from human hepatccellular carcinoma, and comparison of g-GTP with the enzyme from human kidney. Ann NY Acad Sci 1983;417:486-496. in colorectal carcinomas. Clin Chim Acta 11 Huseby N-E, Eide TJ. Variant gamma-ghrtamyltransferases 1983;135:301-307. transpeptidase activity in human breast 12 Bard S, Noel P, Chauvin F, Quash G. gamma-Glutamyl lesions: an unfavourable prognostic sign. Br J Cancer 1986;53:637-642. 0. Diminished lymphocyte and granulocyte gamma-glutamyl 13 Russo SA, Harris MB, Greengard transpeptidase activity in acute lymphocytic leukemia and response to chemotherapy. Am J Hematol 1987;26:67-75. JE, Stefanescu T, Gauhl C, Sayre R, Kleemann B, Mondorf W. Lectin receptors of 14 Scherberich plasma-membranes from human kidney and renal adenocarcinoma. Prot Biol Fluids 1979;27:499-502. K, Yamamoto H, et al. Further investigations on a novel gamma-ghttamyl 15 Hada T, Higashino transpeptidase in human renal carcinoma. Clin Chim Acta 1978;85:267-277. efficiency in discriminating 16 Sacchetti L, Castaldo G, Cimino L, Budillon G, Salvatore F. Diagnostic liver malignancies from cirrhosis by serum gamma-glutamyltransferase isoforms. Clin Chim Acta 1988;177:167-172. B, Freeman J. gamma-Glutamyl transpeptidase activity in human oral squa17 Mock D, Whitestone mous cell carcinoma. Oral Surg 1987,64:197-201. transpeptidase and maintenance 18 Ahmad S, Okine L, Wood R, Aljian J, Vistica DT. gamma-Glutamyl of thiol pools in tumor cells resistant to alkylating agents. J Cell Physiol 1987;131:240-246. hydroly19 Conway JG, Neptun DA, Garvey LK, Popp JA. Carcinogen treatment increases glutathione sis by gamma-glutamyl transpeptidase. Carcinogenesis 1987;8:999-1004. redox cycle in acquired and de novo multidrug 20 Kramer RA, Zakher J, Kim G. Role of the glutathione resistance. Science 1988;241:694-697. 21 Risau W, Hallmann R, Albrecht U. Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier. Dev Biol 1986;117:537-545.
200
22 Mischek U, Meyer J, Galla HJ. Characterization of gamma-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries. Cell Tissue Res 1989;256:221-226. 23 Papandrikopoulou A, Frey A, Gassen HG. Cloning and expression of gamma-glutamyl transpeptidase from isolated porcine brain capillaries. Eur J B&hem 1989;183:693-698. 24 Fischer P, Scherberich JE. Biochemical and immunological differences of gamma-glutamyltransferases (EC 2.3.2.2) from human kidneys and renal cell carcinomas. Cancer Res Clin Oncol 1989;115(Suppl.):S41. 25 Fischer P, Scherberich JE. A monoclonal antibody to gamma-glutamyltransferases of human kidneys and renal tumors. Biol Chem Hoppe-Seyler 1989;370:620. 26 Szewczuk A, Baranowski T. Purification and properties of gamma-glutamyl transpeptidase from beef kidney. Biochem Z 1963;338:317-329. 27 Affinity chromatography. Principles & methods. Uppsala: Pharmacia, 1986. 28 Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76-85. 29 Scherberich JE, Fischer P, Bigalke A, et al. Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and Western blotting of urinary proteins. Electrophoresis 1989;10:58-62. 30 Hess H, Alheid U, Biermann S, et al. Preparation and characterization of monoclonal antibodies against antigens of renal adenocarcinoma. Prot Biol Fluids 1982;29:903-905. 31 Peters JH, Baumgarten H, Schulze M. Monoklonale An&&per. Herstellung und Charakterisierung. Berlin/New York: Springer, 1985. 32 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-497. 33 Gooderham K, Westermeier R. Blotting von Biomolektilen. Freiburg: Pharmacia/LKB, 1988; SD RE-071. 34 Fischer P, Scherberich JE, Albers C, Schoeppe W. Monoclonal antibodies against glycoconjugates of human kidney and renal cancer. Immunobiol 1987;175:287. 35 Maresh GA, Dunbar BS. A simple, inexpensive method for the salt-free concentration of proteins for analysis by two-dimensional gel electrophoresis. Electrophoresis 1988;9:54-57. 36 Yamaguchi N, Kawai K, Ashihara T. Discrimination of gamma-glutamyl transpeptidase from normal and carcinomatous pancreas. Clin Chim Acta 1986;154:133-140. 37 Pawlak A, Lahuna 0, Bulle F, et al. gamma-Glutamyl transpeptidase: a single copy gene in the rat and a multigene family in the human genome. J Biol Chem 1988;263:9913-9916. 38 Tate SS, Khadse V, Wellner D. Renal gamma-glutamyl transpeptidases: structural and immunological studies. Arch Biochem Biophys 1988;262:397-408. 39 Green JA, Cook ND, Manson MM. Monoclonal antibodies against rat kidney gamma-glutamyl transpeptidase show species and tissue specificity. Biochem J 1986;238:913-917. 40 Komatsu T, Yamashita S, Yasuda K. Preparation of monoclonal antibody to rabbit kidney gammaglutamyl transpeptidase and its cross-reactivity analysis in immunohistochemistry. Acta Histochem Cytochem 1987;20:163-176. 41 Yasuda K, Yamashita S, Aiso S, Shiozawa M, Komatsu T. Immunohistochemical study of gammaglutamyl transpeptidase with monoclonal antibodies. I. Preparation and characteristics of monoclonal antibodies to gamma-glutamyl transpeptidase. Acta Histochem Cytochem 1986;19:589-600. 42 Sbiozawa M, Yamashita S, Aiso S, Yasuda K. A monoclonal antibody against human kidney immunochemical, and immunohistochemical chargamma-glutamyl transpeptidase: preparation, acterization. J Histochem Cytochem 1989;37:1053-1061. 43 Taniguchi N, House S, Kuzumaki N, et al. A monoclonal antibody against gamma-glutamyltransferase from human primary hepatoma: Its use in enzyme-linked immunosorbent assay of sera of cancer patients. J Nat1 Cancer Inst 1985;75:841-847. 44 Donati R, Sabolovic N, Wellman M, Artur Y, Siest G. Monoclonal antibodies to human kidney gamma-glutamyltransferase. Clin Chim Acta 1988;174:149-162. 45 Van Dijk J, Oosterwijk E, van Kroonenburgh MJPG, et al. Perfusion of tumor-bearing kidneys as a model for scintigraphic screening of monoclonal antibodies. J Nucl Med 1988;29:1078-1082.
185
CCA 04818
Comparative biochemical and immunological studies on gamma-glutamyltransferases from human kidney and renal cell carcinoma applying monoclonal antibodies Peter Fischer, Jiirgen E. Scherberich and Wilhelm Schoeppe Center of Internal Medicine, Department of Nephrology, Hospital of the J. W. Goethe- University, Frankfurt (FRG) (Received 15 January 1990; revision received 2 July 1990; accepted 5 July 1990) Key words: gamma-Glutamyltransferase (EC 2.3.2.2); Monoclonal antibody; Renal cell carcinoma; Human kidney; Isoenzyme
Summary We have purified gamma-glutamyltransferases (GGT) from human kidneys and renal cell carcinomas, and fractionated them according to different lectin-binding properties of the isoenzymes. Native polyacrylamide gel electrophoresis and isoelectric focusing revealed different GGT-bands (even after desialylation) not only among kidney and renal carcinoma, but also among Con A-affine tumor fractions separated by ion-exchange chromatography. i%4,of native GGTs were between 106 to 161 kDa, the pl ranged from pH 3 to 4 (pH 5 to 6 after desialylation). Monoclonal antibodies to GGT were produced. One of these, of IgG, class and designed 138Hl1, recognizes human kidney GGT and, in addition, GGT from renal cell carcinomas and liver carcinomas. The specificity of mAb 138Hll for GGT was confirmed by Western blotting, by immunohistochemistry and by immunoprecipitation. The potential usefulness of mAb 138Hll in monitoring renal cancer patients and in identification of renal cancer metastases is currently being studied.
Introduction gamma-Glutamyltransferase (GGT, EC 2.3.2.2) of human kidney is a membrane-bound enzyme [ l] that transfers the gamma-glutamyl moiety of gamma-
Correspondence to: Dr. J.E. Scherberich, Center of Internal Medicine, Dept. of Nephrology, University Hospital, Theodor-Stem-Kai 7, D-6000 Frankfurt 70, FRG. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
186
glutamyl compounds such as glutathione to an acceptor which may be an amino acid or a di- or tripeptide or water (for review see [2] and [3]). The principal functions of GGT may be hydrolysis and metabolism of glutathione [4,5]. GGT is of great clinical interest as a marker for several diseases (rev. in [6,7]), for prenatal diagnosis of congenital abnormalities [8], and as a tumor cell marker [9]. Raised activities or novel forms of GGT in several tumors have been described [lo-171. GGT-positive tumor cells have growth advantages and may be resistant towards cytostatic attack due to detoxification properties of GGT together with glutathione [18-201. Furthermore, GGT is used as a marker for the development of the blood-brain barrier [21-231. Our aim was to further investigate GGT as a potential tumor and cell development marker. In the present paper we compare biochemical and immunological properties of GGT isoenzymes purified from human kidney and renal cell carcinoma (RCC). We describe the production and some attributes of novel monoclonal antibodies (mAb) against these human GGT isoenzymes [24,25]. One of these mAbs, designed 138Hl1, was found not only to react with kidney GGT but also to have high affinity towards GGT from renal cell carcinoma. Materials and methods Reagents
Cacodylic acid, bromelaine, gamma-Glu-MNA, Fast Blue B, and 2,2’azinobis(3-ethyl-benzthiazoline-6-sulfonic acid) .2 NH,-salt (ABTS) were obtained from SERVA (Heidelberg), aquacide I, glycylglycin, and polyethylene glycol (PEG) 4000 (for gas chromatography grade) from Merck (Darmstadt), 3-amino-9-ethylcarbazole (AEC) from Ortho (Neckargemtind), GGT-neu Monotest-a from Boehringer (Mannheim), Aerosil (= diatomaceous earth, SiO,) from Degussa (Frankfurt), Antibody-Multiplier ABM-N/S from Linaris (Bettingen), and Ewing Sarcoma Growthfactor (ESG) from Costar (Badhoevedorp, The Netherlands). Rabbit-antimouse Ig (RaM) was obtained from Nordic (Tilburg), rabbit-anti-mouse Ig HRPconjugated from Dakopatts (Hamburg), and biotin/streptavidin system were obtained from Amersham (Braunschweig). Electrophoresis equipment and column material were obtained from Pharmacia/LKB (Freiburg), cell culture and ELISA plates from NUNC (Wiesbaden). All cellculture media were from Biochrom (Berlin). Hybridoma-medium contained 80% RPMI, 20% medium 199, glycine, gentamicin, and various amounts of fetal calf serum (FCS) or Ultroser Y (Gibco, Eggenstein). Phosphate buffered saline (PBS), pH 7.4, contained 0.35 g NaH?PO, .l H,O, 2.68 g Na,HPO, +12 H,O, and 8.48 g NaCl in 1 liter distilled water. 25J-Protein A was a gift from MPI for Biophysics, Frankfurt. Other reagents were of analytical grade. Isolation of GGT from human kidney and RCC
The isolation procedure was based on the method of Szewczuk and Baranowski [26]. Freshly frozen (-75” C) normal human kidney (that could not be used for transplantation) or RCC were thawed, and homogenized in ice-cold PBS with an omnimixer (SORVALL). The homogenate was mixed with bromeiaine (10 g/400 ml
187
for kidney, 10 g/2 1 for RCC), and incubated on a shaker for 3 h at 37’ C. After centrifugation at 40000 X g (30 rnin, 4” C), the supernatant was filtered. The supernatants from kidney and RCC were then further processed in slightly different ways: Kidney The kidney supematant was mixed with aerosil and centrifuged again (40 000 X g, 30 mm, 4’ C) to remove lipid-containing material. The supernatant was then extracted with a half vol of n-butanol. The filtered water-phase was precipitated with 1.5 vol ice-cold acetone. The precipitate was resuspended in a small vol of PBS, and dialyzed four times against water at 4” C. After cent~fugation (40 000 x g, 30 mm, 4“C), the clear, brown supernatant was placed onto a wheat germ-agglutinin (WGA)-Sepharose column. Bound fractions (WGA+) were eluted with 100 g/l N-acetyl glucosamine [27]. The eluates (and the unbound fractions = WGA-) were further separated through gel filtration (Sephacryl S-200). The fractions cont~~ng GGT-activity were pooled, concentrated in a dialysis-tube coated with aquacide, and fractionated by DEAE ion exchange c~omato~aphy 1261. All purified GGT fractions were concentrated with aquacide, steril filtered, and stored at -2OOC. Renal ceil carcinoma RCC supematant was directly extracted with n-butanol without prior aeros~-treatment and precipitated with acetone as described above. Then the RCC-supematant was fractionated with 52% and 70% ~o~umsulfate at 4’C. The 70%pellet was resuspended and dialyzed against water, and centrifuged at 40000 x g for 30 mm at 4’ C. The resulting RCC-supematant was also fractionated using lectin-affinity chromatography, but with concanavalin A (Con A)-Sepharose. Bound glycoproteins (Con A+) were eluted with 100 g/l alphamethyl-~ucopyranosid f27j. The eluates (and unbound fractions = Con A-) were further separated by ion exchange chromatography 1261. Pooled GGT-containing fractions were further purified by gel filtration (Sephacryl S-200). A portion of the resulting (Con A-affine) RCC-GGT was further purified using FPLC-hydrophobic interactivity chromatography (phenyl superose HR 5/5-column). The sample was applicated in a high-salt buffer (1.5 mol/l (NH4@04 and 20 mm01 Tris/HCl, pH 7.5) and bound RCC-GGT was eluted using a linear gradient with decreasing salt concentration of the buffer, Protein and enzyme assays
Total protein was determined according to the method of Smith et al. using bicin~ho~~~ acid 1281.GGT-activity was determined at 37 OC using a standardized enzyme kit with L-g~a-~ut~yl-3-~arboxy-4-~troa~~d as substrate. Electrophoresis
Electrophoreses were performed in the PhastSystem using precast gels. The handling of the gels, silver staining, and electrophoretic transfer of proteins onto ~tr~ell~ose have been described in detail previously 1291.The following modifications were made: native protein fractions (1 pi/slot) and the highMW marker kit
188
were used for native gradient PAGE (8-25%). For isoelectric focusing, IEF-gels of pH 3-9, without buffer strips, and the IEF-calibration kit pH 3-10 were used according to the producer’s manual. For studies with desialylated GGT, 100 ~1 of each GGT fraction were treated with 400 mU neuraminidase for 16 h at 37 * C. Gels were stained for GGT-activity 30-60 rnin at 37°C in 50 ml cacodylate buffer pH 7.2 containing 10 mg gala-L-glutamyl-4-methoxy-2-naphthyla~d~ 10 mg glycylglycin, and 50 mg Fast Blue B salt. The reaction was stopped by rinsing the gels with water. Production ojpoly- and monoclonal antibodies
Female BALB,/c-mice were primed with 0.5-0.8 ml of purified GGT emulsified in 0.5 ml ABM-S adjuvant, followed by repeated booster injections after 14, 28, 38, and 49 days with 0.2 ml GGT containing ABM-N adjuvant and, finally, three days before cell-fusion without adjuvant. Before splenectomy, the mice were anaesthetised with ether and bled. Somatic cell hybridization was based on methods described earlier [30-321. Mouse X63Ag8.653 myeloma-cells were harvested and suspended (8 min, 90 X g) together with twice the number of spleen-cells. The pellet was carefully mixed with 1.5 ml PEG 4000 (10 g/10 ml hyb~doma-medium, without FCS) and after 1 min stepwise diluted with 1, 3, and 16 ml hybridomamedium. After centrifugation (8 min, 90 X g) the cells were resuspended in hybridoma-medium containing 20% FCS and seeded in nine 96-well cellculture-plates (50 pi/well) containing feeder-cells (mouse peritoneal macrophages). After 24 h and during the following 2 wk, hybridoma cells were fed and selected with medium containing hypoxanthine/ a~opte~ne/ thymidine. Positive hybridomas were cloned by picking and by limiting dilution (in medium supplemented with hypoxanthine/ thymidine and ESG without feeder-cells). ELISA
Growing clones were screened for secreting antibodies in a specific ELISA. 96-nop-plates (TSP-system) were coated with 250 &‘well GGT diluted in sodiumbicarbonate-buffer (1.2 ml/100 ml, pH 9.6) at 4°C overnight. Free binding sites were blocked with BSA 100 g/l in PBS, washed with BSA 1 g/l, Tween 20 0.05% in PBS and the plates then incubated in the hybridoma supematants. After washing, the plates were incubated with 125 ~l/lOO ml rabbit-anti-mouse Ig serum, biotin conjugated, washed again and incubated with 200 ,ul/lOO ml biotin-strepta~dinHRP conjugate. For visualization of the peroxidase reaction, ABTS in acetate buffer, pH 4.6, with 50 $/lo0 ml H,Oz (30%) was used. Absorption was measured in an ELISA-reader at 405 nm. ~ete~~natio~
of Ig s~c~~ses
For determination of the antibody subclasses we used an ELISA similar to the one described above. Instead of GGT, Immunoplates were coated with rabbit-antimouse Ig ,antibodies diluted 1: 2 000. Furthermore, subclass-specific anti-mouse-biotin conjugates were used in the detection system.
189
Western blotting
Electrophoretic protein-transfer was monitored by staining the proteins with amido black [29] or with india ink [33]. For specific staining with antibodies, the NC-strips with the blotted proteins were incubated over night with PBS at 37 o C, and blocked for 2 h with PBS containing 10% FCS and 0.5% Tween 20. The strips were incubated overnight at 4O C with the specific antibodies diluted in ‘blocking buffer’ (mAb 1: 2, pAb 1: 250). The strips were then washed twice in PBS containing 0.5% Tween 20 and twice in ‘blocking buffer’, followed by incubation (1 h 37 o C) in rabbit-anti-mouse IgG/IgMserum diluted 1: 500 with 10% FCS/PBS. After washing, the NC strips were incubated with ‘25J-Protein A (lo6 cpm/lane) and the specifically labelled bands were visualized on a X-ray film. Immunoprecipitation
Purified RCC-GGT was incubated over night together with mAb 138Hl1, pAb against GGT or X63-supernatant (control). The suspension was separated in native PAGE without prior centrifugation. Parallel gels were stained for GGT or with silver. Modified double and triple immunodiffusion assay
1% agarose gels were moulded onto glass plates and 3-mm wells were punched into the gels. GGT and mAb were mixed and incubated over night. In the double-immunodiffusion assay, this complex was run against serial dilutions of anti-mouse Ig serum. In the triple-diffusion assay GGT, mAb and anti-mouse Ig serum were applied simultanously to the gel (20 pi/well). After incubation, the gels were rinsed in water for 2 h, dried and stained for GGT as described above. Immunohistochemistry
Cryosections of various human or animal organs (7-10 pm) were incubated 1 h at 25 o C with 50-100 ~1 hybridoma-supematant or diluted pAb (1: 1000 in PBS). Slices were washed in PBS, incubated with rabbit-anti-mouse HRP-conjugated serum, developed with AEC substrate and counterstained with hematoxyline. Supernatants of X63.Ag8 cells and other mAbs from our laboratory [34] served as controls. Staining for GGT-activity was done in the same manner as with native-gels. Kidney sections were incubated l-10 min, RCC-sections up to 30 min. Results Purification of GGT The results of the purification
steps are summarized in Table I. However, a small portion of RCC-GGT was further purified using analytical-scale FPLC-hydrophobic interactivity chromatography, resulting in total recovery of GGT-activity and separation from remaining impurities (native PAGE, silver-stained, not shown) in one single step. Electrophoresis of different GGT fractions
Significant differences among the relative molecular mass (M,) and the isoelectric points (~1) of the GGT fractions of human kidney and RCC became visible in
190 TABLE
1
Purification of GGT from normal human kidney and from renal cell carcinomas Total protein
Total act
Total vol
Spec act
Yield
(mg)
(U)
(ml)
(U/m&
(%)
Total purif. (-fold)
16,850 4,628 3,250 2,402 984 61 27
8,424 3,493 2,534 2,614 2,399 1,525 1,403
500 605 650 88 125 22 90
0.5 0.75 0.78 1.1 2.4 25 52
100 41 30 31 28 18 17
1 1.5 1.6 2.2 4.8 50 104
DEAE, corm.
11
1,091
14
cont.
120
349
21
62,210 33,490 10,115 8,365 4,655 1,854 316 132
4,161 3,716 3,638 3,638 2,662 2,287 1,086 577
1,730 1,300 350 350 350 33 35 17
1 Normal kidney: Bromeiaine sup. Aerosil treated Butanol extract Acetone precip. Diatysed WGAt, cone. Gel filtration
WGA-,
5 Renal cell carcinomw Bromelaine sup. Butanol extract Acetone precip. Dialysed 52% amm.sulf.sup. 70% amm.sulf.sed. Con A+, wnc. All DEAE, wnc. Gel filtration, cone.
47
45h7
24
Con A- (purified)
36
863
97
a
b
,,,,.
.-.(. .___, “‘_.,__f_I.j_ ) ‘.‘
99 2.9
0.07 0.11 0.36 0.43 0.57 1.2 3.4 4.4 9.7 24
13
198
4
5.8
100 89 87 87 64 55 26 14
1 1.6 5.1 6.1 8.1 17 49 63
11
139
21
343
kDa 669 440
232 140
67
.- ..~ 1
2345
,W.’
67
Fig. 1. Native PAGE of GGT &enzymes with Phast-System. Gels were stained for GGT-activity, the standard (S) was stained with silver. a. Untreated GGT-fractions. b. N~~~~ treated GGT-fractions. Equal numbers in Figs. 1 and 2 belong to equal fractions in all four gels: 1, diluted Con A-affine RCC-GGT, 2, Con A+ RCC-GGT not DEAE binding; 3, Con A+ RCC-GGT early DEAE-fraction; 4. Con A+ RCC-GGT, late DEABfraction; 5, RCC-GGT not Con A-affine; 6, normal kidney GGT binding to WGA; 7, normal kidney GGT, WGA-.
191
b
a
PH 9
5
3 S Fig. 2. Isoelectric focussing (IEF) of gamma-glutamyltransferase (GGT’) isoenzymes in polyacrylamide gels with Phast-System. Gels were stained for GGT-activity, the standard (S) was stained with silver. a. Untreated GGT-fractions. b. Neuraminidase treated GGT. (Fractions see Fig. 1.)
native gradient PAGE and IEF, also after desialylation of GGT with neuraminidase. Details are described in the figure legends (Figs. 1 and 2). An overview of the various M, of native GGTs is given in Fig. 3. Immunisation protocol Immunisation with the new adjuvants ABM-S and ABM-N resulted in polyclonal antibodies (pAb) of high titers. PAb against GGT could be diluted at least 1 : 1000 to 1: 10000 in immunohistochemistry or ELISA.
range
of bands norm.GGT, WGK RCC ConA+.DEAE (weak band)
161 kDa early
RCC GGT ConA-; norm.GGT WGA+ (mean value) 135kDa
RCC RCC
ConA+.DEAE,$;;$;,DEAE
RCC
ConA+.DEAE
(main
Fig. 3. Comparison
band)
of the M, of the GGT-isoenzymes
Imta
early 106kDa
separated
in native PAGE.
192
Cell fusion efficiency
From 750 wells, 158 showed growth of hybridomas which were tested in GGTantigen specific ELISA. Absorbance greater than 1.7 X blank was called positive, and the resulting 5 supematants were further screened by immunohistochemistry. Three clones (138Hl1, 146B4, and 146B6) producing mAbs with strong staining of proximal tubules were selected and subcloned. Subclasses of the mAbs
The subclasses of the antibodies as determined 138Hll and IgM for mAbs 146B4 and 146B6.
by ELISA were IgG, for mAb
Antibody specificity
In contrast to the mAbs 146B4 and 146B6, the specificity of mAb 138Hll GGT could be verified in several tests:
for
Western blotting
Purified fractions of human kidney and RCC-GGT were separated in native and IEF gels and electrophoretically transfered to nitrocellulose (NC). Blotted GGTbands were specifically stained by enzyme activity or by mAb 138Hll (autoradiography) in parallel NC-strips (Fig. 4). In case of kidney GGT, the bands of bound mAb 138Hll corresponded to the enzyme-stained GGT bands in native PAGE and IEF respectively (Fig. 4, lanes 1, 3, 5, 7). Tumor GGT was also detected by mAb 138Hl1, but not aII enzymatic active bands were stained after IEF (Fig. 4, lanes 2, 4, 6, 8).
+ 1
2
3
4
56
78
Fig. 4. Immunoblotting with mAb 138Hll; the bands detected by the antibody in native PAGE (lanes 3 and 4) and in IEF (lanes 7 and 8) corresponded to the bands that were stained for GGT activity (lanes 1 and 2 and lanes 5 and 6, respectively) in a parallel run. Lanes 1,3, 5, and 7 contained kidney GGT, lanes 2,4,6,8 RCC-GGT.
193
=GGT
2345678 1 Fig. 5. Native PAGE stained for GGT-activity. Purified RCC-GGT mixed with mAb 138Hll (lane S), GGT antisera (lanes 3 and 4), control antibodies (lanes 1 and Z), X63-supematant (6), or diluted with PBS (7), and GGT alone (8) were incubated overnight and separated in the gel. Normal migration (cf. Fig. 1) was seen for the controls (1, 2, 6-8), total inactivation or precipitation (at the origin, top) in case of the GGT antisera (3 and 4). In contrast, in case of mAb 138Hll (lane 5), the GGT band migrated slower and was stained at a M, of - 322 kDa belonging to a complex of IgG, and GG’I”(arrow).
Suspensions of RCC-GGT mixed with mAb 138Hl1, pAb or controls (after over night-incubation, without centrifugation) were separated in native PAGE and stained for GGT (Fig. 5). Normal electrophoretic migration of the GGT band was seen for the controls (lanes 1, 2, 6-8), whereas the pAb against GGT absolutely precipitated or inactivated the enzyme (only little staining at the origin of start (top), lanes 3, 4). In contrast, in the case of mAb 138Hll the GGT band was stained at a M, of - 322 kDa belonging to a complex of IgG, and RCC-GGT (lane 5, arrow). Modified double and triple immunodiffusion
Because mAbs usually do not precipitate antigens, we used a modified Ouchterlony assay with a second precipitating antiserum. In the double-effusion assay, the preformed complex of mAb 138Hll and GGT (in the inner well) was run against serial dilutions of the anti-mouse serum, resulting in a ring of precipitated but active GGT (Fig. 6a). In the triple-diffusion assay, GGT, mAb 138Hl1, and anti-mouse Ig-serum were applied to the gel at the same time. A pr~ipitation line of active GGT complexed to antibodies was formed between the antibody-wells (Fig. 6b, c).
194
b
Fig. 6. Modified Ouchterlony assay with double and triple immuno-diffusion in an agarose gel stained for GGT activity. Enzymatic active GGT-mAb-complexes could be precipitated by this method: (a) 1, preincubated complex of normal GGT and mAb 138Hll (see Fig. 5); 2-7, 1: 1 to 1: 32 dilutions of rabbit-anti-mouse Ig (RaM) serum; (b) 8, RCC-GGT; 9, RaM serum; 10, mAb 138Hll; (c) 11, normal GGT; 12, RaM serum diluted 1: 8; 13, mAb 138Hll; 14, RaM serum.
Immunohistochemistry Various cryosections of human kidneys, RCC and other GGT-containing human organs and tumors were stained by mAb 138Hll (Table II). Quantitative differences between mAb 138Hll and the polyclonal anti-GGT antibodies were demonstrated for kidneys: different segments of the proximal tubules were stained with decreasing intensity from Sl to S3 by the mAb (Fig. 7) corresponding to enzymatic GGT-staining. No difference in intensity of staining of Sl to S3 was seen with the polyclonal antibodies (not shown).
195 TABLE II Immunohistochemistry Oman
with mAb 138Hll on human organs No. nos./tested
Intensity
Remarks
14/14 3/3 O/l l/l I/I 3/3 I/I O/I O/l O/5 O/l I/I o/3
+++++
proximal tubules proximal tubules -
++t
epithelium -
14/15 I/l 2/2 2/2 o/12 o/2
++/+++++ +++++ +/+++ ++++
tumor cells tumor cells tumor cells crypts _ -
N#tWd
Kidney Fetal kidney Colon Colon polyp Stomach Liver Testicle Arteria Muscle Squamous epithel. Thyroid gland Thyroid struma Lymphatic ganglion TWi&: Renal cell Ca a Hepatocell. Ca Liver metastases Stomach Ca Squamous ceil Ca Pancreas adeno Ca
-I++-!-/+++ f ++++ ++++ -
some epithelium crypts gall canaliculi spermatides _
-
a Ca. carcinoma
Fig. 7. Normal human kidney. Strong staining with mAb 138Hll of the luminal brushborder membrane of the proximal tubule (PT); glomeruli (Glo) and distal tubules (DT) are negative. Intensity of staining varies between the different segments of FT (X 100, counterstained with hematoxyline).
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Fig. 8. Renal cell carcinoma. A GGT-typical, trabecular staining profile is observed. Whereas stroma-cells (S) are GGT free, the tumor cells (arrows) express high GGT levels. Notice that the tumor cells are stained on all sides, in contrast to the polarized normal PT-cells in Fig. 7 ( x 100.hematoxyline).
Each but one of the tested RCCs was stained by mAb 138Hll. In well differentiated clear cell RCCs, a typical staining of tumor cells resembling a trabecular structure was achieved with mAb 138Hll (Fig. 8) similar to enzymatic GGT-staining and to the pAbs. Tumor stroma was not stained (Fig. 8). In human liver, the gall canaliculi were stained by 138Hl1, in colon segments the lumina of the crypts, and in fetal kidneys (20th week of gestation) the forming proximal tubules (Table II) were stained. No reaction of mAb 138Hll was seen on rat, porcine or bovine kidney-sections. Strong staining of proximal tubules in kidneys became evident, but only little staining of some tumor cells in RCC was observed with the mAbs 146B4 and 146B6 (not shown). Discussion Purification of
GGT
The method described here for the purification of GGT appears easier and faster to perform than our method described earlier using plasma membrane-fractions isolated from kidney cortex through differential centrifugation. Butanol extraction and acetone precipitation allowed us to handle great sample volumes also in the case of renal tumors containing low GGT. In contrast, precipitation with aerosil for elimination of lipids and lipoproteins appeared unsuitable for this purpose. Whereas aerosil precipitation functioned well in small test volumes (data not shown), using
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this technique for all of the 500 ml sample resulted in nearly 60% loss of GGT-activity, due to unspecific protein binding capacity of aerosil [35]. WGA was used to further purify kidney GGT due to its affinity for the major portion of renal GGT (WGA also binds RCC-GGT). Because normal kidney does not bind to Con A ( < 2%), this lectin was used to separate a RCC-specific GGT isoform (- 50% binding of total RCC-GGT). However, hydrophobic interactivity chromatography, yet only tested with a small portion of RCC-GGT, will also be helpful for large-scale purification of GGT. Immunisation procedure
Using ABM-N and ABM-S as an adjuvant for challenging mice resulted in serum antibody-titers comparable to that after immunisation with Freund’s adjuvant (unpublished results). However, the liquid ABM solutions were easier to handle and produced no visible side effects, such as abscesses, in the animals. GGT isoforms
Various human GGT isoforms have been found. Whilst kidney GGT gave a single peak in DEAE chromatography, RCC-GGT was eluted in several fractions according to various M, (Fig. la, 3) and multiple isoelectric points (Fig. 2a) as verified in native PAGE and IEF. Differences in 44, were still left after treatment with neuraminidase (Fig. lb), but the pattern of the fractions in IEF appeared to be identical (despite intensity of staining, Fig. 2b). However, normal kidney GGT revealed an exceptional band in the range of pH 5 (Fig. 2b, lane 6). The GGT fractions with various affinity to the lectins Con A and WGA also disclosed various 44, as confirmed by electrophoresis. Whilst Con A- RCC-GGT seems to be identical in M, to normal GGT (Fig. 3), the major Con A+ RCC-GGT fraction (eluted early from DEAE) showed an exclusive M, of 106 kDa, and an extreme p1 of - pH 3. The highest M, of 161 kDa was found for WGA-negative kidney GGT. This GGT-isoform was also demonstrated (as a weak band) for the Con A+ (early DEAE) RCC-GGT fraction. Whereas the appearance of a novel Con A-affine GGT-isoform in RCCs has been described by Hada et al. [15], they did not observe multiple forms of Con A+ GGT in ion-exchange chromatography. However, they described molecular masses of 90 kDa and 130 kDa for normal kidney and RCC-GGT respectively, but they reported faster migration of RCC-GGT in native, nongradient electrophoresis compared to normal GGT, even after neuraminidase treatment. Whilst their latter results correspond to our data, the differences in molecular masses may be explained by the different methods used. The appearance of different elution patterns of tumor and normal GGT-fractions during ion-exchange chromatography have been described by Yamaguchi et al. [36]. In case of normal pancreas GGT, they found two peaks in Mono-Q ion-exchange chromatography compared to a single peak for carcinomatous pancreas GGT. In case of normal kidney GGT they found one single elution peak comparable to our results with DEAE ion-exchange chromatography or Mono-Q chromatography (not shown). The research group did not examine RCC-GGT. Whether these differences among the various observed GGT-isoforms are only
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due to qualitative as well as quantitative differences in glycosilation, or whether there is another gene of the GGT multigene family in the human genome [37] expressed in RCC (real isoenzyme!) still remains to be answered [38]. Monoclonal
antibodies
to GGT
Recently, the production of monoclonal antibodies to GGT from various organisms has been described. Green et al. [39] have generated mAbs to rat kidney GGT, which, when tested on mesenchymal and epithelial kidney tumors, only reacted with some remnant proximal tubules but failed to bind to tumor cells; fetal kidneys were negative, too. Komatsu et al. [40] have described mAbs to rabbit kidney GGT. These mAbs showed no common epitopes with GGT from human kidney. Yasuda, Shiozawa et al. [41] produced mAbs against rat kidney GGT which cross-reacted with human kidney GGT, and, while this paper was in preparation, a mAb against human kidney GGT was produced [42]. Taniguchi et al. [43] produced an IgM-type mAb against GGT of human hepatocellular carcinoma that cross-reacted with human kidney GGT but not with GGT of normal liver. A recent publication from Donati et al. [44] described several mAbs to human kidney GGT. Nevertheless, so far no mAb capable of recognizing GGT from renal cell carcinomas has been described. In contrast, mAb 138Hl1, as selected here, revealed strong reaction with human RCC and some other carcinomas. No cross-reactivity with other species tested so far has been seen-even porcine kidney showed no reaction-but reaction of rnAb 138Hll with differently glycosylated GGT isoenzymes of all tested human organs known to contain GGT was observed. In contrast to the low specific enzymatic activity of RCC-GGT, an intensive staining of the tumor cells from renal clear cell carcinomas and papillomas was obtained with mAb 138Hll. In well differentiated RCC, a regular, trabecular staining pattern of tumor cell membranes was demonstrated. These results present evidence that the 138Hll epitope is strongly expressed on plasma membranes of RCC. It appears possible that in patients with RCC, GGT of tumor origin is shed into urine or into the blood circulation. Different levels of immunoreactive and enzymatic active GGT forms have been obtained for hepatomas [43] and were demonstrated in a pilot study with a competitive ELISA using mAb 138Hll and urine of patients with RCC (unpublished results). This may allow monitoring patients suffering from RCC before and after removal of the tumor in an ELISA using mAb 138Hll. Since no basal membrane barrier is built by RCC in contrast to tubule epithelia, mAb 138Hll is of potential use for identifying tumor cell metastases settled in regional or peripheral lymphnodes or other organs. Work is now in progress for in vitro binding-studies of mAb 138Hll after tumor nephrectomy, similar as reported previously [45]. Acknowledgements
The authors thank Prof. Dr. D. Baron (Boehringer, Penzberg) and Dr. G. Wolf (presently at the University of Philadelphia) for valuable practical advice, Prof. Dr. Dr. H. Fasold (Dept. of Biochemistry, University of Frankfurt) for his continued
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discussion and support of this work, Drs. Koepsell, Seibecke, and Ms. Ollig (MPI of Biophysics, Frankfurt) for their support concerning autoradiography of the blots, Dipl.-Biol. Schindler (Pharmacia, Freiburg) for his support with the FPLC, and Ms. M. Haimerl for excellent technical assistance. This work was supported in part by a grant of the Paul and Ursula Klein Foundation, Frankfurt (Main). References 1 Scherberich JE, Gauhl C, Mondorf W. Biochemical, immunological and ultrastructural studies on brush-border membranes of human kidney. Curr Probl Clin Biochem 1978;8:85-95. 2 Tate SS, Meister A. gamma-Glutamyl transpeptidase from kidney. Methods Enzymol 1985;113:400419. 3 Allison RD. gamma-Glutamyl transpeptidase: kinetics and mechanism. Methods Enzymol 1985;113:419-437. 4 Curthoys NP, Hughhey RP. Processing of the propeptide form of rat renal gamma-glutamyl transpeptidase. Enzyme 1979;24:383-403. 5 Hanigan MN, Pitot HC. gamma-Glutamyl transpeptidase its role in hepatocarcinogenesis. Carcinogenesis 1985;6:165-172. gamma-Glutamyl transpeptidase. Adv Clin Chem 1975;53-107. 6 RosalkiSB. Pathobiochemie und differentialdiagnostische WertigI Kottgen E. Die gamma-Glutamyltransferase. keit. Internist 1980;21:231-235. activity in the amniotic fluid 8 Macek M, Annem G, Gustavson KH, et al. gamma-Glutamyltransferase of fetuses with chromosomal aberrations and inborn errors of metabolism. Clin Genet 1987;32:403408. M, Phares W. gamma-Glutamyl transpeptidase: a tumor cell marker with a pharmaco9 Vanderlaan logical function. Histochem J 1981;13:865-877. T, Nakagen M, Hattori N. Purification of gamma-glutamyl 10 Toya D, Sawabu N, Ozaki K, Wakabayashi transpeptidase (g-GTP) from human hepatccellular carcinoma, and comparison of g-GTP with the enzyme from human kidney. Ann NY Acad Sci 1983;417:486-496. in colorectal carcinomas. Clin Chim Acta 11 Huseby N-E, Eide TJ. Variant gamma-ghrtamyltransferases 1983;135:301-307. transpeptidase activity in human breast 12 Bard S, Noel P, Chauvin F, Quash G. gamma-Glutamyl lesions: an unfavourable prognostic sign. Br J Cancer 1986;53:637-642. 0. Diminished lymphocyte and granulocyte gamma-glutamyl 13 Russo SA, Harris MB, Greengard transpeptidase activity in acute lymphocytic leukemia and response to chemotherapy. Am J Hematol 1987;26:67-75. JE, Stefanescu T, Gauhl C, Sayre R, Kleemann B, Mondorf W. Lectin receptors of 14 Scherberich plasma-membranes from human kidney and renal adenocarcinoma. Prot Biol Fluids 1979;27:499-502. K, Yamamoto H, et al. Further investigations on a novel gamma-ghttamyl 15 Hada T, Higashino transpeptidase in human renal carcinoma. Clin Chim Acta 1978;85:267-277. efficiency in discriminating 16 Sacchetti L, Castaldo G, Cimino L, Budillon G, Salvatore F. Diagnostic liver malignancies from cirrhosis by serum gamma-glutamyltransferase isoforms. Clin Chim Acta 1988;177:167-172. B, Freeman J. gamma-Glutamyl transpeptidase activity in human oral squa17 Mock D, Whitestone mous cell carcinoma. Oral Surg 1987,64:197-201. transpeptidase and maintenance 18 Ahmad S, Okine L, Wood R, Aljian J, Vistica DT. gamma-Glutamyl of thiol pools in tumor cells resistant to alkylating agents. J Cell Physiol 1987;131:240-246. hydroly19 Conway JG, Neptun DA, Garvey LK, Popp JA. Carcinogen treatment increases glutathione sis by gamma-glutamyl transpeptidase. Carcinogenesis 1987;8:999-1004. redox cycle in acquired and de novo multidrug 20 Kramer RA, Zakher J, Kim G. Role of the glutathione resistance. Science 1988;241:694-697. 21 Risau W, Hallmann R, Albrecht U. Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier. Dev Biol 1986;117:537-545.
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