Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum*

Trophozoites the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mM glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate. of molecular weight Gelatin substrate analysis. Soluble parasite extracts (from 9 · 10 6 parasites/lane) and recombinant falcipain-2 (lane 7) were analyzed by 12% gelatin substrate SDS-PAGE. Protease activity is identified as clear bands against a Coomassie blue-stained background. P. falciparum trophozoites dried on a glass slide, fixed with methanol, and evaluated by immunofluorescence microscopy with murine anti-falcipain-2 antiserum. The field is visualized by bright field microscopy (1) and fluorescence (2).


SUMMARY
Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mM glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.

INTRODUCTION
Malaria remains one of the most important infectious diseases in the world (1). A key factor contributing to our continued inability to control this disease is the increasing resistance of malaria parasites to available drugs (2). The identification and characterization of new targets for antimalarial chemotherapy is thus an urgent priority.
Among potential new targets for chemotherapy are proteases that degrade hemoglobin, a principal source of amino acids (3). In Plasmodium falciparum, the most virulent human malaria parasite, erythrocytic parasites transport hemoglobin to an acidic food vacuole, where the protein is hydrolyzed (4,5). Enzymes that appear to participate in hemoglobin degradation include aspartic (6,7), cysteine (8), and metallo (9) proteases.
Enzymes of each of these mechanistic classes are potential chemotherapeutic targets.
However, our understanding of the precise roles of plasmodial proteases in hemoglobin degradation is incomplete. In particular, studies of the roles of cysteine proteases in this process have been limited by the lack of straightforward purification schemes and difficulties with heterologous expression. Further characterization of plasmodial cysteine proteases and their roles in hemoglobin degradation should aid in the development of inhibitors of this process as antimalarial drugs.
Cysteine protease activity was originally identified in extracts of trophozoites, the erythrocytic parasite stage during which most hemoglobin degradation occurs (8,10). A critical role for a cysteine protease hemoglobinase was suggested when it was demonstrated that cultured malaria parasites failed to develop when incubated with cysteine protease inhibitors (8,11,12). Morphological examination of cysteine protease inhibitor-treated parasites revealed abnormally swollen, dark-staining food vacuoles, and biochemical evaluation indicated that the abnormality was caused by an accumulation of undigested hemoglobin in the food vacuole (8,(12)(13)(14)(15). These results suggested a central role for a cysteine protease in early steps in hemoglobin degradation by P. falciparum. However, other studies yielded conflicting results, suggesting that the trophozoite cysteine protease cannot hydrolyze native hemoglobin, but rather that this protease hydrolyzes only denatured hemoglobin fragments produced by the action of vacuolar aspartic proteases (16,17).
Until now, trophozoite cysteine protease activity has been attributed to the product of the single-copy falcipain gene (now renamed falcipain-1) (18). However, although recombinant falcipain-1 was shown to degrade hemoglobin (19), the lack of adequate expression and purification schemes has prevented the definitive characterization of the falcipain-1 gene product. We now report the purification of a major P. falciparum cysteine protease by affinity chromatography. This purification has yielded the unexpected result that the principal trophozoite cysteine protease, as determined with peptide substrates, is not the product of the falcipain-1 gene, but rather the product of a newly identified gene, falcipain-2. Our analysis of native and recombinant falcipain-2 strongly suggests that this protease plays a key role in the hydrolysis of both native and denatured hemoglobin by malaria parasites.
Preparation of parasite and subcellular fractions˙ Infected erythrocytes were washed with ice-cold phosphate buffered saline (PBS), treated with 0.1% saponin in ice-cold PBS for 5 min to lyse erythrocyte membranes (22), centrifuged (12,000 × g, for 10 min at 4°C), and washed three times with ice-cold PBS. For trophozoite extracts, pellets were suspended in extraction buffer (20 mM bis TrisCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenylmethyl-sulfonyl fluoride (PMSF), 10 µM pepstatin, pH 6.0), subjected to two freeze-thaw cycles to lyse parasite membranes, and repeatedly extracted in this buffer until protease activity was undetectable. Extracts were pooled and stored at -70°C.
For preparation of the crude vacuolar and cytosolic fractions, 2 × 10 8 trophozoites were prepared by lysis of erythrocyte membranes as described above, incubated with 5 volumes of 5% D-sorbitol for 10 min on ice and centrifuged at 650 × g for 7 min (16). The pellet was resuspended in extraction buffer and subjected to two freeze-thaw cycles to prepare the crude vacuole extract, which was stored at -70°C; the supernant was the cytosolic fraction. Purification of food vacuoles by density-gradient centrifugation was carried out exactly as previously described (23).
Preparation of affinity column˙ Glycyl-phenyalanyl-glycyl-semicarbazone (25 mg) was dissolved in 1 ml of methanol and combined with 5 ml of pre-swollen cyanogen bromide (CNBr)-activated aminohexyl-Sepharose 4B beads in a final volume of 50 ml of sodium carbonate buffer (100 mM, pH 8.0) as previously described (24). The mixture was rocked overnight at room temperature (RT), the beads were washed with 1 liter each of 50% methanol and deionized water, and unreactive sites were blocked with 6% ethanolamine for 4 h at RT. The beads were then washed with 1 liter each of deionized water and 20 mM TrisCl, pH 7.0, and the column was stored at 4°C.
Purification of native falcipain-2˙ Purification of falcipain-2 was carried out at 4°C using ice-cold buffers. The glycyl-phenylalanyl-glycyl-Sepharose column (~3 ml bed volume) was pre-equilibrated in binding buffer (extraction buffer with 2 mM dithiothreitol (DTT), 100 mM NaCl). Trophozoite extract (from ~3 × 10 10 trophozoites per experiment) was then adjusted to the same concentrations of NaCl and DTT as binding buffer and applied to the column. The flow-through was reapplied to the column, which was then washed sequentially with 10 bed volumes binding buffer and 6 bed volumes of 20 mM sodium acetate buffer (NaOAc), 1 mM EDTA, pH 5.5. For elution, 3 volumes of elution buffer (20 mM TrisCl, 1 mM EDTA, 1 mM 2,2´-dipyridyl disulfide, pH 7.0) was passed though the column, the flow was stopped, and the column was left overnight at 4°C. Elution was resumed with 9 additional bed volumes of elution buffer. The eluted enzyme was concentrated using Centricon 10 concentrators (Amicon) to a final volume of 200-300 µl. An equal volume of glycerol was added, and the enzyme was stored at -20°C.
For sequencing, falcipain-2 was electrophoresed on a 12.5% SDS-PAGE gel, transferred to an Immobilon-P SQ membrane (Millipore) and evaluated by Edman sequencing before or after treatment with CNBr. For mass spectrometry analysis, falcipain-2 was excised from a Coomassie Blue-stained SDS-PAGE gel and digested overnight with trypsin. Peptides were extracted from the gel slices with three 50% acetonitrile/5% formic acid washes. The combined supernatant was desalted with a C 18 ZipTip (Millipore) and analyzed using a PE BioSystems Voyager Elite MALDI-TOF mass spectrometer.
Isolation of DNA and RNA˙ Genomic DNA was isolated from schizont-stage parasites (prepared as discussed above) by treatment with lysis buffer (100 µg/ml proteinase K, 10 mM TrisCl, 100 mM EDTA, 0.5% SDS, pH 8.0) followed by phenol extraction and isopropanol precipitation. Total RNA was extracted from parasites using Large scale refolding was carried out in optimized refolding buffer (100 mM TrisCl, 1 mM EDTA, 20% glycerol, 250 mM L-arginine, 1 mM GSH, 1 mM GSSG, pH 8.0) with a 100-fold dilution of recombinant falcipain-2 (to 10 µg/ml) into 500 ml of the ice-cold buffer, incubation with moderate stirring at 4°C for 24 h, and concentration to 20 ml using a stirred cell with a 10 kDa cut-off membrane (Amicon) at 4°C. The sample was then filtered using a 0.22 µm syringe filter, dialyzed against a 100 × volume of binding buffer, and further purified by affinity chromatography using a glycylphenylalanyl-glycyl-Sepharose column, as described earlier for native falcipain-2.

Assays of falcipain-2 activity˙
For substrate gel analysis, samples were mixed with SDS-PAGE sample buffer lacking 2-mercaptoethanol and electrophoresed in a polyacrylamide gel copolymerized with 0.1% gelatin (8). The gel was then washed twice (30 min, RT) with 2.5% Triton X-100 and incubated overnight at 37°C in 100 mM NaOAc, 10 mM DTT, pH 5.5 before staining with Coomassie Blue.
Fluorimetric assays of native and recombinant falcipain-2 activity and inhibition were carried out as previously described (11,28) in 100 mM NaOAc, 10 mM DTT, pH 5.5 (or with changes in pH or reductant as described) in a final volume of 0.35 ml. In a series of seven purifications, 80-95% of soluble trophozoite cysteine protease activity bound to the affinity ligand. Re-application of flowthrough fractions to the affinity column allowed additional binding. After three applications, binding was complete, and residual cysteine protease activity, measured against the fluorogenic substrate Z-Phe-Arg-AMC, was 7% of initial activity. Whether the unbound activity represented additional falcipain-2 or other proteases is unknown. In any event, falcipain-2, the only protease purified by our affinity approach, appears to be the principal trophozoite cysteine protease. Purified falcipain-2 was stable, retaining greater than 95% of its activity after storage in 50% glycerol at -20°C for 30 days.
Falcipain-2 is concentrated in the parasite food vacuole˙ Crude food vacuole preparations were markedly enriched for cysteine protease activity (Table 1). Falcipain-2 was purified from the crude vacuolar fraction (Fig. 1D), and 85% of protease activity in density-gradient purified food vacuoles bound to the affinity ligand. Falcipain-2 was also present in cytosolic fractions, albeit with a much lower specific activity (Table   1). This activity may have been due to leakage of the protease from vacuoles during their preparation or the presence of the enzyme (possibly as an inactive proform that was activated during preparation) in the cytosol.

Identification and analysis of the falcipain-2 gene˙
The amino-terminal sequence of falcipain-2 matched the deduced amino acid sequence encoded by a 1455 bp ORF from the Sanger Centre P. falciparum genomic sequence database (within fragment pfg11_624). A portion of this ORF also matched a P. falciparum erythrocyticstage expressed sequence tag (Genbank N97987 (32)). The falcipain-2 gene was amplified from P. falciparum W2-strain trophozoite-stage cDNA and genomic DNA using primers spanning the ORF. The PCR products included identical uninterrupted ORFs which differed from the database sequence at 30 nucleotides (Fig. 2). The start codon for the falcipain-2 gene was assigned to the first ATG codon following a stop codon based on the first ATG rule (33,34), the presence of a consensus lower eukaryote initiation sequence (AXX-ATG-G (33, 35)), and markedly increased AT content upstream of the start codon, a feature of non-coding P. falciparum DNA (36).
The falcipain-2 gene predicts a fairly typical papain-family cysteine protease, with a large prodomain, a mature domain of 27 kDa, and conservation of active site amino acids (Fig. 3). A BLAST search showed falcipain-2 to be most similar among GenBank sequences to falcipain-1 (18), although identity between predicted mature domains (37%) was only slightly higher than that between falcipain-2 and many other cysteine proteases, including plant, viral, and mammalian enzymes. Falcipain-2 has some unusual features for a papain-family enzyme (37), including (a) lack of a typical mature protease processing site, (b) more predicted disulfide bonds (four) than many members of this family, (c) a 17 amino acid insert between the mature protease processing site and a highly conserved amino-terminal region, and (d) an insert between catalytic His and Asn residues at the location of a larger insert in falcipain-1.
As also seen with falcipain-1, the falcipain-2 prodomain is unusually large, with minimal amino-terminal sequence conservation, but modest conservation of the most carboxy-terminal sequence (for the 79 carboxy-terminal amino acids of the falcipain-2 prodomain, identity was 34% between falcipain-1 and falcipain-2 and 33% between falcipain-2 and papain). Falcipain-2 lacks a typical signal sequence, but does contain a 20 amino acid hydrophobic stretch that is predicted by the PRED-TMR algorithm to represent a transmembrane domain (26).
Digests of P. falciparum W2 strain genomic DNA were probed with the falcipain-2 gene. The probe hybridized strongly with two DNA fragments generated by each of 5 restriction endonucleases (Fig. 4). Three of these enzymes had no restriction sites within the -35FP2 probe, and for the other two enzymes, Eco RI and Bgl II, which had cleavage sites near the 5' terminus of the probe, a third DNA fragment was 17 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from recognized under lower stringency conditions (not shown). Probing the same blot under high stringency with a falcipain-1 probe yielded a completely different hybridization pattern (not shown). These results suggest the presence of a second gene that is closely related to falcipain-2. The gene, which is not falcipain-1, may be a third putative cysteine protease gene (named falcipain-3) which was also identified in a genomic database search (within Sanger Centre fragment pfg11_624) and has not yet been fully sequenced. Falcipain-3 is more similar in its mature domain sequence to falcipain-2 than is falcipain-1, but its predicted amino-terminal sequence differs from that of falcipain-2, and it was not detected in the cysteine protease activity purified from trophozoites (Fig. 3).

Falcipain-2 is an acidic cysteine protease with unique substrate specificity˙
Under our conditions of study, the hydrolysis of the synthetic peptide substrate Z-Phe-Arg-AMC by native falcipain-2 was optimal at acid pH and was markedly stimulated by reducing agents, as is typical for papain-family proteases (Fig. 7). Falcipain-2 was inhibited by the cysteine protease inhibitors E-64 and leupeptin, but not by inhibitors of other protease classes. These properties were very similar to those determined earlier for the cysteine protease activity of trophozoite extracts (8,11), supporting the conclusion that falcipain-2 is the principal cysteine protease of trophozoites. The properties of recombinant falcipain-2 were very similar to those of the native enzyme ( Fig. 7).
To begin to characterize the substrate specificity of falcipain-2 and to compare it with other papain-family enzymes, we evaluated the hydrolysis of a panel of synthetic peptide substrates. Native falcipain-2 cleaved 10 of 25 tested substrates; all of the cleaved substrates had arginine or lysine at the P 1 position (Table 2). Both native and 19 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from recombinant falcipain-2 showed a marked preference for leucine or phenylalanine at the P 2 position. The P 2 specificity of native falcipain-2 was compared with that of other papain-family proteases, using a panel of Z-X-Arg-AMC substrates (Fig. 8).
Falcipain-2 shared with cathepsin L, papain, and cruzain the preference for hydrophobic but not charged amino acids at the P 2 position. The strong falcipain-2 preference for leucine at P 2 was unique among the proteases tested.  (Fig. 9C). To investigate the subcellular localization of falcipain-2, methanol fixed trophozoites were probed with antiserum against the protease. The antiserum diffusely stained trophozoites, but not host erythrocytes (Fig. 9D). Staining of the parasite food vacuole was evident, although the signal was attenuated in the vicinity of hemozoin. This result suggests that falcipain-2 localizes to the food vacuole, 20 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from as shown biochemically ( Fig. 1 and Table 1), but also that the enzyme is present in the cytosol, probably as an inactive proform.
This activity required reducing agents, but physiological concentrations (1 mM) of GSH that did not appreciably denature hemoglobin (not shown) were adequate to support efficient hydrolysis of hemoglobin (Fig. 10). Denatured globin was more rapidly hydrolyzed than hemoglobin in a reducing environment (not shown), and also was slowly hydrolyzed under nonreducing conditions.

DISCUSSION
A principal cysteine protease of P. falciparum trophozoites was purified using an affinity chromatography protocol, which was adapted from a method for the purification of cathepsin B (24). Our purification provided, for the first time, a definitive amino-terminal sequence of this protease. Surprisingly, this sequence was not encoded by the falcipain-1 gene, but rather by a previously unidentified cysteine protease gene that we have named falcipain-2. In retrospect, it appears that the conclusion that falcipain-1 encodes the principal trophozoite cysteine protease (18) was based on cross-reactive antisera. Indeed, a rabbit antiserum raised against recombinant falcipain-1 reacted with purified falcipain-2 (data not shown). Falcipain-2 was the only protease purified by our affinity technique. Importantly, both sequencing and mass spectrometry analyses of purified protein did not detect the presence of falcipain-1 or falcipain-3. The biological roles of these other putative P. falciparum proteases are unknown. Falcipain-2 constituted at least 93% of soluble trophozoite cysteine protease activity, as measured with the substrate Z-Phe-Arg-AMC, but some activity was not accounted for by this enzyme, and activities may differ against different substrates. It is thus possible that the other plasmodial cysteine proteases are also active in trophozoites, and play ancillary roles in hemoglobin degradation. Falcipain-2 activity was optimal at acidic pH and concentrated in the acidic food vacuole, as would be expected for a trophozoite hemoglobinase.
The facile heterologous expression and refolding of falcipain-2 was somewhat surprising, as the expression of P. falciparum enzymes has often been problematic, and as our expression construct encoded only a small part of the prodomain. An extensive literature has documented the essential nature of protease prodomains in the expression and refolding of active enzymes (43)(44)(45). The prodomains presumably mediate folding and act as protease inhibitors (46)(47)(48). For multiple proteases, including α-lytic prodomains that mediate enzyme inhibition and/or refolding (54)(55)(56).
The refolding of falcipain-2 was accompanied by processing to the mature active protease. This processing was autocatalytic, as is the case with other papain-family proteases including papain (57), cathepsin B (58), cathepsin L (59), cathepsin K (60), and cruzain (61). Surprisingly, the processing occurred in refolding buffer at an alkaline pH (8.0) at which mature falcipain-2 has minimal activity against peptide substrates or hemoglobin. For other papain-family enzymes, acidic conditions appear to be required to unfold the prodomain and allow autoactivation (62). Processing of profalcipain-2 may have been expedited by the fact that our expression construct included a truncated prodomain. This domain was adequate to facilitate refolding, but may not have interacted with the active site in the manner shown for full prodomains of related enzymes (54)(55)(56). Thus, for expressed truncated profalcipain-2, the active site may have been free to cleave the truncated proregion under alkaline conditions. Of note, cleavages during refolding occurred at the native processing site and three sites 4-7 amino acids upstream of the native site, with the majority of molecules cleaved at sites containing P 2 leucine, a feature of the most favored peptide substrates identified for falcipain-2.
Falcipain-2 has some unusual features when compared to other papain-family cysteine proteases, including an unusually large prodomain. Interestingly, the prodomains of falcipain-1 (18) and its analogues from other plasmodial species (63,64) are even larger. Analysis of the specificity of falcipain-2 indicated that, at least with peptide substrates, the amino acid at the P 2 position plays a key role in mediating substrate specificity. This is also the case with other cathepsin L-like papain family proteases, but not with cathepsin B, which accepts chemically disparate P 2 amino acids in its substrates. Falcipain-2 showed a preference for the bulky hydrophobic amino acids leucine and phenylalanine over smaller hydrophobic (valine) or charged (arginine, glutamic acid) P 2 amino acids. The marked preference for leucine and poor acceptance of valine distinguished falcipain-2 from cathepsin L. The P 2 specificity of falcipain-2 was more similar to that of cathepsin K and cathepsin S, two other mammalian papainfamily cysteine proteases (68,69). Although results with peptide substrates may not predict specificity against physiological substrates, these data are consistent with a preference for P 2 leucine in inhibitors with potent antimalarial activity (22,28). The identification of lead falcipain-2 inhibitors may be expedited by efforts to develop cathepsin K inhibitors as treatments for osteoporosis (70).
Multiple P. falciparum food vacuole enzymes, including aspartic, cysteine, and metallo proteases, appear to participate in hemoglobin degradation, although the precise role of each protease is not well understood (5). It has been proposed that hemoglobin degradation is an ordered process in which aspartic proteases initiate hemoglobin hydrolysis, and a cysteine protease subsequently hydrolyzes denatured hemoglobin peptides (5,16). This model is appealing in its simplicity, but it cannot easily explain the observation with cultured parasites that the inhibition of falcipain-2 causes the food vacuole to fill with intact globin (8) falcipain-2 appears to be present in the food vacuole, as erythrocyte cytoplasm, which contains millimolar GSH (71), is transported into the food vacuole in large quantity, and parasites maintain millimolar concentrations of GSH via synthetic pathways (72)(73)(74) and the export of oxidized glutathione (72). Our results agree with earlier findings (17) that falcipain-2 more efficiently cleaves hemoglobin after its denaturation. These data support a model in which falcipain-2 participates in initial cleavages of hemoglobin and plays a key role in the hydrolysis of globin fragments after initial cleavages cause denaturation of the substrate.
Falcipain-2 and other P. falciparum hemoglobinases are promising chemotherapeutic targets. Indeed, inhibitors of both falcipain-2 (12,22,28,75) and the plasmepsins (6,(76)(77)(78) are potent antimalarials, and combinations of inhibitors of both classes of proteases yield synergistic antimalarial activity in vitro (14,79) and in vivo (79). The availablility of large quantities of active recombinant falcipain-2 offers hope that additional biochemical and structural characterization of the protease will benefit ongoing drug discovery efforts directed toward this enzyme.
Acknowledgments ˙ We thank Chi Chang, Wendy Kang, Andrey Semenov, and Belinda Lee for expert technical assistance, Jiri Gut for assistance with immunofluorescence microscopy, Elizabeth Hansell, David Tew and Robert Smith for the gifts noted, and Richard Jacob for assistance with mass spectrometric analysis. This work was supported by grants from the National Institutes of Health. were aligned using the DNASTAR program (CLUSTAL method). Numbering is for the known or predicted mature sequence of each protease, dashes represent gaps required for optimal alignment, conserved active site amino acids are highlighted, and identities with falcipain-2 are shaded. were analyzed by 12% gelatin substrate SDS-PAGE. Protease activity is identified as clear bands against a Coomassie blue-stained background. D. Localization of falcipain-2. Erythrocytes infected with P. falciparum trophozoites were dried on a glass slide, fixed with methanol, and evaluated by immunofluorescence microscopy with murine anti-falcipain-2 antiserum. The same field is visualized by bright field microscopy (1) and fluorescence (2). Parasites within erythrocytes are visible with both modalities.
Labeling of parasites, but not infected or uninfected erythrocyte cytosol, is apparent.