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Isolation by zinc-affinity chromatography of the histidine–proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase
Biochimica et Biophysica Acta 1645 (2003) 81 – 88 www.bba-direct.com
Isolation by zinc-affinity chromatography of the histidine–proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase Evidence that the formation of a protein–protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme Maria Ranieri-Raggi a, Daniela Martini a, Antonietta R.M. Sabbatini a, Arthur J.G. Moir b, Antonio Raggi a,* a
Dipartimento di Scienze dell’Uomo e dell’Ambiente, Chimica e Biochimica Medica, Universita` di Pisa, via Roma 55, 56126 Pisa, Italy b Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2UH, UK Received 17 April 2002; received in revised form 23 October 2002; accepted 11 November 2002
Abstract The histidine – proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn2 +-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn2 + from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95 000 and the N-terminal sequence L-T-P-T-D-XK-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70 000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-TD-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme. D 2002 Elsevier Science B.V. All rights reserved. Keywords: AMP deaminase structure; Histidine – proline-rich glycoprotein; Immobilized metal ion affinity chromatography
1. Introduction Histidine –proline-rich glycoprotein (HPRG) is present at a relatively high concentration in the plasma of vertebrates, where it has been implicated in a number of processes, including blood coagulation and fibrinolysis, immune response and transport of metal ions [1]. Although the
Abbreviations: HPRG, histidine – proline-rich glycoprotein; PGF, peptide-N-glycosidase F * Corresponding author. Tel.: +39-050-561912; fax: +39-050-550241. E-mail address: [email protected] (A. Raggi).
physiological role of this protein remains unclear, it has been suggested that one of its functions may be to bring two or more ligands together [2]. In a previous paper, we reported that denaturation of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) in acidic medium allows the chromatographic separation from the enzyme of a peptide with an amino acid composition significantly different from that derived from the available AMP deaminase cDNAs [3]. N-terminal sequence analysis of the fragments liberated by limited proteolysis revealed a striking similarity of the novel protein to rabbit plasma HPRG [2] although, in comparison with mature HPRG, the AMP deaminase-associated variant probably contains a unique N-terminal extension [3]. In a
1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 7 0 - 9 6 3 9 ( 0 2 ) 0 0 5 2 7 - 7
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recent paper, we demonstrated that an antibody against human plasma HPRG reacts with an AMP deaminase preparation from human skeletal muscle [4]. A selective binding of the anti-HPRG antibody to type IIB fibers was detected, suggesting the preferential association of muscle HPRG to the AMP deaminase isoenzyme contained in the fast-twitch glycolytic fibers. We report here that the specific binding of HPRG to a Zn-affinity column can be used for the one-step isolation of this protein from purified rabbit skeletal muscle AMP deaminase. The separation process induces a marked reduction in solubility of the catalytic subunit of the enzyme, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.
suggested by the manufacturer. The column was charged with 0.1 M ZnCl2 in 0.5 M NaCl/50 mM sodium acetate (pH 4.0) and washed with the same buffer without ZnCl2. After equilibration of the resin with 0.5 M NaCl/20 mM sodium phosphate (pH 7.0), containing 0.1% h-mercaptoethanol and 3 M urea (buffer A), a sample of AMP deaminase denatured by overnight dialysis against buffer A was applied to the column that was washed with buffer A. Following the followthrough peak, the column was eluted with a linear gradient of imidazole (2– 100 mM in 50 ml) in buffer A. After having washed the column with buffer A, a stripping elution was made with 0.5 M NaCl/20 mM sodium phosphate (pH 7.2), containing 0.1% h-mercaptoethanol and 50 mM EDTA. Similar separations were carried out in the absence of urea as described in the Results and Discussion section.
2. Materials and methods
2.4. Deglycosylation
2.1. Reagents
Samples (0.1 ml) containing 25 Ag of the affinity-purified HPRG component of AMP deaminase dialysed against 0.25 M sodium phosphate (pH 8.6) were denatured by heating at 100 jC in the presence of 0.1% SDS and 1% h-mercaptoethanol. After SDS denaturation, 0.4% Triton X-100 was added to avoid inactivation of PGF by SDS. The deglycosylation reaction (45 h at 37 jC) was started with the addition of 5 units of PGF to the incubation mixture. The extent of HPRG glycosylation was estimated by comparing the mobility on SDS/PAGE of treated HPRG with that of the protein incubated in the same mixture but in the absence of PGF.
Peptide-N-glycosidase F (PGF) from Flavobacterium meningosepticum was purchased from Roche Diagnostics (Mannheim, Germany). Chelating Fast Flow Sepharose was from Pharmacia LKB Technology (Uppsala, Sweden). Phosphocellulose resin (P-11) was supplied by Whatman International Ltd. (Maidstone, UK). All chemicals and other reagents used were of analytical grade. 2.2. Enzyme AMP deaminase was prepared as described previously [5] from fresh muscle dissected from the back and hind leg of rabbits. Because the enzyme undergoes progressive fragmentation with storage, homogenization of the muscle and phosphocellulose purification of the enzyme was carried out using at each step a buffer system containing 5 mM NaN3 to reduce the rate of proteolytic processes. This modification did not change the specific activity of the purified enzyme (1100 Amol of AMP deaminated per min/ mg of protein when assayed in 50 mM imidazole/HCl (pH 6.5)/100 mM KCl/2 mM AMP at 20 jC). The AMP deaminase activity was determined spectrophotometrically as previously described in a Shimadzu UV-260 spectrophotometer [6]. Protein concentrations of the various enzyme fractions were determined spectrophotometrically by using A2801%,1 cm values of 9.1 and 8.2, respectively, for AMP deaminase and its isolated HPRG component, which were calculated on the basis of protein determinations by the method described in Ref. [7], using bovine serum albumin as standard. The molecular mass of AMP deaminase was taken as 309 kDa [8].
2.5. SDS/PAGE Electrophoresis in the presence of 0.1% (w/v) SDS was carried out under reducing conditions on 10% or 13.5% (w/v) polyacrylamide slab gels in 0.1 M Tris/0.1 M Bicine (pH 8.3). Protein standards (Sigma) and prestained proteins (Bio-Rad, Richmond, CA) were used to determine molecular weights (MWs). 2.6. Electroblotting and sequence analysis Electroblotting was performed by the method of LeGendre and Matsudaira [9] using 10 mM 3-(cyclohexylamino)-1propane-sulfonic acid (pH 11) containing 10% (v/v) methanol. Transfers were performed for 60 –90 min at 400 mA. N-terminal sequencing was performed using an Applied Biosystems model 476A protein sequencer.
3. Results and discussion
2.3. Immobilized Zn2+-affinity chromatography
3.1. Acidic treatment of AMP deaminase isolates its HPRG component with a MW of 95 000 on SDS/PAGE
A mini-column of 2 ml bed volume was prepared with Chelating Fast Flow Sepharose according to the procedure
We previously demonstrated that limited proteolysis of rabbit skeletal muscle AMP deaminase with trypsin, remov-
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ing the 95-residue N-terminal fragment of the enzyme, converts the native 85-kDa enzyme subunit to an approx. 70-kDa core, that is resistant to further proteolysis and which is present as a minor component in the SDS/PAGE electrophoretogram of the native enzyme [10]. The observed 85- to 70-kDa transition also occurred on storage of AMP deaminase, as well as of the muscle used for the enzyme preparation [11]. When the freshly prepared enzyme was subjected to automated Edman degradation, in addition to low amount of the sequences corresponding to fragments beginning at Phe-4 and Pro-2 of the known catalytic subunit of AMP deaminase, a low yield of the sequence L-T-P-T-DX was obtained, corresponding to the N-terminal sequence of rabbit plasma HPRG [3]. Electroblotting and sequencing of the 70-kDa SDS/PAGE band of the trypsinized enzyme revealed two sequences in equal amounts, one of which corresponded to a fragment starting at Leu-96 of the AMP deaminase subunit, whereas the other (L-T-P-T-D-X-K) was identical with the N-terminal sequence of rabbit plasma HPRG [2]. Further analysis by SDS/PAGE of several fresh preparations of the enzyme has now revealed the presence of a variable amount of a minor component with an apparent molecular mass of 95 kDa in addition to the main 85-kDa protein band and the minor 70-kDa proteolyzed component (Fig. 1, lane b). The addition of 1 Al of 1 M HCl to 0.1 ml of native AMP deaminase (1 mg/ml) caused protein precipita-
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tion. Analysis by SDS/PAGE of the precipitate revealed a strong similarity with the enzyme used as starting material except for the absence of the 95-kDa component (Fig. 1, lane c). By electroblotting and sequencing analysis, the main 85-kDa band of the starting material and of the precipitate yielded no N-terminal sequences, confirming the previous suggestion that the N terminus of rabbit skeletal muscle AMP deaminase is modified [3]. SDS/ PAGE of the supernatant revealed the presence of only one component, which could be identified as the 95-kDa component (Fig. 1, lane d). Electroblotting and sequencing of the 95-kDa band revealed the sequence L-T-P-T-D-X-KT-T-K-P-L corresponding to the N-terminal sequence of rabbit plasma HPRG [2] which is consistent with the identification of the sequence in freshly prepared enzyme as a minor component. It should be noted that rabbit plasma HPRG migrates in SDS/PAGE with an apparent molecular mass of 90 or 94 kDa [2,12,13] higher than that deduced from its sequence (58 kDa) or that calculated taking account of its 17.5% carbohydrate content (70 kDa) [2]. The higher intensity of the isolated 95-kDa band (Fig. 1, lane d) compared to that of the 95-kDa minor component of the whole enzyme (Fig. 1, lane a) suggests that acidic treatment promotes some conformational change in the HPRG component of the enzyme that gives rise to a species with a lower mobility on SDS/PAGE resulting in an 85- to 95-kDa shift of the apparent molecular mass of the protein. 3.2. Specific binding of the HPRG component of AMP deaminase to a Zn2+-charged metal-affinity column
Fig. 1. Effect of acidic treatment on SDS/PAGE of rabbit skeletal muscle AMP deaminase. The addition of 2.5 Al of 1 M HCl to 0.25 ml of AMP deaminase (1 mg/ml) caused protein precipitation. The precipitate was separated by centrifugation and was redissolved in 0.25 ml of 75 mM Tris/ HCl (pH 8.0) containing 15 mM h-mercaptoethanol and 9 M urea. Samples were denatured in SDS and run on 13.5% (w/v) polyacrylamide slab gel. (a) Prestained molecular weight standards; (b) 10 Ag of native AMP deaminase; (c) 10 Al of the solution of redissolved precipitate; (d) 10 Al of the supernatant obtained with the centrifugation of acid-treated AMP deaminase.
Histidine forms stable complexes with transition metals. Taking advantage of this property, a procedure for purifying proteins containing exposed histidine residues has been developed, which is known as immobilized metal ion affinity chromatography [14]. Rabbit plasma HPRG contains 53 histidine residues, of which 34 are located in the histidine –proline-rich domain containing 15 repeats of the sequence (H/P)(H/P)PHG [2]. These histidine residues have been shown to mediate interactions with transition metals [12]. Because the HPRG component of rabbit skeletal muscle AMP deaminase contains 10 mol of histidine residues per 10 000 g of protein [3], we exploited metal affinity chromatography in an attempt to separate the novel component of the enzyme from the catalytic subunit. Fig. 2 shows a chromatographic profile of rabbit skeletal muscle AMP deaminase (2.9 mg) denatured by overnight dialysis against 0.5 M NaCl/20 mM sodium phosphate (pH 7.0), containing 0.1% h-mercaptoethanol and 3 M urea (buffer A), and applied to a metal-affinity column charged with Zn2 + and equilibrated with buffer A. The results show that the 95-kDa component of AMP deaminase was specifically retained on the column while the 85- and 70-kDa components passed through and emerged in the void volume (Fig. 2, peak A) so that analysis by SDS/PAGE of the A280 nm peak fraction revealed a strong similarity with the
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Fig. 2. Zn-affinity chromatographic analysis of rabbit skeletal muscle AMP deaminase. A sample of AMP deaminase (2.9 mg) denatured by overnight dialysis against buffer A was chromatographed on a Zn-affinity column as described in the Materials and Methods section and the A280 nm peak fractions were analyzed by 10% (w/v) SDS/PAGE.
enzyme used as starting material except for the absence of the 95-kDa minor component migrating as the slower band in the electrophoretogram of whole AMP deaminase. This component was retained by the column during the elution with a linear gradient of 2– 100 mM imidazole in buffer A and was only eluted when the column was washed with 0.5 M NaCl/20 mM sodium phosphate (pH 7.2), containing 0.1% h-mercaptoethanol and 50 mM EDTA (buffer B) which strips the metal ions from the gel and causes elution of the bound proteins (Fig. 2, peak B). Electroblotting and sequencing of the 95-kDa peptide isolated by zinc-affinity chromatography revealed the sequence L-T-P-T-D-X-K-T-TK-P-L-A-E-K-A-L-D-L-I, corresponding to the rabbit plasma HPRG N terminus [2]. SDS/PAGE of the starting material and the two pooled peaks is shown in Fig. 3, lanes a – c. In three different experiments using three different AMP deaminase preparations, the recovery of the total material applied to the column in terms of mass was 73 F 8%, 39 F 12% and 36 F 8% being accounted for by peaks A and B, respectively. These data strengthen the hypothesis that the species migrating in SDS/ PAGE as a 95-kDa band actually derives from some modification of the 85-kDa component of the enzyme. The protein that was not adsorbed during application of AMP deaminase to the Zn-charged column precipitated immediately after its elution in the flow-through. In contrast,
the isolated 95-kDa component of the enzyme, that was completely devoid of AMP deaminase activity even after removal by dialysis of the EDTA used for the stripping procedure and the addition of 10 AM ZnCl2 to the assay mixture, which causes an instantaneous and complete reactivation of AMP deaminase inactivated by chelating agents [15,16], was quite stable even after several months storage at 20 jC and retained its solubility after several freezing and thawing procedures. This allowed its prolonged incubation with PGF to examine the extent of its glycosylation on the basis of the effect of the treatment on its mobility on SDS/PAGE. Following 45 h incubation with PGF in the presence of SDS, HPRG was subjected to SDS/PAGE which showed a reduction of the apparent MW from 95 to 70 kDa (Fig. 4). This observation which characterizes the HPRG component of AMP deaminase as a N-glycosylated protein is also in agreement with the results of the carbohydrate structure analysis of rabbit plasma HPRG showing that treatment with PGF reduces the apparent MW of this protein from 93 to 67 kDa [13]. 3.3. Association with the HPRG component is critical for a stable AMP deaminase activity In further experiments, AMP deaminase was loaded on to the Zn-charged column under nondenaturing conditions.
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Fig. 3. SDS/PAGE of the components of AMP deaminase separated by Znaffinity chromatography under denaturing or nondenaturing conditions. The enzyme used as starting material and the pools of the protein peaks resolved by the Zn-affinity chromatographic analysis in denaturing (lanes a – c) or nondenaturing (lanes d – f) conditions, were run on 10% (w/v) polyacrylamide slab gel. (a) Eight micrograms sample of AMP deaminase dialysed against buffer A; (b) 4 Ag sample of AMP deaminase not retained by the Zn-charged column as in Fig. 2; (c) 5 Ag sample of the HPRG component eluted with 50 mM EDTA from the Zn-charged column as in Fig. 2; (d) 8 Ag sample of freshly prepared AMP deaminase applied to the Zn-charged column equilibrated with 1 M KCl, pH 7.0; (e) 6 Ag sample of AMP deaminase not retained by the Zn-charged column as in (d); (f) 4 Ag sample of the protein eluted with 50 mM EDTA from the column as in (d).
When 0.8 mg enzyme in 1 M KCl, pH 7.0, was applied to the column equilibrated with the same medium, the results were apparently similar to those obtained with the denatured enzyme because 45% of the protein was not retained by the column and started to precipitate immediately after its collection in the flow-through, whereas 40% of the protein was accounted for by the eluate obtained by washing the column with buffer B (results not shown). However, analysis by SDS/PAGE (Fig. 3, lanes d – f) showed that both pooled peaks as well as the enzyme used as starting material contained as a prevalent component an 85-kDa band which yielded no N-terminal sequence. The specific AMP deaminase activity of the sample that was retained by the column, measured in the presence of 10 AM ZnCl2 after removal by dialysis of the EDTA used for the stripping procedure, was less than one tenth of that of the native enzyme. This observation suggests that even under nondenaturing conditions, Zn-affinity chromatography almost totally separates from the catalytic subunit of AMP deaminase, which is not retained by the resin, the HPRG component, which in this case maintains its native state, that is, N-terminally modified 85 kDa molecular species. This hypothesis is strengthened
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by the finding that on storage at 4 jC, an apparent 85- to 95kDa transition occurred in the EDTA-eluted protein, as indicated by the slow increase with time of the intensity of the 95-kDa band present as a minor component (see Fig. 3, lane f) in the material freshly eluted with buffer B. By electroblotting and sequencing analysis, the 95-kDa band revealed the single sequence L-T-P-T-D-X-K-T-T-K-P-L. The yield of the sequence increased after storage for few days at 4 jC indicating that the HPRG component of AMP deaminase has a blocked N terminus and that it undergoes a proteolytic process starting with its isolation. Interestingly, 1 h incubation of the EDTA-eluted protein solution with 1% h-mercaptoethanol at room temperature before analysis by SDS/PAGE caused the same 85- to 95-kDa transition that was observed on storage (results not shown). It seems likely, on the basis of this observation, that the shift in the migration on SDS/PAGE is due to a change in conformation consequent to the reduction of a disulfide bond present in freshly isolated HPRG. The presence in the HPRG component of AMP deaminase of a disulfide bridge homologous to that supposed to connect Cys-6 and Cys-497 in rabbit plasma HPRG was inferred on the basis of electroblotting and sequencing analysis of the SDS/PAGE bands corresponding to presumably disulfide-linked fragments liberated by trypsin cleavage [3]. It was also observed that the reduction of that disulfide bridge in rabbit plasma HPRG
Fig. 4. Analysis by SDS/PAGE of the affinity-purified 95-kDa component of AMP deaminase before and after deglycosylation with PGF. The 95-kDa component of AMP deaminase isolated by Zn-affinity chromatography was treated with PGF as described under the Materials and Methods section and 2 Ag samples of the digestion mixture were run on 10% (w/v) polyacrylamide slab gel. (a) Sample analyzed at zero time incubation; (b) sample analyzed after 45 h incubation; (c) sample analyzed after 45 h incubation in the absence of PGF.
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required prior denaturation and that even after reduction, the separation of the N- and C-terminal domains of the protein by ion-exchange chromatography was difficult, because of the hydrophobicity of the contact area [2]. On this basis, we may reasonably assume that on SDS/PAGE of the whole enzyme under reducing conditions, the migration of the HPRG component of AMP deaminase as an 85-kDa species is probably due to the interaction of HPRG with the catalytic subunit of the enzyme further reducing the approachability of h-mercaptoethanol to the disulfide bridge. These results demonstrate that ageing as well as protein denaturation by overnight dialysis of AMP deaminase against 0.1% hmercaptoethanol and 3 M urea promotes the truncation of the HPRG component of AMP deaminase at its N terminus followed by a conformational change which is the basis of the 85- to 95-kDa band transition. The observed precipitation of the protein not retained by the Zn-affinity column (presumably the catalytic subunit of the enzyme) could be imputed to a possible chelation of the active site zinc during elution from the column due to a partial displacement of chelated Zn ions from the iminodiacetic acid groups as the HPRG is adsorbed. However, in our hand, none of the various procedures adopted for the preparation of the apoenzyme resulted in any change of protein solubility [15]. It is more likely that protein precipitation should be ascribed to a disruption of the association between the two components of the enzyme due to the selective binding of HPRG to the resin. A quite different observation was made when the enzyme in 0.5 M NaCl/20 mM sodium phosphate (pH 7.0) was applied to the Zn-charged column equilibrated with the same buffer (results not shown). Under these conditions, the whole protein was retained by the column. The application of a buffer composed of 0.5 M KCl/50 mM imidazole/HCl (pH 6.5) resulted in the slow elution of a fraction (40% of the total protein) with relatively high AMP deaminase activity that by SDS/PAGE analysis gave rise to an electrophoretogram similar to that of the applied enzyme (i.e. a major 85-kDa band and a minor 70-kDa band). Subsequent washing with buffer B eluted from the column a sharp protein peak with low enzyme activity (30% of the total protein, but accounting for about 1% of the enzyme activity applied to the column). Analysis by SDS/PAGE showed that this peak contained the same components as had been obtained with the previous elution. These observations indicate that in the presence of phosphate, a competitive inhibitor of skeletal muscle AMP deaminase [17,18] that has been reported to stabilize the tetrameric structure of the enzyme even in the absence of K+ [19], a protein – protein complex is formed between the catalytic subunit and the HPRG component which, in contrast to what is observed in the absence of phosphate, maintains stability even after the adsorption of HPRG to the Zn-charged column. It is not possible to deduce from the SDS/PAGE analysis the stoichiometric association of the two proteins in the complexes yielded by the two elution steps, because both the catalytic subunit and the HPRG component
behave as 85-kDa species. However, the protein fraction eluted with the imidazole buffer showed a loss of AMP deaminase activity with time of storage at 4 jC with a halftime of a week, whereas the activity of the EDTA-eluted fraction remained constant during the first 10 days of storage. The two protein fractions also showed different kinetic behaviour (Fig. 5) that may be ascribed to the presence of a different stoichiometry of association between HPRG and the catalytic subunit. The freshly imidazole-eluted enzyme shows a sigmoid substrate-saturation curve (Hill coefficient, h = 1.6; Km = 0.6 mM AMP) and is therefore quite distinct from the EDTA-eluted enzyme that follows hyperbolic kinetics (h = 1.0; K m = 1.0 mM AMP), similar to that observed with the starting material (h = 1.1; Km = 0.4 mM AMP). A plausible interpretation of this data is that a minor molar ratio HPRG/AMP deaminase that probably is at the basis of the observed lower affinity for the Zn-charged column, could also be responsible for inactivation of the imidazole-eluted enzyme at low substrate concentration, in keeping with the described dilution-induced apparent homotropic cooperativity in rabbit skeletal muscle AMP deaminase [20]. The observation that this phenomenon does not occur with the EDTA-eluted enzyme, although it probably contains a much more dilute catalytic subunit, may be explained by the protection against loss of activity exerted by its high HPRG content. Altogether, the above observations suggest a role of the HPRG component of skeletal muscle AMP deaminase in giving stability to the enzyme molecular structure. 3.4. Functional implications of the possible participation of the HPRG component in the assembly and maintenance of skeletal muscle AMP deaminase quaternary structure The tetrameric nature of skeletal muscle AMP deaminase is well documented in the literature. However, the molecular mass of the enzyme from various species ranges from 238 to 326 kDa [8,21 –30]. In the light of the observation that the rabbit enzyme undergoes fragmentation on storage at 4 jC, the different reports on the molecular mass of skeletal muscle AMP deaminase have been explained by the inherent instability of the enzyme, especially during the prolonged extraction step at room temperature [11]. This view has been confirmed by the conversion in vitro of the 80-kDa subunit into a 66-kDa form of the rat enzyme during incubation of muscle extract at 37 jC [31] and the 10kDa reduction in subunit molecular mass of human AMP deaminase recombinant protein after 3 months storage at 4 jC [25]. Our determination by sedimentation-equilibrium analysis of the molecular mass of freshly prepared rabbit skeletal muscle AMP deaminase in 1 M KCl, pH 7.0, indicated the presence of two species of 173 and 309 kDa, which were interpreted as consistent with the existence of a dimer – tetramer equilibrium [8]. The data of the present paper confirm our recent observation that purified skeletal muscle AMP deaminase contains two different protein species, one
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Fig. 5. Substrate-versus-velocity curves of the components of AMP deaminase yielded by elution of the enzyme adsorbed to a Zn-affinity column under nondenaturing conditions in the presence of phosphate. A sample of AMP deaminase (2 mg) was chromatographed on a Zn-affinity column equilibrated with 0.5 M NaCl/20 mM sodium phosphate (pH 7.0) and eluted with 0.5 M KCl/50 mM imidazole/HCl (pH 6.5) followed by a stripping elution with the EDTAcontaining buffer B. The AMP deaminase activity was assayed by addition of 10 Al samples of the imidazole-eluted (.) or EDTA-eluted (D) fractions to the assay mixture (1 ml) containing 50 imidazole/HCl (pH 6.5)/100 mM KCl and the reported AMP concentrations. The activity of native AMP deaminase (o) was determined by diluting 100-fold in the assay mixture an enzyme solution having approximately the same concentration (0.15 mg/ml) of the two separated AMP deaminase fractions.
of them being the catalytic subunit and the other showing similarity with plasma HPRG [3]. Therefore, the heterogeneity observed in sedimentation-equilibrium centrifugation of the native enzyme should be interpreted as being due to the presence of HPRG/AMP deaminase protein –protein complexes with different molar ratio, the observed 309-kDa molecular mass determined for the heavier component being in agreement with a model for AMP deaminase quaternary structure in which two 85-kDa catalytic subunits assemble with two approx. 70-kDa HPRG subunits (assuming a carbohydrate content similar to that of the plasma protein). A recent paper on the quaternary structure of wild-type human AMP deaminase (AMPD1) recombinant protein showed that this enzyme exists as a large complex (>2000 kDa), whereas the expression of a N-truncated cDNA (DL96AMPD1) produced a stable recombinant enzyme with a molecular mass of 305 kDa [25]. In our opinion, the claimed novel aspects of tetramer assembly of the wild-type enzyme revealed by recombinant expression of human AMPD1 should be rejected. First, the kinetic properties of the recombinant enzyme sharply differ from those established in the literature for native skeletal muscle AMP deaminase (e.g. eight times higher Km, strong activation by ATP). Secondly, the observation that the same enzyme elutes in the void volume of a column packed with a resin with an exclusion volume >2000 kDa has been arbitrarily attributed
to the formation of aggregates of tetramers on the basis of the observation that following extended storage at 4 jC, the recombinant protein generates an apparent tetramer assembly formed by N-truncated 76-kDa subunits. Thirdly, the data of the present paper, showing that the association with the HPRG component is critical for a stable AMP deaminase activity, strongly suggest that a possible explanation for the abnormal behaviour of the recombinant enzyme may well be due to the instability of the isolated native 85-kDa catalytic subunit resulting in an aggregation-induced precipitation. Until evidence can be obtained in vivo for the association that we have observed in vitro between the AMP deaminase catalytic subunit and the skeletal muscle isoform of HPRG, it is premature to assign any physiological significance to the observed interaction. It should be noted, however, that our recent paper [4] showed that an antibody against human plasma HPRG reacts with an AMP deaminase preparation from human skeletal muscle. A clear positive reaction of the anti-HPRG antibody was also detected at the level of type IIB fibers, which are well known to contain the highest level of AMP deaminase among muscle fibers. Moreover, the available data permit to assert that the two components of the enzyme manifest as a common property the ability to interact with zinc ions. The full-length cDNA sequence of plasma human and murine HPRG and a partial cDNA sequence of the rabbit protein have been reported [2,32,33]. Alignment of
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the predicted amino acid sequences indicates that all HPRG species share an overall domain structure comprising an Nterminal domain containing two cystatin-like repeats, a histidine – proline-rich domain and a C-terminal domain. Alignment of our partial amino acid sequence data of the rabbit skeletal muscle AMP deaminase-associated variant of HPRG with the predicted sequence of rabbit plasma HPRG indicates that the rabbit muscle and plasma forms of HPRG share high identity, diverging only at 5 amino acid residues among the 78 sequenced (93.6% identity), that span all the three proposed overall domains of HPRG (A.J.G. Moir and A. Raggi, unpublished work). The 34 histidine residues located in the histidine – proline-rich domain of rabbit plasma HPRG have been shown to mediate interactions with transition metals, although no evidence has been given of the existence of any specific metal binding site [2]. Because the HPRG component of rabbit skeletal muscle AMP deaminase contains 10 mol of histidine residues per 10 000 g of protein [3], we exploited metal-affinity chromatography in an attempt to separate the novel component of the enzyme from the catalytic subunit. As has been shown in the present paper, the isolation of the HPRG component from the purified enzyme under denaturing conditions was achieved by zincaffinity chromatography. This clearly indicates the presence in rabbit muscle HPRG of a metal binding site, which permits one to envisage the addition of HPRG into the family of metal trafficking proteins called metallochaperones [34]. The characterization of skeletal muscle AMP deaminase as a zinc metalloenzyme was reported for the rat enzyme [15] as well as for the rabbit enzyme [16] on the basis of its interaction with chelating agents and metal ions. In this view, HPRG may enhance the stability of AMP deaminase in vivo through insertion of zinc or modulation of intracellular zinc availability.
Acknowledgements This research was supported by a grant from the Italian MURST. We thank Mr. Piero Bertelli for skilled technical assistance.
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[2] D.B. Borza, F.M. Tatum, W.T. Morgan, Biochemistry 35 (1996) 1925 – 1934. [3] M. Ranieri-Raggi, U. Montali, F. Ronca, A. Sabbatini, P.E. Brown, A.J.G. Moir, A. Raggi, Biochem. J. 326 (1997) 641 – 648. [4] A.R.M. Sabbatini, M. Ranieri-Raggi, L. Pollina, P. Viacava, J.R. Ashby, A.J.G. Moir, A. Raggi, J. Histochem. Cytochem. 47 (1999) 255 – 260. [5] M. Ranieri-Raggi, A. Raggi, FEBS Lett. 102 (1979) 59 – 63. [6] D. Martini, M. Ranieri-Raggi, A.R.M. Sabbatini, A. Raggi, Biochim. Biophys. Acta 1544 (2001) 123 – 132. [7] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265 – 275. [8] M. Ranieri-Raggi, A. Raggi, Biochem. J. 272 (1990) 755 – 759. [9] N. LeGendre, P. Matsudaira, in: P. Matsudaira (Ed.), A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, New York, 1989, pp. 49 – 69. [10] F. Ronca, M. Ranieri-Raggi, P.E. Brown, A.J.G. Moir, A. Raggi, Biochim. Biophys. Acta 1209 (1994) 123 – 129. [11] M. Ranieri-Raggi, A. Raggi, Biochem. J. 189 (1980) 367 – 368. [12] W.T. Morgan, Biochemistry 24 (1985) 1496 – 1501. [13] S. Mori, M. Nishibori, K. Yamaoka, M. Okamoto, Arch. Biochem. Biophys. 383 (2000) 191 – 196. [14] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Nature 258 (1975) 598 – 599. [15] A. Raggi, M. Ranieri, G. Taponeco, S. Ronca-Testoni, G. Ronca, C.A. Rossi, FEBS Lett. 10 (1970) 101 – 104. [16] C.L. Zielke, C.H. Suelter, J. Biol. Chem. 246 (1971) 2179 – 2186. [17] S. Ronca-Testoni, A. Raggi, G. Ronca, Biochim. Biophys. Acta 198 (1970) 101 – 112. [18] T.J. Wheeler, J.M. Lowenstein, J. Biol. Chem. 254 (1979) 8994 – 8999. [19] M. Ranieri-Raggi, A. Raggi, Ital. J. Biochem. 33 (1984) 155 – 176. [20] R.M. Hemphill, C.L. Zielke, C.H. Suelter, J. Biol. Chem. 246 (1971) 7237 – 7240. [21] Y.P. Lee, J. Biol. Chem. 227 (1957) 993 – 998. [22] R.D. Currie, H.L. Webster, Biochim. Biophys. Acta 64 (1962) 30 – 40. [23] R. Wolfenden, Y. Tomozawa, B. Bamman, Biochemistry 7 (1968) 3965 – 3970. [24] S. Ronca-Testoni, M. Ranieri, A. Raggi, G. Ronca, Ital. J. Biochem. 19 (1970) 262 – 276. [25] D.K. Mahnke-Zizelman, P.C. Tullson, R.L. Sabina, J. Biol. Chem. 273 (1998) 35118 – 35125. [26] L.K. Ashman, J.L. Atwell, Biochim. Biophys. Acta 258 (1972) 618 – 625. [27] C.J. Coffee, W.A. Kofke, J. Biol. Chem. 250 (1975) 6653 – 6658. [28] A. Boosman, O.P. Chilson, J. Biol. Chem. 251 (1976) 1847 – 1852. [29] N. Ogasawara, H. Goto, Y. Yamada, M. Yoshino, Biochem. Biophys. Res. Commun. 79 (1977) 671 – 676. [30] B.A. Barshop, C. Frieden, J. Biol. Chem. 259 (1984) 60 – 66. [31] R. Marquetant, R.L. Sabina, E.W. Holmes, Biochemistry 28 (1989) 8744 – 8749. [32] M.D. Hulett, C.R. Parish, Immunol. Cell Biol. 78 (2000) 280 – 287. [33] T. Koide, D. Foster, S. Yoshitake, E.W. Davie, Biochemistry 25 (1986) 2220 – 2225. [34] T.V. O’Halloran, V.C. Culotta, J. Biol. Chem. 275 (2000) 25057 – 25060.
Isolation by zinc-affinity chromatography of the histidine–proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase Evidence that the formation of a protein–protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme Maria Ranieri-Raggi a, Daniela Martini a, Antonietta R.M. Sabbatini a, Arthur J.G. Moir b, Antonio Raggi a,* a
Dipartimento di Scienze dell’Uomo e dell’Ambiente, Chimica e Biochimica Medica, Universita` di Pisa, via Roma 55, 56126 Pisa, Italy b Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2UH, UK Received 17 April 2002; received in revised form 23 October 2002; accepted 11 November 2002
Abstract The histidine – proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn2 +-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn2 + from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95 000 and the N-terminal sequence L-T-P-T-D-XK-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70 000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-TD-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme. D 2002 Elsevier Science B.V. All rights reserved. Keywords: AMP deaminase structure; Histidine – proline-rich glycoprotein; Immobilized metal ion affinity chromatography
1. Introduction Histidine –proline-rich glycoprotein (HPRG) is present at a relatively high concentration in the plasma of vertebrates, where it has been implicated in a number of processes, including blood coagulation and fibrinolysis, immune response and transport of metal ions [1]. Although the
Abbreviations: HPRG, histidine – proline-rich glycoprotein; PGF, peptide-N-glycosidase F * Corresponding author. Tel.: +39-050-561912; fax: +39-050-550241. E-mail address: [email protected] (A. Raggi).
physiological role of this protein remains unclear, it has been suggested that one of its functions may be to bring two or more ligands together [2]. In a previous paper, we reported that denaturation of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) in acidic medium allows the chromatographic separation from the enzyme of a peptide with an amino acid composition significantly different from that derived from the available AMP deaminase cDNAs [3]. N-terminal sequence analysis of the fragments liberated by limited proteolysis revealed a striking similarity of the novel protein to rabbit plasma HPRG [2] although, in comparison with mature HPRG, the AMP deaminase-associated variant probably contains a unique N-terminal extension [3]. In a
1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 7 0 - 9 6 3 9 ( 0 2 ) 0 0 5 2 7 - 7
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recent paper, we demonstrated that an antibody against human plasma HPRG reacts with an AMP deaminase preparation from human skeletal muscle [4]. A selective binding of the anti-HPRG antibody to type IIB fibers was detected, suggesting the preferential association of muscle HPRG to the AMP deaminase isoenzyme contained in the fast-twitch glycolytic fibers. We report here that the specific binding of HPRG to a Zn-affinity column can be used for the one-step isolation of this protein from purified rabbit skeletal muscle AMP deaminase. The separation process induces a marked reduction in solubility of the catalytic subunit of the enzyme, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.
suggested by the manufacturer. The column was charged with 0.1 M ZnCl2 in 0.5 M NaCl/50 mM sodium acetate (pH 4.0) and washed with the same buffer without ZnCl2. After equilibration of the resin with 0.5 M NaCl/20 mM sodium phosphate (pH 7.0), containing 0.1% h-mercaptoethanol and 3 M urea (buffer A), a sample of AMP deaminase denatured by overnight dialysis against buffer A was applied to the column that was washed with buffer A. Following the followthrough peak, the column was eluted with a linear gradient of imidazole (2– 100 mM in 50 ml) in buffer A. After having washed the column with buffer A, a stripping elution was made with 0.5 M NaCl/20 mM sodium phosphate (pH 7.2), containing 0.1% h-mercaptoethanol and 50 mM EDTA. Similar separations were carried out in the absence of urea as described in the Results and Discussion section.
2. Materials and methods
2.4. Deglycosylation
2.1. Reagents
Samples (0.1 ml) containing 25 Ag of the affinity-purified HPRG component of AMP deaminase dialysed against 0.25 M sodium phosphate (pH 8.6) were denatured by heating at 100 jC in the presence of 0.1% SDS and 1% h-mercaptoethanol. After SDS denaturation, 0.4% Triton X-100 was added to avoid inactivation of PGF by SDS. The deglycosylation reaction (45 h at 37 jC) was started with the addition of 5 units of PGF to the incubation mixture. The extent of HPRG glycosylation was estimated by comparing the mobility on SDS/PAGE of treated HPRG with that of the protein incubated in the same mixture but in the absence of PGF.
Peptide-N-glycosidase F (PGF) from Flavobacterium meningosepticum was purchased from Roche Diagnostics (Mannheim, Germany). Chelating Fast Flow Sepharose was from Pharmacia LKB Technology (Uppsala, Sweden). Phosphocellulose resin (P-11) was supplied by Whatman International Ltd. (Maidstone, UK). All chemicals and other reagents used were of analytical grade. 2.2. Enzyme AMP deaminase was prepared as described previously [5] from fresh muscle dissected from the back and hind leg of rabbits. Because the enzyme undergoes progressive fragmentation with storage, homogenization of the muscle and phosphocellulose purification of the enzyme was carried out using at each step a buffer system containing 5 mM NaN3 to reduce the rate of proteolytic processes. This modification did not change the specific activity of the purified enzyme (1100 Amol of AMP deaminated per min/ mg of protein when assayed in 50 mM imidazole/HCl (pH 6.5)/100 mM KCl/2 mM AMP at 20 jC). The AMP deaminase activity was determined spectrophotometrically as previously described in a Shimadzu UV-260 spectrophotometer [6]. Protein concentrations of the various enzyme fractions were determined spectrophotometrically by using A2801%,1 cm values of 9.1 and 8.2, respectively, for AMP deaminase and its isolated HPRG component, which were calculated on the basis of protein determinations by the method described in Ref. [7], using bovine serum albumin as standard. The molecular mass of AMP deaminase was taken as 309 kDa [8].
2.5. SDS/PAGE Electrophoresis in the presence of 0.1% (w/v) SDS was carried out under reducing conditions on 10% or 13.5% (w/v) polyacrylamide slab gels in 0.1 M Tris/0.1 M Bicine (pH 8.3). Protein standards (Sigma) and prestained proteins (Bio-Rad, Richmond, CA) were used to determine molecular weights (MWs). 2.6. Electroblotting and sequence analysis Electroblotting was performed by the method of LeGendre and Matsudaira [9] using 10 mM 3-(cyclohexylamino)-1propane-sulfonic acid (pH 11) containing 10% (v/v) methanol. Transfers were performed for 60 –90 min at 400 mA. N-terminal sequencing was performed using an Applied Biosystems model 476A protein sequencer.
3. Results and discussion
2.3. Immobilized Zn2+-affinity chromatography
3.1. Acidic treatment of AMP deaminase isolates its HPRG component with a MW of 95 000 on SDS/PAGE
A mini-column of 2 ml bed volume was prepared with Chelating Fast Flow Sepharose according to the procedure
We previously demonstrated that limited proteolysis of rabbit skeletal muscle AMP deaminase with trypsin, remov-
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ing the 95-residue N-terminal fragment of the enzyme, converts the native 85-kDa enzyme subunit to an approx. 70-kDa core, that is resistant to further proteolysis and which is present as a minor component in the SDS/PAGE electrophoretogram of the native enzyme [10]. The observed 85- to 70-kDa transition also occurred on storage of AMP deaminase, as well as of the muscle used for the enzyme preparation [11]. When the freshly prepared enzyme was subjected to automated Edman degradation, in addition to low amount of the sequences corresponding to fragments beginning at Phe-4 and Pro-2 of the known catalytic subunit of AMP deaminase, a low yield of the sequence L-T-P-T-DX was obtained, corresponding to the N-terminal sequence of rabbit plasma HPRG [3]. Electroblotting and sequencing of the 70-kDa SDS/PAGE band of the trypsinized enzyme revealed two sequences in equal amounts, one of which corresponded to a fragment starting at Leu-96 of the AMP deaminase subunit, whereas the other (L-T-P-T-D-X-K) was identical with the N-terminal sequence of rabbit plasma HPRG [2]. Further analysis by SDS/PAGE of several fresh preparations of the enzyme has now revealed the presence of a variable amount of a minor component with an apparent molecular mass of 95 kDa in addition to the main 85-kDa protein band and the minor 70-kDa proteolyzed component (Fig. 1, lane b). The addition of 1 Al of 1 M HCl to 0.1 ml of native AMP deaminase (1 mg/ml) caused protein precipita-
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tion. Analysis by SDS/PAGE of the precipitate revealed a strong similarity with the enzyme used as starting material except for the absence of the 95-kDa component (Fig. 1, lane c). By electroblotting and sequencing analysis, the main 85-kDa band of the starting material and of the precipitate yielded no N-terminal sequences, confirming the previous suggestion that the N terminus of rabbit skeletal muscle AMP deaminase is modified [3]. SDS/ PAGE of the supernatant revealed the presence of only one component, which could be identified as the 95-kDa component (Fig. 1, lane d). Electroblotting and sequencing of the 95-kDa band revealed the sequence L-T-P-T-D-X-KT-T-K-P-L corresponding to the N-terminal sequence of rabbit plasma HPRG [2] which is consistent with the identification of the sequence in freshly prepared enzyme as a minor component. It should be noted that rabbit plasma HPRG migrates in SDS/PAGE with an apparent molecular mass of 90 or 94 kDa [2,12,13] higher than that deduced from its sequence (58 kDa) or that calculated taking account of its 17.5% carbohydrate content (70 kDa) [2]. The higher intensity of the isolated 95-kDa band (Fig. 1, lane d) compared to that of the 95-kDa minor component of the whole enzyme (Fig. 1, lane a) suggests that acidic treatment promotes some conformational change in the HPRG component of the enzyme that gives rise to a species with a lower mobility on SDS/PAGE resulting in an 85- to 95-kDa shift of the apparent molecular mass of the protein. 3.2. Specific binding of the HPRG component of AMP deaminase to a Zn2+-charged metal-affinity column
Fig. 1. Effect of acidic treatment on SDS/PAGE of rabbit skeletal muscle AMP deaminase. The addition of 2.5 Al of 1 M HCl to 0.25 ml of AMP deaminase (1 mg/ml) caused protein precipitation. The precipitate was separated by centrifugation and was redissolved in 0.25 ml of 75 mM Tris/ HCl (pH 8.0) containing 15 mM h-mercaptoethanol and 9 M urea. Samples were denatured in SDS and run on 13.5% (w/v) polyacrylamide slab gel. (a) Prestained molecular weight standards; (b) 10 Ag of native AMP deaminase; (c) 10 Al of the solution of redissolved precipitate; (d) 10 Al of the supernatant obtained with the centrifugation of acid-treated AMP deaminase.
Histidine forms stable complexes with transition metals. Taking advantage of this property, a procedure for purifying proteins containing exposed histidine residues has been developed, which is known as immobilized metal ion affinity chromatography [14]. Rabbit plasma HPRG contains 53 histidine residues, of which 34 are located in the histidine –proline-rich domain containing 15 repeats of the sequence (H/P)(H/P)PHG [2]. These histidine residues have been shown to mediate interactions with transition metals [12]. Because the HPRG component of rabbit skeletal muscle AMP deaminase contains 10 mol of histidine residues per 10 000 g of protein [3], we exploited metal affinity chromatography in an attempt to separate the novel component of the enzyme from the catalytic subunit. Fig. 2 shows a chromatographic profile of rabbit skeletal muscle AMP deaminase (2.9 mg) denatured by overnight dialysis against 0.5 M NaCl/20 mM sodium phosphate (pH 7.0), containing 0.1% h-mercaptoethanol and 3 M urea (buffer A), and applied to a metal-affinity column charged with Zn2 + and equilibrated with buffer A. The results show that the 95-kDa component of AMP deaminase was specifically retained on the column while the 85- and 70-kDa components passed through and emerged in the void volume (Fig. 2, peak A) so that analysis by SDS/PAGE of the A280 nm peak fraction revealed a strong similarity with the
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Fig. 2. Zn-affinity chromatographic analysis of rabbit skeletal muscle AMP deaminase. A sample of AMP deaminase (2.9 mg) denatured by overnight dialysis against buffer A was chromatographed on a Zn-affinity column as described in the Materials and Methods section and the A280 nm peak fractions were analyzed by 10% (w/v) SDS/PAGE.
enzyme used as starting material except for the absence of the 95-kDa minor component migrating as the slower band in the electrophoretogram of whole AMP deaminase. This component was retained by the column during the elution with a linear gradient of 2– 100 mM imidazole in buffer A and was only eluted when the column was washed with 0.5 M NaCl/20 mM sodium phosphate (pH 7.2), containing 0.1% h-mercaptoethanol and 50 mM EDTA (buffer B) which strips the metal ions from the gel and causes elution of the bound proteins (Fig. 2, peak B). Electroblotting and sequencing of the 95-kDa peptide isolated by zinc-affinity chromatography revealed the sequence L-T-P-T-D-X-K-T-TK-P-L-A-E-K-A-L-D-L-I, corresponding to the rabbit plasma HPRG N terminus [2]. SDS/PAGE of the starting material and the two pooled peaks is shown in Fig. 3, lanes a – c. In three different experiments using three different AMP deaminase preparations, the recovery of the total material applied to the column in terms of mass was 73 F 8%, 39 F 12% and 36 F 8% being accounted for by peaks A and B, respectively. These data strengthen the hypothesis that the species migrating in SDS/ PAGE as a 95-kDa band actually derives from some modification of the 85-kDa component of the enzyme. The protein that was not adsorbed during application of AMP deaminase to the Zn-charged column precipitated immediately after its elution in the flow-through. In contrast,
the isolated 95-kDa component of the enzyme, that was completely devoid of AMP deaminase activity even after removal by dialysis of the EDTA used for the stripping procedure and the addition of 10 AM ZnCl2 to the assay mixture, which causes an instantaneous and complete reactivation of AMP deaminase inactivated by chelating agents [15,16], was quite stable even after several months storage at 20 jC and retained its solubility after several freezing and thawing procedures. This allowed its prolonged incubation with PGF to examine the extent of its glycosylation on the basis of the effect of the treatment on its mobility on SDS/PAGE. Following 45 h incubation with PGF in the presence of SDS, HPRG was subjected to SDS/PAGE which showed a reduction of the apparent MW from 95 to 70 kDa (Fig. 4). This observation which characterizes the HPRG component of AMP deaminase as a N-glycosylated protein is also in agreement with the results of the carbohydrate structure analysis of rabbit plasma HPRG showing that treatment with PGF reduces the apparent MW of this protein from 93 to 67 kDa [13]. 3.3. Association with the HPRG component is critical for a stable AMP deaminase activity In further experiments, AMP deaminase was loaded on to the Zn-charged column under nondenaturing conditions.
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Fig. 3. SDS/PAGE of the components of AMP deaminase separated by Znaffinity chromatography under denaturing or nondenaturing conditions. The enzyme used as starting material and the pools of the protein peaks resolved by the Zn-affinity chromatographic analysis in denaturing (lanes a – c) or nondenaturing (lanes d – f) conditions, were run on 10% (w/v) polyacrylamide slab gel. (a) Eight micrograms sample of AMP deaminase dialysed against buffer A; (b) 4 Ag sample of AMP deaminase not retained by the Zn-charged column as in Fig. 2; (c) 5 Ag sample of the HPRG component eluted with 50 mM EDTA from the Zn-charged column as in Fig. 2; (d) 8 Ag sample of freshly prepared AMP deaminase applied to the Zn-charged column equilibrated with 1 M KCl, pH 7.0; (e) 6 Ag sample of AMP deaminase not retained by the Zn-charged column as in (d); (f) 4 Ag sample of the protein eluted with 50 mM EDTA from the column as in (d).
When 0.8 mg enzyme in 1 M KCl, pH 7.0, was applied to the column equilibrated with the same medium, the results were apparently similar to those obtained with the denatured enzyme because 45% of the protein was not retained by the column and started to precipitate immediately after its collection in the flow-through, whereas 40% of the protein was accounted for by the eluate obtained by washing the column with buffer B (results not shown). However, analysis by SDS/PAGE (Fig. 3, lanes d – f) showed that both pooled peaks as well as the enzyme used as starting material contained as a prevalent component an 85-kDa band which yielded no N-terminal sequence. The specific AMP deaminase activity of the sample that was retained by the column, measured in the presence of 10 AM ZnCl2 after removal by dialysis of the EDTA used for the stripping procedure, was less than one tenth of that of the native enzyme. This observation suggests that even under nondenaturing conditions, Zn-affinity chromatography almost totally separates from the catalytic subunit of AMP deaminase, which is not retained by the resin, the HPRG component, which in this case maintains its native state, that is, N-terminally modified 85 kDa molecular species. This hypothesis is strengthened
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by the finding that on storage at 4 jC, an apparent 85- to 95kDa transition occurred in the EDTA-eluted protein, as indicated by the slow increase with time of the intensity of the 95-kDa band present as a minor component (see Fig. 3, lane f) in the material freshly eluted with buffer B. By electroblotting and sequencing analysis, the 95-kDa band revealed the single sequence L-T-P-T-D-X-K-T-T-K-P-L. The yield of the sequence increased after storage for few days at 4 jC indicating that the HPRG component of AMP deaminase has a blocked N terminus and that it undergoes a proteolytic process starting with its isolation. Interestingly, 1 h incubation of the EDTA-eluted protein solution with 1% h-mercaptoethanol at room temperature before analysis by SDS/PAGE caused the same 85- to 95-kDa transition that was observed on storage (results not shown). It seems likely, on the basis of this observation, that the shift in the migration on SDS/PAGE is due to a change in conformation consequent to the reduction of a disulfide bond present in freshly isolated HPRG. The presence in the HPRG component of AMP deaminase of a disulfide bridge homologous to that supposed to connect Cys-6 and Cys-497 in rabbit plasma HPRG was inferred on the basis of electroblotting and sequencing analysis of the SDS/PAGE bands corresponding to presumably disulfide-linked fragments liberated by trypsin cleavage [3]. It was also observed that the reduction of that disulfide bridge in rabbit plasma HPRG
Fig. 4. Analysis by SDS/PAGE of the affinity-purified 95-kDa component of AMP deaminase before and after deglycosylation with PGF. The 95-kDa component of AMP deaminase isolated by Zn-affinity chromatography was treated with PGF as described under the Materials and Methods section and 2 Ag samples of the digestion mixture were run on 10% (w/v) polyacrylamide slab gel. (a) Sample analyzed at zero time incubation; (b) sample analyzed after 45 h incubation; (c) sample analyzed after 45 h incubation in the absence of PGF.
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required prior denaturation and that even after reduction, the separation of the N- and C-terminal domains of the protein by ion-exchange chromatography was difficult, because of the hydrophobicity of the contact area [2]. On this basis, we may reasonably assume that on SDS/PAGE of the whole enzyme under reducing conditions, the migration of the HPRG component of AMP deaminase as an 85-kDa species is probably due to the interaction of HPRG with the catalytic subunit of the enzyme further reducing the approachability of h-mercaptoethanol to the disulfide bridge. These results demonstrate that ageing as well as protein denaturation by overnight dialysis of AMP deaminase against 0.1% hmercaptoethanol and 3 M urea promotes the truncation of the HPRG component of AMP deaminase at its N terminus followed by a conformational change which is the basis of the 85- to 95-kDa band transition. The observed precipitation of the protein not retained by the Zn-affinity column (presumably the catalytic subunit of the enzyme) could be imputed to a possible chelation of the active site zinc during elution from the column due to a partial displacement of chelated Zn ions from the iminodiacetic acid groups as the HPRG is adsorbed. However, in our hand, none of the various procedures adopted for the preparation of the apoenzyme resulted in any change of protein solubility [15]. It is more likely that protein precipitation should be ascribed to a disruption of the association between the two components of the enzyme due to the selective binding of HPRG to the resin. A quite different observation was made when the enzyme in 0.5 M NaCl/20 mM sodium phosphate (pH 7.0) was applied to the Zn-charged column equilibrated with the same buffer (results not shown). Under these conditions, the whole protein was retained by the column. The application of a buffer composed of 0.5 M KCl/50 mM imidazole/HCl (pH 6.5) resulted in the slow elution of a fraction (40% of the total protein) with relatively high AMP deaminase activity that by SDS/PAGE analysis gave rise to an electrophoretogram similar to that of the applied enzyme (i.e. a major 85-kDa band and a minor 70-kDa band). Subsequent washing with buffer B eluted from the column a sharp protein peak with low enzyme activity (30% of the total protein, but accounting for about 1% of the enzyme activity applied to the column). Analysis by SDS/PAGE showed that this peak contained the same components as had been obtained with the previous elution. These observations indicate that in the presence of phosphate, a competitive inhibitor of skeletal muscle AMP deaminase [17,18] that has been reported to stabilize the tetrameric structure of the enzyme even in the absence of K+ [19], a protein – protein complex is formed between the catalytic subunit and the HPRG component which, in contrast to what is observed in the absence of phosphate, maintains stability even after the adsorption of HPRG to the Zn-charged column. It is not possible to deduce from the SDS/PAGE analysis the stoichiometric association of the two proteins in the complexes yielded by the two elution steps, because both the catalytic subunit and the HPRG component
behave as 85-kDa species. However, the protein fraction eluted with the imidazole buffer showed a loss of AMP deaminase activity with time of storage at 4 jC with a halftime of a week, whereas the activity of the EDTA-eluted fraction remained constant during the first 10 days of storage. The two protein fractions also showed different kinetic behaviour (Fig. 5) that may be ascribed to the presence of a different stoichiometry of association between HPRG and the catalytic subunit. The freshly imidazole-eluted enzyme shows a sigmoid substrate-saturation curve (Hill coefficient, h = 1.6; Km = 0.6 mM AMP) and is therefore quite distinct from the EDTA-eluted enzyme that follows hyperbolic kinetics (h = 1.0; K m = 1.0 mM AMP), similar to that observed with the starting material (h = 1.1; Km = 0.4 mM AMP). A plausible interpretation of this data is that a minor molar ratio HPRG/AMP deaminase that probably is at the basis of the observed lower affinity for the Zn-charged column, could also be responsible for inactivation of the imidazole-eluted enzyme at low substrate concentration, in keeping with the described dilution-induced apparent homotropic cooperativity in rabbit skeletal muscle AMP deaminase [20]. The observation that this phenomenon does not occur with the EDTA-eluted enzyme, although it probably contains a much more dilute catalytic subunit, may be explained by the protection against loss of activity exerted by its high HPRG content. Altogether, the above observations suggest a role of the HPRG component of skeletal muscle AMP deaminase in giving stability to the enzyme molecular structure. 3.4. Functional implications of the possible participation of the HPRG component in the assembly and maintenance of skeletal muscle AMP deaminase quaternary structure The tetrameric nature of skeletal muscle AMP deaminase is well documented in the literature. However, the molecular mass of the enzyme from various species ranges from 238 to 326 kDa [8,21 –30]. In the light of the observation that the rabbit enzyme undergoes fragmentation on storage at 4 jC, the different reports on the molecular mass of skeletal muscle AMP deaminase have been explained by the inherent instability of the enzyme, especially during the prolonged extraction step at room temperature [11]. This view has been confirmed by the conversion in vitro of the 80-kDa subunit into a 66-kDa form of the rat enzyme during incubation of muscle extract at 37 jC [31] and the 10kDa reduction in subunit molecular mass of human AMP deaminase recombinant protein after 3 months storage at 4 jC [25]. Our determination by sedimentation-equilibrium analysis of the molecular mass of freshly prepared rabbit skeletal muscle AMP deaminase in 1 M KCl, pH 7.0, indicated the presence of two species of 173 and 309 kDa, which were interpreted as consistent with the existence of a dimer – tetramer equilibrium [8]. The data of the present paper confirm our recent observation that purified skeletal muscle AMP deaminase contains two different protein species, one
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Fig. 5. Substrate-versus-velocity curves of the components of AMP deaminase yielded by elution of the enzyme adsorbed to a Zn-affinity column under nondenaturing conditions in the presence of phosphate. A sample of AMP deaminase (2 mg) was chromatographed on a Zn-affinity column equilibrated with 0.5 M NaCl/20 mM sodium phosphate (pH 7.0) and eluted with 0.5 M KCl/50 mM imidazole/HCl (pH 6.5) followed by a stripping elution with the EDTAcontaining buffer B. The AMP deaminase activity was assayed by addition of 10 Al samples of the imidazole-eluted (.) or EDTA-eluted (D) fractions to the assay mixture (1 ml) containing 50 imidazole/HCl (pH 6.5)/100 mM KCl and the reported AMP concentrations. The activity of native AMP deaminase (o) was determined by diluting 100-fold in the assay mixture an enzyme solution having approximately the same concentration (0.15 mg/ml) of the two separated AMP deaminase fractions.
of them being the catalytic subunit and the other showing similarity with plasma HPRG [3]. Therefore, the heterogeneity observed in sedimentation-equilibrium centrifugation of the native enzyme should be interpreted as being due to the presence of HPRG/AMP deaminase protein –protein complexes with different molar ratio, the observed 309-kDa molecular mass determined for the heavier component being in agreement with a model for AMP deaminase quaternary structure in which two 85-kDa catalytic subunits assemble with two approx. 70-kDa HPRG subunits (assuming a carbohydrate content similar to that of the plasma protein). A recent paper on the quaternary structure of wild-type human AMP deaminase (AMPD1) recombinant protein showed that this enzyme exists as a large complex (>2000 kDa), whereas the expression of a N-truncated cDNA (DL96AMPD1) produced a stable recombinant enzyme with a molecular mass of 305 kDa [25]. In our opinion, the claimed novel aspects of tetramer assembly of the wild-type enzyme revealed by recombinant expression of human AMPD1 should be rejected. First, the kinetic properties of the recombinant enzyme sharply differ from those established in the literature for native skeletal muscle AMP deaminase (e.g. eight times higher Km, strong activation by ATP). Secondly, the observation that the same enzyme elutes in the void volume of a column packed with a resin with an exclusion volume >2000 kDa has been arbitrarily attributed
to the formation of aggregates of tetramers on the basis of the observation that following extended storage at 4 jC, the recombinant protein generates an apparent tetramer assembly formed by N-truncated 76-kDa subunits. Thirdly, the data of the present paper, showing that the association with the HPRG component is critical for a stable AMP deaminase activity, strongly suggest that a possible explanation for the abnormal behaviour of the recombinant enzyme may well be due to the instability of the isolated native 85-kDa catalytic subunit resulting in an aggregation-induced precipitation. Until evidence can be obtained in vivo for the association that we have observed in vitro between the AMP deaminase catalytic subunit and the skeletal muscle isoform of HPRG, it is premature to assign any physiological significance to the observed interaction. It should be noted, however, that our recent paper [4] showed that an antibody against human plasma HPRG reacts with an AMP deaminase preparation from human skeletal muscle. A clear positive reaction of the anti-HPRG antibody was also detected at the level of type IIB fibers, which are well known to contain the highest level of AMP deaminase among muscle fibers. Moreover, the available data permit to assert that the two components of the enzyme manifest as a common property the ability to interact with zinc ions. The full-length cDNA sequence of plasma human and murine HPRG and a partial cDNA sequence of the rabbit protein have been reported [2,32,33]. Alignment of
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the predicted amino acid sequences indicates that all HPRG species share an overall domain structure comprising an Nterminal domain containing two cystatin-like repeats, a histidine – proline-rich domain and a C-terminal domain. Alignment of our partial amino acid sequence data of the rabbit skeletal muscle AMP deaminase-associated variant of HPRG with the predicted sequence of rabbit plasma HPRG indicates that the rabbit muscle and plasma forms of HPRG share high identity, diverging only at 5 amino acid residues among the 78 sequenced (93.6% identity), that span all the three proposed overall domains of HPRG (A.J.G. Moir and A. Raggi, unpublished work). The 34 histidine residues located in the histidine – proline-rich domain of rabbit plasma HPRG have been shown to mediate interactions with transition metals, although no evidence has been given of the existence of any specific metal binding site [2]. Because the HPRG component of rabbit skeletal muscle AMP deaminase contains 10 mol of histidine residues per 10 000 g of protein [3], we exploited metal-affinity chromatography in an attempt to separate the novel component of the enzyme from the catalytic subunit. As has been shown in the present paper, the isolation of the HPRG component from the purified enzyme under denaturing conditions was achieved by zincaffinity chromatography. This clearly indicates the presence in rabbit muscle HPRG of a metal binding site, which permits one to envisage the addition of HPRG into the family of metal trafficking proteins called metallochaperones [34]. The characterization of skeletal muscle AMP deaminase as a zinc metalloenzyme was reported for the rat enzyme [15] as well as for the rabbit enzyme [16] on the basis of its interaction with chelating agents and metal ions. In this view, HPRG may enhance the stability of AMP deaminase in vivo through insertion of zinc or modulation of intracellular zinc availability.
Acknowledgements This research was supported by a grant from the Italian MURST. We thank Mr. Piero Bertelli for skilled technical assistance.
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