Title: Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)
Abstract: The cDNA encoding the precursor of an aspartic proteinase from the flowers of the cardoon, Cynara cardunculus, was expressed in Pichia pastoris, and the recombinant, mature cyprosin that accumulated in the culture medium was purified and characterized. The resultant mixture of microheterogeneous forms was shown to consist of glycosylated heavy chains (34 or 32 kDa) plus associated light chains with molecular weights in the region of 14,000–18,000, resulting from excision of most, but not all, of the 104 residues contributed by the unique region known as the plant specific insert. SDS-polyacrylamide gel electrophoresis under non-reducing conditions indicated that disulfide bonding held the heavy and light chains together in the heterodimeric enzyme forms. In contrast, when a construct was expressed in which the nucleotides encoding the 104 residues of the plant specific insert were deleted, the inactive, unprocessed precursor form (procyprosin) accumulated, indicating that the plant-specific insert has a role in ensuring that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature, active proteinase. Kinetic parameters were derived for the hydrolysis of a synthetic peptide substrate by wild-type, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were mapped using a systematic series of synthetic inhibitors. The significance is discussed of the susceptibility of cyprosin to inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme. The cDNA encoding the precursor of an aspartic proteinase from the flowers of the cardoon, Cynara cardunculus, was expressed in Pichia pastoris, and the recombinant, mature cyprosin that accumulated in the culture medium was purified and characterized. The resultant mixture of microheterogeneous forms was shown to consist of glycosylated heavy chains (34 or 32 kDa) plus associated light chains with molecular weights in the region of 14,000–18,000, resulting from excision of most, but not all, of the 104 residues contributed by the unique region known as the plant specific insert. SDS-polyacrylamide gel electrophoresis under non-reducing conditions indicated that disulfide bonding held the heavy and light chains together in the heterodimeric enzyme forms. In contrast, when a construct was expressed in which the nucleotides encoding the 104 residues of the plant specific insert were deleted, the inactive, unprocessed precursor form (procyprosin) accumulated, indicating that the plant-specific insert has a role in ensuring that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature, active proteinase. Kinetic parameters were derived for the hydrolysis of a synthetic peptide substrate by wild-type, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were mapped using a systematic series of synthetic inhibitors. The significance is discussed of the susceptibility of cyprosin to inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme. Sequences have been elucidated recently for genes encoding aspartic proteinases of plant origin, e.g. barley (1Runeberg-Roos P. Törmäkangas K. Östman A. Eur. J. Biochem. 1991; 202: 1021-1027Crossref PubMed Scopus (104) Google Scholar), rice (2Asakura T. Watanabe H. Abe K. Arai S. Eur. J. Biochem. 1995; 232: 77-83Crossref PubMed Scopus (89) Google Scholar), tomato (3Schaller A. Ryan C.A. Plant Mol. Biol. 1996; 31: 1073-1077Crossref PubMed Scopus (82) Google Scholar), and oilseed rape and Arabidopsis (4D'Hondt K. Stack S. Gutteridge S. Vanderkerckhove J. Krebbers E. Gal S. Plant Mol. Biol. 1997; 33: 187-192Crossref PubMed Scopus (37) Google Scholar). All of these sequences predict that, by comparison with the well studied aspartic proteinases from mammals, fungi, yeasts likeSaccharomyces (5Van den Hazel H.B. Wolff A.M. Kielland-Brandt M.C. Winther J.R. Biochem. J. 1997; 326: 339-344Crossref PubMed Scopus (16) Google Scholar) and Candida (6Monod M. Togni G. Hube B. Sanglard D. Mol. Microbiol. 1994; 13: 357-368Crossref PubMed Scopus (218) Google Scholar), parasites likePlasmodium falciparum (7Dame J.B. Reddy G.R. Yowell C.A. Dunn B.M. Kay J. Berry C. Mol. Biochem. Parasitol. 1994; 64: 177-190Crossref PubMed Scopus (106) Google Scholar), and viruses such as HIV 1The abbreviations used are: HIV, human immunodeficiency virus; ACHPA, 4-amino-5-cyclohexyl-3-hydroxypentanoic acid; AHPPA, 4-amino-3-hydroxy-5-phenylpentanoic acid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s) (8Wlodawer A. Vondrasek J. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 249-284Crossref PubMed Scopus (636) Google Scholar), approximately 100 extra amino acids are introduced into the C-terminal domain of the newly synthesized plant polypeptides. The function of this region or plant-specific insert (9Ramalho-Santos M. Verissı́mo P. Cortes L. Samyn B. van Beeumen J. Pires E. Faro C. Eur. J. Biochem. 1998; 255: 133-138Crossref PubMed Scopus (68) Google Scholar) is currently unknown, but, as it does share considerable sequence identity with a group of mammalian sphingolipid activator proteins known as saposins (10Guruprasad K. Törmäkangas K. Kervinen J. Blundell T.L. FEBS Lett. 1994; 352: 131-136Crossref PubMed Scopus (79) Google Scholar, 11O'Brien J.S. Kretz K.A. Dewji N. Wenger D.A. Esch F. Fluharty A.L. Science. 1988; 241: 1098-1101Crossref PubMed Scopus (211) Google Scholar), it has been postulated to bind selectively to certain lipids and thus direct the precursor form of the aspartic proteinase into the appropriate cytomorphological compartment in the plant cell (10Guruprasad K. Törmäkangas K. Kervinen J. Blundell T.L. FEBS Lett. 1994; 352: 131-136Crossref PubMed Scopus (79) Google Scholar). Alignment of the sequences of the inserts predicted by the plant aspartic proteinase genes with those of saposins correctly positions all six cysteine residues and a glycosylation site and also maintains the pattern of hydrophobic residues described previously for all known saposins (12Ponting C.P. Russell R.B. Trends Biochem. Sci. 1995; 20: 179-180Abstract Full Text PDF PubMed Scopus (105) Google Scholar). However, each plant-specific insert does not consist of a single saposin domain but appears to correspond to the C-terminal portion of one saposin domain linked to the N-terminal portion of a second saposin domain. On this basis, the plant-specific inserts have been described as swaposins (12Ponting C.P. Russell R.B. Trends Biochem. Sci. 1995; 20: 179-180Abstract Full Text PDF PubMed Scopus (105) Google Scholar), indicating that they are likely to have a structure similar to that of the saposins, but with N- and C-terminal halves interchanged in sequence order. Relatively little information has been reported to date on aspartic proteinases originating from plant tissues, but, in the few enzymes that have been isolated, this swaposin domain is not present and appears to have been excised by post-translational processing. Nothing is known of the enzyme(s) responsible in planta, but the excision of each insert is imprecise, resulting in the generation of a complex mixture of heterogeneous, mature aspartic proteinases within the tissues of each of the plants that has been studied, e.g. from seeds of barley (13Sarkkinen P. Kalkkinen N. Tilgmann C. Siuro J. Kervinen J. Mikola L. Planta. 1992; 186: 317-323Crossref PubMed Scopus (104) Google Scholar), pumpkin (14Hiraiwa N. Kondo M. Nishimura M. Hara-Nishimura I. Eur. J. Biochem. 1997; 246: 133-141Crossref PubMed Scopus (89) Google Scholar), and Arabidopsis thaliana (15Mutlu A. Pfeil J.E. Gal S. Phytochemistry. 1998; 47: 1453-1459Crossref PubMed Scopus (28) Google Scholar), from rice (16Asakura T. Watanabe H. Abe K. Arai S. J. Agric. Food Chem. 1997; 45: 1070-1075Crossref Scopus (51) Google Scholar) and from flowers of the cardoon, Cynara cardunculus (9Ramalho-Santos M. Verissı́mo P. Cortes L. Samyn B. van Beeumen J. Pires E. Faro C. Eur. J. Biochem. 1998; 255: 133-138Crossref PubMed Scopus (68) Google Scholar, 17Heimgartner U. Pietrzak M. Geertsen R. Brodelius P.E. da Silva Figueirdo A.C. Pais M.S.S. Phytochemistry. 1990; 29: 1405-1410Crossref Scopus (168) Google Scholar, 18Verissı́mo P. Faro C. Moir A.J.G. Lin Y. Tang J. Pires E. Eur. J. Biochem. 1996; 235: 762-768Crossref PubMed Scopus (145) Google Scholar). Commonly, the enzymes thus generated are heterodimers with molecular weights in the region of 40,000–45,000. This complexity of natural isoforms is compounded even further by the expression of several genes in the plant tissues, each encoding closely related enzymes so that physicochemical and enzymatic characterization of naturally occurring aspartic proteinases isolated directly from plants has been made rather difficult. A recombinant approach was thus employed in attempts to gain an initial insight into the significance of the plant-specific insert and to examine the activity and specificity of an aspartic proteinase from the plant kingdom. We have expressed in the methylotrophic yeast, Pichia pastoris, the cDNA encoding the precursor of one aspartic proteinase from the flowers of the cardoon, C. cardunculus (19Cordeiro M. Xue Z.-T. Pietrzak M. Pais M.S. Brodelius P.E. Plant Mol. Biol. 1994; 24: 733-741Crossref PubMed Scopus (74) Google Scholar). Extracts of these flowers have been documented previously to contain a number of isoforms of aspartic proteinases called cyprosins and cardosins (9Ramalho-Santos M. Verissı́mo P. Cortes L. Samyn B. van Beeumen J. Pires E. Faro C. Eur. J. Biochem. 1998; 255: 133-138Crossref PubMed Scopus (68) Google Scholar, 17Heimgartner U. Pietrzak M. Geertsen R. Brodelius P.E. da Silva Figueirdo A.C. Pais M.S.S. Phytochemistry. 1990; 29: 1405-1410Crossref Scopus (168) Google Scholar,18Verissı́mo P. Faro C. Moir A.J.G. Lin Y. Tang J. Pires E. Eur. J. Biochem. 1996; 235: 762-768Crossref PubMed Scopus (145) Google Scholar) and have been used for centuries as coagulants in traditional cheese-making in regions of southern Europe, particularly the Iberian peninsula (20Wiklund A. Botan. J. Linnean Soc. 1992; 109: 75-123Crossref Scopus (89) Google Scholar). A cDNA clone encompassing the full-length precursor of a cyprosin was isolated by courtesy of Dr. M. Pietrzak (Basel, Switzerland) by rescreening of a cDNA library prepared from flower buds of C. cardunculus, as described previously (19Cordeiro M. Xue Z.-T. Pietrzak M. Pais M.S. Brodelius P.E. Plant Mol. Biol. 1994; 24: 733-741Crossref PubMed Scopus (74) Google Scholar, 21Brodelius P.E. Cordeiro M. Mercke P. Domingos A. Clemente A. Pais M.S. James M.N.G. Aspartic Proteinases. Plenum Press, New York1998: 435-439Google Scholar). This was amplified by PCR using Vent DNA polymerase (New England Biolabs) with appropriate forward and reverse primers (5′-GG AAT TCC GGA TCC TCA CCT ACT GCA TTT TCG GTC-3′ and 5′-GA ATT CCG GGA TCC TCA AGC TGC TTC TGC AAA-3′, respectively; purchased from Amersham Pharmacia Biotech, Cambridge, United Kingdom (UK)) and subcloned into the pUC18 vector. In turn, this recombinant pUC18 was used as template DNA for overlapping PCR mutagenesis reactions, as described previously (22Tigue N.J. Kay J. J. Biol. Chem. 1998; 273: 26441-26446Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Mutations were introduced at appropriate locations by two initial and one subsequent PCR reaction. To modify the sequence connecting the propart region to the mature cyprosin enzyme, a 241-bp fragment spanning from the pUC18 vector to the desired, mutated cleavage junction was amplified using appropriate forward and reverse primers (5′-GTT GGG TAA CGC CAG GG-3′ = F1 and 5′-CCT CAG AAA AGC AGC GAA GCC-3′, respectively). The second PCR amplified a 478-bp fragment using a forward primer (5′-GGC TTC GCT GCT TTT CTG AGG-3′) and a reverse primer (5′-GTT CTT GAA CAA GAC CTT G-3′ = R1) complementary to the sequence located downstream from the region into which the mutations were to be introduced and downstream from a BglII restriction site. The purified fragments were combined and used as template DNA in a final PCR using the F1 and R1 flanking primers. The resultant 698-bp amplicon was digested with the restriction endonucleases HindIII and BglII and subcloned to replace the corresponding wild-type segment in the original procyprosin construct. The region encoding the 104 residues of the plant specific insert was excised by an identical strategy. The primer pairs were 5′-GG AAT TCC GGA TCC TCA CCT ACT GCA TTT TCG GTC-3′ = F2 and 5′-CTG GAT GAG AGG TAC CGC ACC AAT TGC ATG ATT GAT TTC-3′; and 5′-GTA CCT CTC ATC CAG GGA GAA TCA GCA GTA GAC TGC AAC-3′ and 5′-GA ATT CCG GGA TCC TCA AGC TGC TTC TGC AAA-3′ = R2. The resultant 911- and 296-bp fragments were combined and used in the final reaction using the F2/R2 combination of primers. The 1192-bp amplicon thus generated was digested with BamHI, ligated into a similarly treated, dephosphorylated pUC18 vector, and the reaction mixture was used to transform competent Escherichia coli (DH5α) cells. The authenticity of all manipulations was verified by dideoxy sequencing of both DNA strands. Restriction digestion withBamHI enabled subcloning of each desired cDNA into the pET-3a expression vector (AMS Biotechnology, Witney, UK). Expression of this recombinant plasmid was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside (0.4 mm final concentration) when the cells had reached anA 600 of 0.6. After 3 h, the cells were harvested and lysed, and the resultant insoluble material was washed at 4 °C for 4 h with 100 mm Tris-HCl, pH 11.0, containing 50 mm β-mercaptoethanol. After centrifugation at 16,000 × g for 30 min, the resultant pellet was solubilized by stirring at 25 °C for 16 h in 6 murea in 100 mm Tris-HCl buffer, pH 8.0, supplemented with 1 mm glycine, 1 mm EDTA, and 50 mmβ-mercaptoethanol. The cDNA encoding each procyprosin (wild-type or mutants) was subcloned into the expression vector pPICZα C (Invitrogen, Leek, Netherlands) and used to transformE. coli (Top10F′) cells. Selection of transformants containing the pPICZα C vector was made on low salt LB agar containing zeocin (50 μg ml−1). Plasmid DNA was purified from selected colonies, linearized by digestion with the restriction endonuclease PmeI, and electroporated into P. pastoris (KM71) cells according to the manufacturer's instructions (Invitrogen). Yeast colonies that had undergone the appropriate recombination events to incorporate the procyprosin gene into the host chromosome were used to inoculate BMGY medium (100 ml) containing ampicillin (100 μg/ml). The flasks (250 ml) were shaken at 30 °C until the cells attained anA 600 of 5.0. After harvesting by centrifugation at 3000 × g for 5 min at room temperature, the cell pellets were resuspended in 20-ml aliquots of BMMY induction medium containing ampicillin (100 μg/ml) in 100-ml flasks and shaken at 30 °C over a period of 7 days. Expression was induced over this time period by the addition of methanol to a final concentration of 0.5% every 24 h. Samples of medium from induced Pichiacells were analyzed by SDS-PAGE followed by staining with Coomassie Blue or by Western blotting using an anti-cyprosin antiserum that had been raised in rabbits (17Heimgartner U. Pietrzak M. Geertsen R. Brodelius P.E. da Silva Figueirdo A.C. Pais M.S.S. Phytochemistry. 1990; 29: 1405-1410Crossref Scopus (168) Google Scholar). Detection of immunoreactive bands used a goat anti-rabbit IgG-alkaline phosphatase conjugate as described previously (22Tigue N.J. Kay J. J. Biol. Chem. 1998; 273: 26441-26446Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Proteinase activity was monitored during purification steps using the substrate Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu 2The nomenclature system ∼P6-P5-P4-P3-P2-P1*P1′-P2′-P3′∼ is used to depict amino acids adjacent to the residues in the P1 and P1′ positions that contribute the scissile peptide bond (indicated by an asterisk). at pH 4.0, with the cleavage being monitored by fast protein liquid chromatography using a Pep-RPC reverse phase column (22Tigue N.J. Kay J. J. Biol. Chem. 1998; 273: 26441-26446Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Aliquots of conditioned medium were diluted by 10-fold prior to each assay in order to circumvent the complications caused by the increasing amounts of a yellow pigment that is released by the yeast cells into the growth medium. This absorbs in the UV region of the spectrum and thus interferes with detection of the products of peptide substrate digestion. Samples of conditioned medium harvested after 6 days were adjusted to pH 4.0 by the addition of 1 m sodium formate buffer, pH 3.0, and dialyzed at 4 °C against 10 mm sodium formate buffer, pH 4.0, containing 50 mm sodium chloride with five changes of buffer. Dialysates were applied to a Hi-Trap SP column fitted into a fast protein liquid chromatography instrument (Amersham Pharmacia Biotech), and elution was continued with the same pH 4.0 buffer. Under these conditions, the yellow Pichia-derived pigment was not retained by the column. After extensive washing, recombinant cyprosin was eluted using a linear gradient of 50–500 mm NaCl in the sodium formate buffer at pH 4.0. Fractions were monitored for activity and combined as appropriate. Samples for N-terminal sequencing were subjected to SDS-PAGE under reducing or non-reducing conditions and blotted onto polyvinylidene difluoride membrane, and appropriate bands were subjected to automated Edman degradation, as described previously (23Hill J. Montgomery D.S. Kay J. FEBS Lett. 1993; 326: 101-104Crossref PubMed Scopus (29) Google Scholar). Attempts to derive C-terminal sequence on relevant bands were carried out (24Arnold D. Keilholz W. Schild H. Dumrese T. Stevanovic S. Rammensee H.-G. Biol. Chem. 1997; 378: 883-891Crossref PubMed Scopus (9) Google Scholar), using a Hewlett-Packard G1009A C-terminal sequencing system. Deglycosylation reactions were carried out with N-glycosidase F (Roche Molecular Biochemicals, Mannheim, Germany) in 250 mmTris-HCl buffer, pH 8.8, at 37 °C for 1 h. A naturally occurring preparation of isoform 3 of cyprosin was purified to homogeneity from the dried flowers of C. cardunculus as described previously (17Heimgartner U. Pietrzak M. Geertsen R. Brodelius P.E. da Silva Figueirdo A.C. Pais M.S.S. Phytochemistry. 1990; 29: 1405-1410Crossref Scopus (168) Google Scholar). Kinetic parameters for hydrolysis of the chromogenic substrate Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu were derived spectrophotometrically as described previously (23Hill J. Montgomery D.S. Kay J. FEBS Lett. 1993; 326: 101-104Crossref PubMed Scopus (29) Google Scholar). Values of k cat were derived from the equation V max =k cat × [E t], where the concentration of active enzyme E t was derived by active site titration using preparations of isovaleryl-pepstatin of precisely defined concentration (23Hill J. Montgomery D.S. Kay J. FEBS Lett. 1993; 326: 101-104Crossref PubMed Scopus (29) Google Scholar). Inhibition constants were derived at pH 5.0, and the estimated error on all measurements was always less than ±15%. However, it was necessary to use final concentrations of cyprosin in the assay cuvettes of approximately 5 nm, and so K i values for tight-binding inhibitors (whereK i ≪ 0.1 × E t) are given as best estimates, indicated by the < symbol. The synthetic inhibitors of HIV proteinase, Saquinavir and Indinavir, were generously supplied by Dr. J. A. Martin (Roche Products Ltd., Welwyn Garden City, Herts, UK), Ritonavir by Dr. D. Kempf (Abbott), and HBY-793 by Dr. J. Knolle (Hoechst AG, Frankfurt, Germany), respectively. All of the other compounds used were the kind gifts of Dr. D. F. Veber, formerly of Merck, Sharp and Dohme Research Laboratories, West Point, PA. Protein inhibitors from potato, Ascaris lumbricoides, and Saccharomyces cerevisiae were the respective gifts of Drs. J. Brzin and B. Strukelj (J. Stefan Institute, Ljubljana, Slovenia), Dr. R. J. Peanasky (formerly of the University of South Dakota, Vermillion, SD), and Dr. L. H. Phylip (School of Biosciences, Cardiff University, Cardiff, Wales). The nucleotide sequence of the procyprosin clone isolated by rescreening of the cDNA library from flower buds of C. cardunculus (19Cordeiro M. Xue Z.-T. Pietrzak M. Pais M.S. Brodelius P.E. Plant Mol. Biol. 1994; 24: 733-741Crossref PubMed Scopus (74) Google Scholar, 21Brodelius P.E. Cordeiro M. Mercke P. Domingos A. Clemente A. Pais M.S. James M.N.G. Aspartic Proteinases. Plenum Press, New York1998: 435-439Google Scholar) has been deposited in the EMBL/GenBank data bases under the accession number X81984. The amino acid sequence predicted by this clone is aligned with that of human procathepsin D in Fig. 1. From this, it is apparent that (i) the plant gene encodes an insert of 104 residues within the C-terminal domain that is not present in the mammalian enzyme, and (ii) both proteins are predicted to have one common glycosylation motif (at Asn67-Gly68-Thr69); the other known site of carbohydrate attachment in cathepsin D (at Asn183-Val184-Thr185) is not present in the cyprosin sequence, although an additional glycosylation motif (at Asn83I-Glu84I-Thr85I) 3In keeping with IUB convention, putative plant-specific insert residues have been designated with the suffixI, and prosegment residues are assigned the suffixP. is present in the plant-specific insert of cyprosin (Fig. 1 and see Introduction). Excluding the plant-specific insert, the sequence of the mature enzyme region of this cyprosin shares 52% identity with that of cathepsin D; in contrast, the two prosegments have very little similarity to one another. As described under "Materials and Methods," the cDNA encoding this wild-type form of procyprosin was introduced into pET-3a. Expression in E. coli strain BL21 (DE3) pLysS resulted in the accumulation of recombinant protein at a high level (estimated to be 10–20 mg/liter of culture) but this was insoluble and misfolded. Despite extensive efforts, suitable conditions to prepare significant amounts of properly folded protein could not be established. Consequently a different expression system was required and the methylotrophic yeast, P. pastoris, was selected since this has been used previously to produce an aspartic proteinase (precursor) in a soluble, properly folded form (25Yamada M. Azuma T. Matsuba T. Iida H. Suzuki H. Yamamoto K. Kohli Y. Hori H. Biochim. Biophys. Acta. 1994; 1206: 279-285Crossref PubMed Scopus (24) Google Scholar). The procyprosin construct was subcloned into the pPICZαC plasmid, and appropriate recombinants of the P. pastoris cells (KM71 strain) were selected as described under "Materials and Methods." Expression of the procyprosin gene resulted in the appearance in the culture medium of a cluster of immunoreactive bands in the 32–34-kDa region, together with a second cluster in the region between 14 and 18 kDa (Fig. 2 A). The relative proportions of each band in each of the clusters varied from batch-to-batch of induced culture medium so the clusters are referred to as heavy and light chains, respectively. The heavy chain (32–34 kDa) cluster was apparent as early as the second day of induction, while the light chains (14–18 kDa) became visible after about day 5. The recombinant protein was purified from medium harvested after 6 days, as described under "Materials and Methods." Acidification and dialysis of the medium at pH 4.0, followed by chromatography on a HiTrap SP column, successfully removed the yellow pigment that is a persistent contaminant released into the culture medium of inducedPichia cells. The recombinant protein that was eluted by the salt gradient from the HiTrap SP column did not emerge in a sharp peak but rather was a broad smear of material that absorbed at 280 nm and that reacted with the cyprosin antiserum (data not shown). Early fractions of material contained a mixture of 34- and 32-kDa heavy chains in which the 34-kDa band was predominant (Fig.3 A, lane 1), while in the later fractions the 32-kDa band was most abundant (Fig. 3 A, lane 2). All of the fractions also contained light chains migrating in the 14–18-kDa range, which stained only weakly with Coomassie Blue. The 34- and 32-kDa heavy chains and the light chains were all immunoreactive with anti-cyprosin antiserum (data not shown). Thus, it was apparent that all of the contaminating proteins had been removed and that the only remaining proteins were derived from recombinant (pro)cyprosin. The yield of (total) purified protein was approximately 1 mg/liter of culture medium. However, microheterogeneity was clearly evident.Figure 3Analysis by SDS-PAGE of recombinant proteins and naturally occurring cyprosin. A, early (lane 1) and late (lane 2) fractions eluted from chromatography of recombinant cyprosin on a Hi Trap SP column, were resolved under reducing conditions. B, a late fraction aliquot of recombinant wild-type cyprosin (lanes 3 and 5) and a sample of naturally occurring cyprosin (isoform 3; lanes 4and 6) were subjected to electrophoresis under reducing (lanes 3 and 4) and non-reducing (lanes 5 and 6) conditions.C, aliquots of recombinant wild-type cyprosin before (lane 7) and after (lane 8) treatment with N-glycosidase F were analyzed under reducing conditions. Lane 9 depicts under reducing conditions a sample of the mutant procyprosin in which the plant-specific insert was deleted and the sequence between propart and mature enzyme regions was altered to ∼Phe-Ala-Ala-Phe∼.Lanes 1–6 and 9 were stained with Coomassie Blue after electrophoresis, whereas lanes 7 and 8 were revealed by Western blotting with an anti-cyprosin antiserum, demonstrating that the antibodies are capable of recognizing protein epitopes. Markers of M rapproximately 43,000, 29,000, 18,000, and 14,000 migrated as indicated. Since the cyprosin light chain bands were difficult to visualize (panel A), only the heavy chain regions of the gels are depicted in lanes 3–9.View Large Image Figure ViewerDownload (PPT) The 34- and 32-kDa heavy chains were resolved from one another by SDS-PAGE under reducing conditions and blotted onto polyvinylidene difluoride membrane. N-terminal sequencing by Edman degradation revealed that both were identical and contained overlapping sequences (in the ratio 40:60).SEQUENCES 1 And 2 The distinction in the sizes of these heavy chains must therefore be accounted for by differences at their C termini. The N termini identified for the recombinant heavy chains are displaced further upstream by three and one residue, respectively, from the Asp residue (Fig. 1) that was suggested to be at the N terminus of the heavy chain of a cyprosin isoform isolated from flowers of C. cardunculus (21Brodelius P.E. Cordeiro M. Mercke P. Domingos A. Clemente A. Pais M.S. James M.N.G. Aspartic Proteinases. Plenum Press, New York1998: 435-439Google Scholar). Processing of the recombinant procyprosin precursor had thus taken place to remove the prosegment by cleavage at two adjacent sites in the sequence, resulting in microheterogeneity at the N termini of the resultant (34/32 kDa) heavy chains of mature cyprosin. After SDS-PAGE under reducing conditions and blotting onto polyvinylidene difluoride, attempts were also made at N-terminal sequencing of the 14–18-kDa light chains. In all cases, however, multiple residues were detected in every cycle, making assignment difficult, but indicating that the array of light chains observed was likely to have arisen from microheterogeneity at their N termini. However, when samples of recombinant cyprosin were subjected to SDS-PAGE under non-reducing conditions, a different effect was observed. A typical example is depicted in Fig. 3 B(compare lanes 3 and 5). This preparation of recombinant cyprosin consisted almost exclusively of 32-kDa heavy chain under reducing conditions but in the absence of reducing agent, the dominant band detected was at ∼46 kDa. N-terminal analysis of this 46-kDa band gave the same microheterogeneous pair of sequences, as was detected for the 34- and 32-kDa bands from the reduced SDS gel. In addition, however, a third sequence was detected in the non-reduced sample.SEQUENCES 3 This sequence is likely to have been derived from a light chain that had remained attached to the heavy chains under the non-reducing conditions, thereby indicating that the recombinant 34/32-kDa heavy chains and the light chains may be linked by disulfide bonds formed between sequences of the plant-specific insert that remain attached at the C- and N termini of the heavy and light chains, respectively, after excision of the bulk of the plant-specific insert by thePichia cells. The presence of Cys97Iin the N-terminal sequence of this light chain indicated its availability for disulfide bonding. No data were obtained from attempts at C-terminal sequencing of the 34-kDa heavy chain, but, for the 32-kDa heavy chain, leucine was identified as the C-terminal residue, with threonine occupying the penultimate position. This ∼Thr-Leu combination occurs only once in the sequence Cys3I-Lys4I-Thr5I-Leu6Inear the predicted N terminus of the plant-specific insert region (Fig.1). Proteolytic processing after this leucine residue would thus result in Cys3I remaining within the heavy chain, which would be predicted to have an M r of 29,000 on SDS-PAGE under reducing conditions. Treatment (of a different preparation of recombinant cyprosin which contained both 34- and 32-kD