Analysis of glyoxalase-I from normal and tumor tissue

Biochimica et Biophysica Acta, 1182(1993) 311-316

311

© 1993 Elsevier Science Publishers B.V. All rights reserved 0925-4439/93/$06.00

BBADIS 61308

Analysis of glyoxalase-I from normal and tumor tissue from human colon Sulabha Ranganathan

and Kenneth

D. Tew

Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, PA (USA)

(Received 9 September 1992) (Revised manuscript received 14 May 1993)

Key words: GlyoxalaseI; Glutathione-S-transferase;Carcinoma;Tumor marker; Colon; (Human) Glyoxalase-I (Gly-I) is part of the glyoxalase system which converts methylglyoxal to D-lactic acid via an S-D-lactoylglutathione intermediate. This glutathione (GSH)-binding protein was purified from human colon tumors and corresponding normal tissue. The GSH-affinity purified fraction from normal human colon tissue showed enzyme activity of 30.6 + 11.5 /xmol/min per mg protein, with methylglyoxal as substrate. Corresponding fractions from carcinomas showed significantly elevated Gly-I activity of 54.5 _+ 15 p~mol/min per mg protein. Polyclonal antibodies made against human Gly-I cross-reacted weakly with mouse liver Gly-I but not with yeast Gly-I. Isoelectric points of Gly-I from human, mouse and yeast were determined to be 4.6, 4.9 and 7.0, respectively, by horizontal IEF. Immunohistochemical analysis confirmed the increase of Gly-! in human colon carcinoma in 16 out of 21 samples when compared to corresponding normal tissue. The elevated levels of Gly-I in colon tumors may be an indicator of the enhanced proliferative status of the neoplastic condition.

Introduction

The glyoxalase system, which consists of two enzymes, glyoxalase-I (Gly-I) and glyoxalase-II (Gly-II) plus reduced glutathione (GSH) catalyses the detoxification of methylglyoxal [1]. Gly-I, using GSH as a cofactor, converts methylglyoxal to S-D-lactoylglutathione. Gly-II catalyses the hydrolysis of S-Dlactoylglutathione to D-lactic acid and GSH [1-3]. The glyoxalase system is present in the cytosolic fraction of cells and has been identified in several organisms including many plants, bacteria, yeast, mouse, rat, pig, cow and humans [2]. The glyoxalase system is reported to be expressed throughout embryogenesis in humans [4]. Increased levels of Gly-I were reported during liver regeneration after partial hepatectomy in rats and mice [5,6]. Gly-I appears to have higher activity in immature proliferating tissues and low activity in mature, differentiated tissues [7]. A previous study utilizing tumor tissues demonstrated decreased levels of Gly-I when compared to the autopsied normal tissue [8]. Gly-II was absent from several tumor tissues [9]. Ayoub et al. [10] demonstrated in-

Correspondence to: S. Ranganathan, Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

crease in the Gly-I activity and decrease in Gly-II activity in several tumor cell lines when compared to non-malignant quiescent cells. Also, studies on cell lines demonstrated a decrease in Gly-I and increase in Gly-II as a result of drug-induced differentiation and activation [11,12]. Preliminary studies of glutathione S-transferases of human colon in our laboratory suggested an increase in accompanied levels of Gly-I in tumor tissue when compared to normal mucosa [13]. In order to investigate the significance of this increase in colon tumors, polyclonal antibodies to Gly-I have been prepared and Gly-I from matched pair samples of tumor and normal tissue analyzed by immunohistochemical and biochemical methods. Materials and M e t h o d s

Isolation o f glyoxalase-I

Normal and tumor tissue specimens from human colon were obtained from patients after surgery at the Fox Chase Cancer Center and stored at - 8 0 ° C until use. To isolate the enzyme, tissue was homogenized in 10 mM Tris-HC1 (pH 7.8) and centrifuged at 10 000 × g for 20 min. The supernatant was further centrifuged at 100000 X g for 1 h. The resulting supernatant was dialysed and concentrated overnight [13], and applied to a column of S-hexylglutathione coupled to epoxyactivated agarose (Sigma, St. Louis, MO). Enzyme was

312 TABLE l Purification of human colon glyoxalase-I * Fraction Normal: Tissue homogenate Supernatant after 10000 x g spin Supernatant after 100000 x g spin S-hexylglutathione column fraction, dialyzed and concentrated Tumor: Tissue homogenate Supernatant after 10000 x g spin Supernatant after 100000x g spin S-hexylglutathione column fraction, dialyzed and concentrated

Volume (ml)

Total protein (mg)

Total activity (p~mol/min)

10 12 11

243.0 146.4 130.9

136.1 121.5 112.6

0.323

10 12 11 0.348

0.103

0.56 0.83 0.86

2.2

307 182.4 190.3

Specific activity ( / z m o l / m i n per mg)

20.9

230.3 189.4 192.0

0.117

0.75 1.04 1.01

6.7

57.6

* Purification steps shown here are for tissue samples for patient No. 10.

eluted from the column with 5 mM S-hexylglutathione as previously described [14]. The concentration of protein was estimated by the Bradford method [15]. Mouse liver Gly-1 was purified similarly. Yeast Gly-I was purchased from Sigma (St. Louis, MO).

Analysis and purification by SDS-PAGE SDS-PAGE was performed according to the method of Laemmli [16], using 12% acrylamide. Gels were silver-stained by the method of Wray et al. [17] to visualize the proteins. In order to purify the Gly-I from the gel, 12% acrylamide gels were run as above. Nanogram quantities of protein were transferred onto polyvinylidene (PVDF) membranes for 5 rain by capillary action. PVDF membranes were stained with aurodye forte reagent from Amersham International plc (Amersham, UK). Stain was further enhanced with Intense BL silver stain from Amersham. Stained membranes were aligned with the unstained gel, using pre-stained molecular weight markers on the gel. Areas containing Gly-I were cut and protein was isolated from the gel by the following method [18]. Briefly, gel bands were cut into 3-5 mm pieces and soaked in 1 mM DTT for 15 min. Then, the gel pieces were crushed in elution buffer with a composition of 0.1% SDS, 0.05 M Tris, 0.1 mM EDTA, 5 mM DTT, 0.2 M NaCI (pH 7.9) and left at room temperature on a rocker for at least 2 h to elute. Crushed gel pieces were pelleted and supernatant containing Gly-I was dialyzed extensively against 10 mM Tris-HC1 (pH 7.8) and concentrated in a Centricon (2 mi) concentrator (Amicon, Danvers, MA).

Antibody production Female New Zealand white rabbits were injected with approx. 50/.~g purified Gly-I protein (as described above) using standard procedures and boosted twice

with protein before isolating antisera using routine methods.

Immunoblot analysis 12% polyacrylamide SDS-PAGE was performed as described above. Proteins were transferred onto nitrocellulose membranes electrophoretically [19]. Membranes were blocked with TBS-containing BSA, stained with Gly-I primary antibody (1 : 500), peroxidase conjugated secondary antibody and developed with H20 2 and 4-chloro-l-naphthol. Stained gels and Western blots were scanned by laser densitometer.

Enzyme assay Glyoxalase-I activity was measured by a modified method of Racker [1] according to Oray and Norton [20]. The assay was performed in 200 mM imidazole HC1 (pH 7.0); 16 mM MgSO4 buffer. Final concentrations of methylglyoxal and glutathione were 7.90 mM T A B L E II Glyoxalase-I enzyme activities in patients * Patient No.

1 2 3 4 5 6 7 8 9 10

Normal tissue

T u m o r tissue

Crude lysate

GSH-affinity purified fraction

Crude lysate

GSH-affinity purified fraction

0.86 1.60 0.47 0.42 1.58 0.30 0.28 0.73 0.51 0.56

30.01 49.60 23.6 17.22 47.92 21.70 34.56 32.25 28.56 20.9

1.74 2.55 0.74 0.69 2.34 0.42 0.35 1.02 0.67 0.75

65.52 63.14 45.9 24.84 85.52 39.81 58.90 47.94 66.18 57.6

* Glyoxalase-I activity was measured by using methylglyoxal as a substrate. Activities are in / x m o l / m i n per mg protein.

313

kD

1

2

3

4

5 ®

95

Isoelectric focusing The isoelectric points of Gly-I proteins were determined by horizontal IEF gels. Affinity purified samples from human colon and mouse liver, along with yeast Gly-I (Sigma, St. Louis, MO) were separated on a 1% agarose gel containing pH 3-10 ampholines and Coomassie stained. Immunohistochemical analysis Normal and tumor tissues were fixed overnight at 4°C in 90% ethanol:10% formalin solution. Speciments were processed, embedded in paraffin and 5 /x thick sections were cut. Sections were stained with glyoxalase-I antibodies (1 : 50 dilution) by methodology described previously [21].

55 43

Results

36 Purification of Gly-I from normal and tumor tissue is shown in Table I. This table shows that no major differences in recoveries were found for enzymes in

29

pl 9.3 8.6 m

1

2

3

4

5

®

6.8 5.9

Fig. 1 (A) Silver stain of SDS-PAGE gel showing Gly-I from GSH affinity-purified samples. Lanes: 1. weight markers; 2, yeast; 3, mouse liver; 4, human colon tumor; 5, normal colon. Affinity-purified samples were boiled for 5 min and separated on a 12% polyacrylamide gel. Arrows indicate glyoxalase-I in each lane. Other bands in the gels represent other GSH-binding proteins including glutathione S-transferases. (B) lmmunostain of corresponding blot of the above gel with Gly-I antibodies (1:500). Lanes: indicated as above. Affinity-purified proteins were separated on a gel as above and transferred onto nitrocellulose membrane prior to immunostaining. The antibody recognizes mouse liver and human colon glyoxalases and lacks cross reactivity with yeast glyoxalase-l.

and 1 mM, respectively, per reaction. Increase in absorbance at 240 nm due to the formation of S-Dlactoylglutathione was measured with different fractions collected during purification. One unit of glyoxalase-I catalyzes the formation of 1 /~mol of S-Dlactoylglutathione per min.

5.1 4.6

3.6

Fig. 2. Coomassie stain of horizontal isoelectric focusing gel to determine the p I values of Gly-I. Lanes: 1, IEF markers; 2, human colon; 3, mouse liver; 4, yeast. Affinity-purified proteins were separated on an agarose gel with the pH ranging from 3 to 10. Arrows indicate glyoxalase-I. Other glutathione-binding proteins can also be identified based on their p I values, e.g., human GST pi-4.8, mouse liver GST p I approx. 8.0. Yeast Gly-I pI was previously reported to be 7.0 [31].

314 both tissues. Table II shows the enzyme activities for normal and tumor samples in crude lysates and affinity purified fractions. These data show that both in crude lysates and purified fractions, tumor tissue from each patient showed elevated levels of enzyme activity when compared to corresponding normal tissue. This table also shows the interindividual variations among patients. Affinity-purified glutathione-binding proteins were SDS-PAGE separated and silver-stained (Fig. 1A). The Gly-I enzyme in these gels appeared to be 2 - 4 times more prevalent in tumor in comparison with normal colon tissue. Western blots stained with the Gly-I antibody confirmed this observation (Fig. 1B). Antibodies against colon Gly-I had no cross-reactivity

with yeast Gly-I and only weak reactivity with mouse liver Gly-I. From the gels, the molecular weight of the colon Gly-I was approx. 21 kDa as a monomer compared to mouse liver Gly-I which was 21.5 kDa. Gly-I exists as a dimer in mammals whereas yeast Gly-I exists as an approximate 32 kDa monomer. From the IEF gels, the isoelectric points of Gly-I from human colon, mouse liver and yeast were determined to be 4.6, 4.9 and 7.0, respectively (Fig. 2). Figs. 3A and B show the enzyme distribution in different colon cells. Tumors sections showed a more intense staining of the enzyme when compared to normal tissue. Most of the stain was located in the epithelial cells lining the intestinal glands and lumen.

Fig. 3. Sections of normal human colon tissue (A) and colon carcinoma (B) stained with glyoxalase I antibodies. Increase in stain (DAB) intensity can be seen from normal to tumor tissue ( x 125). Most of the stain is localized in epithelial cells. L, lumen; LP, lamina propria; G, goblet cell; IG, intestinal gland; E, epithelium.

315 These immunohistochemical analyses showed an elevation of Gly-I stain in 76% (16/21) of tumors compared to pair-matched normal tissues. The other five samples showed either no change [2] or a decrease [3] in stain intensity. Interestingly, these five samples were all from the sigmoid colon.

The elevation in both enzyme activity and protein (judged both by Western blot and immunohistology) plus transcript levels [30] would be consistent with the increased proliferative status of the tumor and may suggest a plausible role for this enzyme as a marker for growth rate a n d / o r neoplasia.

Discussion

Acknowledgements

The fact that Gly-I is found in essentially all organisms indicates an evolutionarily important biological function for this enzyme. Yet, the precise biological importance of Gly-I remains unclear. Increased activity of Gly-I in proliferating tissues such as embryonic [7,22] and regenerating liver [5] suggests a function during cell growth. Increased levels of Gly-I in tumor tissue, which is highly proliferative, could also be cell growth related. The reason for the presence of methylglyoxal in biological systems is presumably related to some aspect of intermediary metabolism. From recent studies, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate appear to be predominant sources of methylglyoxal [23]. It was suggested that methylglyoxal can act as a growth inhibitor of cells [24]; thus, enzyme systems which can detoxify such intermediary metabolites may be crucial to a maintained high prolferative capacity. Previous studies have shown that the growth of ascites tumors in mice, rats and hamsters can be inhibited by methylglyoxal [25]. However, methylglyoxal, which is effective only by peritumoral administration, was shown to cause respiratory and cardiac impairment [26]. It was proposed that the glyoxalase system evolved to protect mammals by detoxifying the methylglyoxal produced by intestinal bacteria [27]. Gly-I also has broad substrate specificity for a-oxoaldehydes [28]. SD-lactoylglutathione, the product of Gly-I catalysis, was shown to potentiate phorbol ester induced secretion of cytoplasmic granules from human neutrophils [3]. It was also shown that the glyoxalase system is modified in patients with diabetes mellitus, where increased blood concentrations of methylglyoxal were found [29]. Therefore, it is apparent that regulation and expression of this enzyme is critical to a number of distinct disease states. A previous study by Jerzykowski [8] used autopsied material as normal tissue to compare the Gly-I levels with tumor tissues. The present study uses pair-matched normal tissue from the same individual and tissue. As can be seen from this study, there is a lot of interindividual variation in Gly-I activity levels. This could be the reason for the other group not being able to detect the increase in enzyme activity. Also, a study comparing non-malignant cells with tumor cell lines has shown a lot of variation in Gly-I activities plus an increase in Gly-I activity in tumor cell lines [10].

We would like to thank the Surgical Oncology Department at the Fox Chase Cancer Center for the tissue specimens and Ms. Donna Bunch for typing the manuscript. This work was supported by a drug resistance grant from Bristol-Squibb Co.

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