Journal of Biochemwal and Biophysical Methods, 22 (1991) 159-165 © 1991 Elsevier Science Publishers B.V. 0165-022X/91/$03.50 ADONIS 0165022X9100057H

159

JBBM00862

The human multicatalytic proteinase: affinity purification using a rnonoclonal antibody Klavs B. Hendil and Wolfgang Uerkvitz August Krogh Instttute, 13 Universitetsparken, 2100 Copenhagen O, Denmark (Received 24 August 1990) (Accepted 25 September 1990)

Summary A monoclonal antibody, coupled to Sepharose CL-4B, was used for the rapid purification of the human muhicatalytic proteinase in a single chromatographic step under mild conditions. The enzyme was homogeneous as judged by nondeuaturing polyacrylamide gel electrophoresis. Electrophoresas under dissociating and reducing conditions revealed at least ten components with molecular masses in the range 22-34 kDa. Affinity-purified enzyme was identical to conventionally purified enzyme with respect to enzymatic properties, molecular mass and subunit composition. Key words: Multicatalytic proteinase; Prosome; Proteasome; Immunoaffinity chromatography

Introduction

A nonlysosomal multicatalytic proteinase (MCP) that seems to be ubiquitous among eukaryotic cells [1,5] has attracted attention because of its complex structure and unusual catalytic properties. The enzyme is a high molecular mass oligomeric protein (740 kDa) which gives rise to a characteristic pattern of at least ten bands (22-34 kDa) on SDS-polyacrylamide gels. The enzyme cleaves peptide bonds on the carboxyl side of basic, hydrophobic, and acidic amino acids and has at least two distinct active sites [2-6].

Correspondence address: W. Uerkvitz, August Krogh Institute, 13 Universitetsparken, 2100 Copenhagen 0, Denmark. Abbreviations: MCP, multicatalytic proteinase; NHMec, 7-amino-4-methylcoumarin; fl-NA, fl-naphthylamine; PBS, phosphate-buffered saline; PEG, poly(ethylene glycol)6000; Suc, succinyl; Z, benzyloxycarbonyl; SDS, sodium dodecyl sulphate.

160

The physiological role of the proteinase in intraceltular proteotysis remains to be elucidated. Evidence has been presented that the proteinase is the catalytic core in a larger protein complex catalysing the ATP-dependent degradation of ubiquitin-protein conjugates [3,7-91. Here we describe a procedure for immunoaffinity purification of the complex enzyme from human placenta under nondenaturing conditions.

Materials and Methods

Materials Materials were obtained from the folIowing sources: Suc-Ala-Ala-Phe-NHMec, Z-Ala-Arg-Arg-NHMec and Z-Leu-Leu-Glu-fiNA (Bachem, Switzerland or Cambridge Research Chemicals); Sepharose CL-4B, Protein A-Sepharose, and heparinSepharose (Pharmacia-LKB); DEAE-cellulose DE-52 (Whatman); hydroxyapatite Bio-Gel H T P (Bio-Rad). The following buffers were used: PBS (137 mM NaC1, 2.7 m M KC1, 4.1 mM N a 2 H P O 4, 0.73 mM K H 2 P Q , p H 7.4); buffer A (154 mM NaC1, 0.1 mM EDTA, 1.5 mM N a H a P O 4, 8.5 mM Na2HPO4, 0.05% Tween 20, pH 7.5). The monoclonal antibodies to human MCP sh'all be described elsewhere (Kaltoft, Koch, Uerkvitz, and Hendil, unpublished data). Monoclonal antibody MCP-21 was purified from peritoneal fluid from hybridoma-bearing mice on protein A-Sepharose according to standard techniques, and coupled to CNBr activated Sepharose CL-4B [101. Assays Assay mixtures (0.2 ml) containing 0.1 m M Suc-Ala-Ala-Phe-NHMec, Z-AlaArg-Arg-NHMec or Z-Leu-Leu-Glu-flNA in 50 mM Tris-HC1 (pH 7.5), 0.1% Brij 35 and enzyme were incubated for 30 rain at 20 o C. The reaction was stopped by adding 0.1 ml of 10% SDS and 1.0 ml of 0.1 M sodium borate (pH 9.1). The N H M e c liberated was measured fluorometrically (excitation 380 nm, emission 460 nm). Liberated fi-NA was diazotised and measured spectrophotometrically at 520 nm [11,12]. The assays were linear with respect to time and concentration of MCP. As used here one unit of enzyme activity is defined as the amount hydrolyzing one nmole substrate per min at 20 o C. Concentrations of proteins were determined from A280 with the following ~1% ~280 values: MCP, 9.61 [13]; IgG, 14 [14]; impure proteins, 10. Extraction of the proteinase All operations were performed at 0 - 4 o C. Enzyme activity was monitored with Suc-Ala-Ala-Phe-NHMec as a substrate. Fresh human placentas were homogenized for 3 × 30 s in a Waring blender with 2 vol. of 5 mM Tris-HC1, p H 7.4, 1 mM EDTA, 5 m M mercaptoethanol and centrifuged at 20000 × g for 45 rain (crude homogenate). A 5-14% (w/v) P E G precipitate was prepared and dissolved in 20 m M Tris-HC1, p H 7.4, 5 mM mercaptoethanol, 1 m M EDTA and 20% glycerol

161 (PEG fraction, 62 mg protein and 2.3 enzyme units per g placenta). It contained 65-75% of the enzyme activity in the homogenate. This extract was clarified by centrifugation (27000 x g for 45 rain) and used either for immunoaffinity or conventional purification of MCP. In the conventional procedure the MCP was purified by sequential chromatography on DEAE-cellulose, Sephacryl S300, hydroxyapatite and heparin-Sepharose CL-6B, essentially as described [15]. The yield was 4-10 gg MCP protein per g placenta.

Results and Discussion

To find conditions for the dissociation of antibody-antigen complexes, six monoclonal antibodies to the proteinase were screened using an enzyme-linked immunosorbent assay. Antibodies MCP-21 and MCP-25 formed complexes with MCP that were relatively easy to dissociate (Fig. 1). Neither urea nor buffers with low pH could be used in a purification step, since they inactivated the enzyme. However, chaotropic salts and, with MCP-21, even NaC1 caused release of the antigen. NaC1 inhibits MCP [2,6,13] but the inhibition was completely reversible (results not shown). We prepared a column of monoclonal antibody MCP-21 immobilized on Sepharose CL-4B (1.6 × 6.7 cm; 2.5 mg l g G / m l gel). The column was equilibrated with 25 mM Tris-HC1, pH 7.5, before it was loaded with PEG fraction (60 ml, 103 mg protein and 3.8 units of MCP per ml, flow rate 48 ml/h). The flow-through fraction exhibited a gradual increase in enzyme activity. This enzyme leakage was independent of the flow rates tested (2-48 ml/h). Loading was stopped when the activity of the flow-through fractions approached that of the applied PEG fraction. The column was washed with 25 mM Tris-HC1 buffers: pH 7.5 (280 ml); pH 8.0 with 50% (v/v) ethylene glycol (60 ml); pH 8.0 with 50 ml NaC1 (60 ml) and, finally, pH 8.0 with 0.2 M NaC1 (180 ml). The proteinase was eluted with 2 M NaC1 in 25 mM Tris-HC1, pH 8.0, and desalted by gelfiltration (PD 10 column, Pharmacia). A typical experiment yielded 10.5 mg of proteinase protein, which seems to be the maximum binding capacity of the column. The recovery of the applied enzyme activity was low (about 20%) under these conditions. However, the recovery could be increased considerably by recycling the active flow-through fractions in a second (or third) chromatographic step with the affinity column. Desalted active fractions containing less than 10 mg of MCP protein could be rerun on the affinity column with recoveries exceeding 90%. The affinity column was used more than 50-times without detectable loss of binding capacity. The crude homogenate (see Materials and Methods) was also a suitable sample for affinity purification, yielding about 10 mg of proteinase per column run and with purity and enzymatic properties being the same as those found with enzyme purified from PEG fractions. However, although a significant increase in total enzyme recovery was obtained, the life-time of the affinity column was reduced.

162 MCP2

(.~ ¢-

c~ c¢

MCP16

100 otl,l,lllll Ill,IIII1 MC~20

MCP21

lOCfllll,lllll c i.,.,..Ill, MCP25

MCP28

100

12345678910

Buffer

,I.IIII 34567

910

number

Fig. 1. Dissociation of multicatalytic proteinase-antibody complexes. NUNC maxisorp microtiter plates were coated with MCP by overnight adsorption with 3/xg MCP/ml PBS foUowed by skim milk (50 g fat-free skim milk powder per 1 PBS, 30 man). After three washes in buffer A, the plates were incubated overnight at 4 ° C with 100 ~1 aliquots of growth media from the hybridoma cultures as indicated and washed again three times in buffer A. Each wetl was then washed once with 200 bd of the dissociation buffer to be tested before it was incubated for 15 rain with fresh dissociation buffer. The plates were then washed three times in buffer A and incubated for 2 h with peroxidase-coupled rabbit antibody to mouse immunoglobulin (Dakopatts, Copenhagen, 1 : 1000 m 1% calf serum in buffer A, 100/~l/well). The plates were washed four times in buffer A before bound peroxidase was detected with o-phenylenediamine/H202 [14]. Colour development was read at 490 nm in a microtiter plate photometer. Dissociation buffers: Control: PBS (100%). 1, 0.1 M glycine, pH 2.8; 2, 3 M urea; 3, 3 M KSCN, pH 7; 4, 0.1 M citrate phosphate, pH 4.0; 5, 50 mM Tris-HC1, pH 6.0, 4 M MgC12; 6, 50 mM Tris-HC1, pH 7.5, 2 M NaI; 7, distilled H20; 8, 50 mM Tris-HCl, pH 7.5, 50% ethylene glycol; 9, 50 mM Tris-HC1, pH 7.5, 2 M NaC1; 10, 50 mM Tris-HC1, pH 8.5, 2 M NaC1.

T h e p u r i f i e d e n z y m e w a s h o m o g e n e o u s as j u d g e d b y P A G E u n d e r n o n d e n a t u r i n g c o n d i t i o n s (Fig. 2C). A b o u t 0.2% o f t h e p r o t e i n was f o u n d at t h e b u f f e r f r o n t , p r o b a b l y as a u t o l y t i c d e g r a d a t i o n p r o d u c t s o f t h e M C P b u t n o o t h e r i m p u r i t i e s c o u l d b e d e t e c t e d b y d e n s i t o m e t r i c s c a n n i n g of gels w i t h 50 /~g p r o t e i n (Fig. 2C). P A G E u n d e r d i s s o c i a t i n g c o n d i t i o n s s h o w e d t h e p r e s e n c e o f at l e a s t t e n c o m p o n e n t s w i t h m o l e c u l a r m a s s e s r a n g i n g f r o m 2 2 - 3 4 k D a (Fig. 2 D ) . S c a n n i n g o f t h e s t a i n e d b a n d s of a f f i n i t y p u r i f i e d a n d c o n v e n t i o n a l l y p u r i f i e d e n z y m e r e v e a l e d s u p e r i m p o s a b l e p r o f i l e s (Fig. 2 A a n d B).

163

A 1 G

D

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67 w

D

g

43"" B

20- - - -

~TD TOP

TD

Fig. 2. Electrophoresis of multicatalytic proteinase. (A) and (B) Densitometric scans (600 nm) of Coomassie blue-stained protein from SDS-PAGE of affinity-purified (A) and conventionally purified (B) enzyme. (C) and (D) PAGE of affinity-purified multicatalytic proteinase in nondenaturing gel (C: 5% acrylamide, pH 8.3 [21]) and SDS-PAGE of the same preparation and of molecular mass markers (D: 12% acrylamide [22]). Fifty /~g of multicatalytic proteinase was applied to each lane. TOP: Origin of separating gel, TD: Tracking dye. Molecular mass markers for lane D (top to bottom): Phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and a-lactaibumin.

In comparing the enzyme preparations, three types of synthetic peptide substrate were chosen, taking into account the catalytic activities referred to as trypsin like (Z-Ala-Arg-Arg-NHMec), chymotrypsin like (Suc-Ala-Ala-Phe-NHMec) and peptidylglutamyl peptide bond (Z-Leu-Leu-Glu-fl-NA) hydrolysing activity [11]. The specific activity measured with Z-Ala-Arg-Arg-NHMec was always about 50% higher for the affinity purified enzyme than for the conventionally purified enzyme, whereas the other two proteolytic activities did not differ (Table 1). The trypsin-like activity resides with an active site that is distinct from the chymotrypsin-like active TABLE 1 Specific activities of affinity and conventionally purified multicatalyttc proteinase (activities and protein were determined as descrtbed in Materials and Methods) Tripeptide substrate

Suc-Ala-Ala-Phe-NHMec Z-Ala-Arg-Arg-NHMec Z-Leu-Leu-Glu-fl-NA

nmol/(min × mg protein) Affinity purified

Conventionally purified

4.0 5.1 1.9

4.3 3.3 2.0

164 site [2,4-6,13,161 so the difference in specific activity b e t w e e n the two types of e n z y m e p r e p a r a t i o n with respect to the t r y p s i n - l i k e activity m a y stem f r o m p a r t i a l i n a c t i v a t i o n of the t r y p s i n - l i k e activity d u r i n g c o n v e n t i o n a l purification. T h e affinity c h r o m a t o g r a p h y d e s c r i b e d here p r o v i d e s a p u r i f i c a t i o n p r o c e d u r e for M C P that is s u p e r i o r to those p r e v i o u s l y used with respect to time a n d yield [2,13,15-201. T h e overatl recovery d e p e n d s o n the choice of extract a p p l i e d to the affinity c o l u n m (crude h o m o g e n a t e or P E G fraction) a n d the n u m b e r of recyclings of the active f l o w - t h r o u g h fractions. Therefore, the overall r e c o v e r y a t t a i n e d is u l t i m a t e l y a m a t t e r of c o s t - b e n e f i t considerations. T h e MCP-21 a n t i b o d y also reacts w i t h M C P from r a b b i t tissues (results n o t shown) a n d m a y therefore p r o v e useful for p u r i f i c a t i o n of M C P f r o m o t h e r species.

Acknowledgments T h e D e p a r t m e n t of Obstetrics a n d G y n e c o l o g y , T h e U n i v e r s i t y H o s p i t a l , C o p e n h a g e n , k i n d l y d o n a t e d raw m a t e r i a l for the e n z y m e purification. Mrs. A n n e M a r i e B. L a u r i d s e n a n d Mrs. K a r e n Dissing p r o v i d e d excellent technical assistance, a n d P r o f e s s o r Peter L e t h J o r g e n s e n gave helpful c o m m e n t s on the m a n u s c r i p t . This s t u d y was s u p p o r t e d b y the C a r l s b e r g F o u n d a t i o n .

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165 11 Wilk, S. and Orlowski, M. (1983) Evidence that pituitary cation-sensitive neutral endopeptldase is a multicatalytic protease complex. J. Neurochem. 40, 842-849. 12 Barrett, A.J. (1976) An improved color reagent for use in Barrett's assay of catliepsin B. Analyt. Biochem. 76, 374-376. 13 Zolfaghari, R., Baker, C.R.F., Amirgholami, A., Canizaro, P.C. and Behal, F.J. (1987) A multicatatytic high-molecular-weight neutral endopeptidase from human kidney. Arch. Biochem. Biophys. 258, 42-50. 14 Hudson, L. and Hay, F.C. (1980) Practical Immunology, 2nd Edn. Blackwell, Oxford. 15 Tanaka, K., Ii, K., Ichihara, A., Waxman, L. and Goldberg, A.L. (1986) A high molecular weight protease in the cytosol of rat liver. J. Biol. Chem. 261, 15197-15203. 16 Dahlmann, B., Kuehn, L., Rutschmann, M. and Reinauer, H. (1985) Purification and characterization of a high-molecular-mass proteinase from rat skeletal muscle. Biochem. J. 228, 161-170. 17 Ray, K. and Harris, H. (1985) Purification of neutral lens endopeptidase: close similarity to a neutral proteinase in pituitary. Proc. Natl. Acad. Sci. USA 82, 7545-7549. 18 McGuire, M.J. and DeMartino, G.N. (1986) Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes. Biochim. Biophys. Acta 873, 279-289. 19 Nojima, M., Ishiura, S., Yamamoto, T., Okuyama, T., Furuya, H. and Sugita, H. (1986) Punhcation and characterization of a high-molecular-weight protease, ingensin, from human placenta. J. Biochem. (Tokyo) 99, 1605-1611. 20 Hough, R., Pratt, G. and Rechsteiner, M. (1987) Purification of two tngh molecular weight proteases from rabbit reticulocyte lysate. J. Biol. Chem. 262, 8303-8313. 21 Davis, B.J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. NY Acad. Sci. 121, 404-427. 22 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.