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 and the of residues for all known saposins Biochem. 1995; PubMed Scopus Google Scholar). plant-specific insert does not consist of a domain but to to the C-terminal of domain to the of a this the plant-specific inserts have been as Biochem. 1995; PubMed Scopus Google Scholar), indicating that are to have a to that of the but with and C-terminal in sequence has been to aspartic proteinases from plant but, in the that have been this domain is not and to have been by is known of the in but the excision of insert is resulting in the of a mixture of mature aspartic proteinases the of of the that has been e.g. from of barley P. N. C. J. Kervinen J. L. PubMed Scopus (104) Google Scholar), N. M. M. Eur. J. Biochem. 1997; PubMed Scopus (89) Google Scholar), and Arabidopsis A. Gal S. 1998; PubMed Scopus Google Scholar), from rice T. Watanabe H. Abe K. Arai S. J. 1997; Scopus Google Scholar) and from flowers of the cardoon, Cynara (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, M. Scopus Google Scholar, P. Faro C. J. Pires E. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar). the thus are with molecular weights in the region of of is by the of genes in the plant encoding that and of aspartic proteinases from has been recombinant was thus in to an into the significance of the plant-specific insert and to the and of an aspartic proteinase from the plant have expressed in the Pichia pastoris, the cDNA encoding the precursor of aspartic proteinase from the flowers of the cardoon, C. M. M. Plant Mol. Biol. 1994; PubMed Scopus Google Scholar). of these flowers have been to a of of aspartic proteinases and (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, M. Scopus Google P. Faro C. J. Pires E. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar) and have been used for as in in of particularly the A. J. Scopus (89) Google Scholar). cDNA the precursor of a cyprosin was by of M. by of a cDNA from of C. cardunculus, as M. M. Plant Mol. Biol. 1994; PubMed Scopus Google Scholar, M. P. A. A. Scholar). was by using polymerase with appropriate and and from and into the In this recombinant was used as for as Kay J. J. Biol. 1998; PubMed Scopus Google Scholar). were introduced at appropriate by and the sequence the region to the mature cyprosin a from the to the was using appropriate and and The a using a and a to the sequence from the region into which the were to introduced and from a The purified were and used as in a using the and The resultant was with the and and to the in the The region encoding the 104 residues of the plant specific insert was by an The were and and and The resultant and were and used in the using the of The thus was with into a and the mixture was used to The of all was by of of cDNA into the of this recombinant was by the of when the of the were and and the resultant was at for with 100 pH at for the resultant was by at for in in 100 pH with and The cDNA encoding or was into the and used to of the was was purified from by with the and into P. to the that the appropriate to the into the were used to medium The were at the of by at for at the cell were in of medium in and at a of was this by the of to a of of medium from were by by with or by using an that been in M. Scopus Google Scholar). of used a as Kay J. J. Biol. 1998; PubMed Scopus Google Scholar). was using the substrate is used to amino acids to the residues in the and positions that the peptide by an at pH with the being by using a Kay J. J. Biol. 1998; PubMed Scopus Google Scholar). of medium were by to in to the by the of a that is by the into the in the region of the and thus with of the of peptide substrate of medium were to pH by the of pH and at against pH with of were to a into a and was with the pH these the was not by the recombinant cyprosin was using a of in the at pH were for and as for were to under or non-reducing conditions and and appropriate were to as J. Kay J. FEBS Lett. 326: PubMed Scopus Google Scholar). to C-terminal sequence were D. H. T. S. Biol. 1997; PubMed Scopus Google Scholar), using a C-terminal were with in pH at for of of cyprosin was purified to from the flowers of C. as M. Scopus Google Scholar). Kinetic parameters for hydrolysis of the substrate were derived as J. Kay J. FEBS Lett. 326: PubMed Scopus Google Scholar). of were derived from the the of active enzyme was derived by active using of of J. Kay J. FEBS Lett. 326: PubMed Scopus Google Scholar). were derived at pH and the all was it was to of cyprosin in the of approximately and values for inhibitors are as indicated by the The synthetic inhibitors of HIV and were by J. A. by D. and by J. All of the used were the of D. F. of and inhibitors from and were the of J. and B. J. of the of and L. H. of The sequence of the by of the cDNA from of C. M. M. Plant Mol. Biol. 1994; PubMed Scopus Google Scholar, M. P. A. A. Scholar) has been in the under the The amino sequence predicted by this is with that of human in it is that the plant an insert of 104 residues the C-terminal domain that is not in the mammalian and proteins are predicted to have the known of in is not in the cyprosin an with plant-specific insert residues have been with the and residues are the is in the plant-specific insert of cyprosin and the plant-specific the sequence of the mature enzyme region of this cyprosin identity with that of in contrast, the have to under and the cDNA encoding this form of was introduced into in E. in the of recombinant at a to of but this was and conditions to of properly folded not a was and the P. pastoris, was this has been used to an aspartic proteinase in a properly folded form M. T. T. H. H. K. H. Biophys. 1994; PubMed Scopus Google Scholar). The construct was into the and appropriate of the P. were as under and of the in the in the culture medium of a of in the together with a in the region and The of in of the from of culture medium the are to as heavy and light The heavy chain kDa) was as as the of the light chains kDa) The recombinant was purified from medium as under and and of the medium at pH by a the that is a into the culture medium of The recombinant that was by the from the not in a but was a of that at and that with the cyprosin not of a mixture of and heavy chains in which the was in the the was All of the light chains in the which with The and heavy chains and the light chains were all with not it was that all of the proteins been and that the proteins were derived from recombinant The of purified was approximately of culture was by of recombinant proteins and and from of recombinant cyprosin a were under a of recombinant cyprosin and and a of cyprosin were to electrophoresis under and and non-reducing and of recombinant cyprosin and with were under under conditions a of the in which the plant-specific insert was and the sequence and mature enzyme was to and were with and were by with an that the are capable of of and as the cyprosin light chain were to the heavy chain of the are in The and heavy chains were from by under conditions and by that were and sequences the The in the of these heavy chains for by at The for the recombinant heavy chains are by and from the that was to at the of the heavy chain of a cyprosin from flowers of C. M. P. A. A. Scholar). of the recombinant precursor thus to the by at in the resulting in at the of the resultant kDa) heavy chains of mature under conditions and were at of the light In all residues were in but indicating that the of light chains was to have from at when of recombinant cyprosin were to under non-reducing a was is in and of recombinant cyprosin of heavy chain under conditions but in the of the was at of this the microheterogeneous of as was for the and from the In a sequence was in the sequence is to have been derived from a light chain that to the heavy chains under the non-reducing indicating that the recombinant heavy chains and the light chains by disulfide sequences of the plant-specific insert that at the and of the heavy and light excision of the of the plant-specific insert by The of the sequence of this light chain indicated for disulfide were from at C-terminal of the heavy but, for the heavy was as the C-terminal with the in the sequence the predicted of the plant-specific insert region this thus in the heavy which predicted to have an of under a of recombinant cyprosin which and heavy with in a in the for the heavy chains from and 32 to approximately 32 and and The thus of the heavy with the of the and with that are M. Scopus Google Scholar). disulfide the of the heavy chains and the of the light chain for the kDa) of the recombinant cyprosin under non-reducing conditions cysteine residues are predicted to the plant-specific insert of and these are all in the insert sequences of aspartic proteinase from (1Runeberg-Roos P. Törmäkangas K. Östman A. Eur. J. Biochem. 1991; 202: 1021-1027Crossref PubMed Scopus (104) Google Scholar, T. Watanabe H. Abe K. Arai S. Eur. J. Biochem. 1995; 232: 77-83Crossref PubMed Scopus (89) Google Scholar, A. Ryan C.A. Plant Mol. Biol. 1996; 31: 1073-1077Crossref PubMed Scopus (82) Google Scholar, K. Stack S. Gutteridge S. Vanderkerckhove J. Krebbers E. Gal S. Plant Mol. Biol. 1997; 33: 187-192Crossref PubMed Scopus (37) Google Scholar). of these into and has been Biochem. 1995; PubMed Scopus Google Scholar) the of the All of these the of the in of the in P. the and mature cyprosin with at the of the heavy as but not all, of the plant specific insert region is but in the predicted of the plant-specific insert and heavy chains that at at a of the of the insert a mixture of light chains that by disulfide bonding to the heavy chains to generate a series of of mature In contrast, the heavy and light chains of a cyprosin from flowers are not held together by disulfide this heterodimeric was into heavy and light chains under non-reducing conditions and sequence was for the light chain of this but the sequence was at the of the C. and P. E. that been as M. Scopus Google Scholar) from the mixture of enzyme forms from the flowers of the sequence at of the plant-specific insert and is from in to the plant-specific insert have at a from those in P. pastoris, from which to the C-terminal of the plant-specific insert. C-terminal sequence has been for the heavy chain of but the heavy chain has been recently (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) to have at to at the of the plant-specific the residues and that in at to those in from the at the of the plant-specific insert. in Pichia was by of the aspartic proteinase in the culture medium of a in of the and to and but not in the of a in with that of the unprocessed this was S. Kervinen J. M. D.A. A. J. J. Biol. 1998; PubMed Scopus Google Scholar) the of the cDNA encoding the precursor of the aspartic from barley using In this the precursor accumulated in the it to purified and to under to was shown to capable of to generate of and as by under by to of approximately and was at the of the but the residues were and at an to that with recombinant in the In the was of the of the these of when the pH was S. Kervinen J. M. D.A. A. J. J. Biol. 1998; PubMed Scopus Google Scholar). In the medium of the Pichia was well pH it was mature cyprosin and not the precursor that accumulated in the the plant proteinases are the of pH values the of the in it that the of mature cyprosin that accumulated in the medium been as a of of the recombinant is that the of cell proteinases at that were not to under the conditions to in but to under the conditions that in as in the with recombinant and forms of the of recombinant from those of the enzyme from barley S. Kervinen J. M. D.A. A. J. J. Biol. 1998; PubMed Scopus Google Scholar). of aspartic proteinases the of proteinases to generate the mature such as 1998; PubMed Scopus Google Scholar). In the of which is the mammalian enzyme to cyprosin and cysteine proteinases are for to the of the cell in under conditions to generate an as in which of the residues of the have been B.M. Dunn B.M. J. Biol. 1996; PubMed Scopus Google Scholar). In to of all residues in it was to an sequence at the the and mature enzyme region of human B.M. Dunn B.M. J. Biol. 1996; PubMed Scopus Google Scholar). was introduced into the sequence by the residues the to the of the chain of mature cyprosin from to of this construct in Pichia in the of mature enzyme that was in and from cyprosin not In to some into the significance of the plant specific a was introduced into this construct by the nucleotides encoding the plant-specific insert of and the residues the plant insert by the residues from human which are known from the to form a in this region of the human enzyme S. J. A.M. S. A. PubMed Scopus Google Scholar). of this construct in Pichia in the of of recombinant The that was a molecular which to that of and it was against the synthetic peptide it that, at in the those to in plant the residues of the plant-specific insert to to that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature in forms from the that are the flowers of the plant (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, M. Scopus Google Scholar, S. Kervinen J. M. D.A. A. J. J. Biol. 1998; PubMed Scopus Google Scholar). Kinetic parameters were for the hydrolysis of a synthetic peptide substrate by a purified of wild-type, recombinant cyprosin in which the heavy chain of the form values by were the pH the values by in but pH and at pH as has been for aspartic proteinases from J. Dunn B.M. J. Google Scholar). were for the hydrolysis of the substrate at pH by of the of purified from the flowers of the plant as M. Scopus Google Scholar). was from that for the recombinant form of cyprosin in Pichia for the was the derived at pH for the recombinant form of the enzyme. for plant aspartic proteinases the of to aspartic proteinases for which have been by indicated that the plant-specific insert residues are to to the active (10Guruprasad K. Törmäkangas K. Kervinen J. Blundell T.L. FEBS Lett. 1994; 352: 131-136Crossref PubMed Scopus (79) Google Scholar). the for and recombinant cyprosin at pH were and it that the residues excision of the plant-specific insert by the Pichia have a but have substrate have been for to S. J. A.M. S. A. PubMed Scopus Google Scholar) and from a comparison of recombinant and forms of an aspartic proteinase from the P. K. J. G. J. D. E. and C. for parameters for the hydrolysis of by recombinant pH at at pH in a The temperature of the parameters for substrate hydrolysis by the recombinant cyprosin was The in a at but was at The of the recombinant proteinase at was at pH and and of and were cyprosin thus a at to and at pH values as as The recombinant plant cyprosin was not by a plant derived from which is a of S. M. P. J. A. B. 1997; PubMed Scopus Google Scholar). the inhibitors of and proteinase from the A. Kay J. T. Dunn B.M. J. Scopus Google Scholar) and S. T. Kay J. P. Dunn B.M. Biochem. J. PubMed Scopus Google Scholar), not the recombinant cyprosin to In contrast, the peptide which a as the the positions in the J. J. Biol. PubMed Google Scholar), subnanomolar as an of recombinant cyprosin in this have been as of an aspartic proteinase in Scholar), a was as as an of the in with the sequence of of has a chain in by in a in inhibitors and in In inhibitors of by in a of inhibitors and in by the a in a with to that of the was as the the positions of and the of in positions was of synthetic inhibitors with recombinant cyprosin and human used are: ACHPA, 4-amino-5-cyclohexyl-3-hydroxypentanoic acid; are from used are: ACHPA, 4-amino-5-cyclohexyl-3-hydroxypentanoic acid; are from Dunn B.M. G. J. S. J. D. Kay J. Biochem. J. PubMed Scopus Google in a of synthetic inhibitors from to with recombinant cyprosin and human are from were at pH and are in AHPPA, 4-amino-3-hydroxy-5-phenylpentanoic are from Dunn B.M. G. J. S. J. D. Kay J. Biochem. J. PubMed Scopus Google in a were at pH and are in AHPPA, 4-amino-3-hydroxy-5-phenylpentanoic In the of the by of a in an in recombinant cyprosin inhibitors and in of the with an an in resulting in an with a of and in that the in the of into a bonding with an in the enzyme that is not with the in the the of by has an in a inhibitors and in of with a a in the in an that cyprosin as that for In to this in the of with the of the resulting in an in and in a in of of In the of by the chain of in cyprosin by an of and of the chain of with the of an with subnanomolar to that of and with the chain of in the of recombinant cyprosin the of this with the enzyme was that of were that an not using the and In the of by in a in and in by the a the of the by the chain of in a in of and recombinant the of the in and it that the to an of recombinant that the active of the plant enzyme is In this it has been held that, the aspartic has of the active in requirements Dunn B.M. 1994; PubMed Scopus Google Scholar). the of the values that have Dunn B.M. G. J. S. J. D. Kay J. Biochem. J. PubMed Scopus Google Scholar) for the of this human enzyme with the of are in and these it is that from to to recombinant cyprosin to human with the of and which an in the The of cyprosin for a in the Dunn B.M. G. J. S. J. D. Kay J. Biochem. J. PubMed Scopus Google Scholar) for human renin, and and E. In contrast, human a for as a Dunn B.M. G. J. S. J. D. Kay J. Biochem. J. PubMed Scopus Google Scholar). The inhibitors in and were synthesized as inhibitors of human renin, which is not specific proteinase substrate to generate and have been to specific such as those in and as all the that inhibitors the of the sequence of residues in by this specific enzyme in the mammalian to against a proteinase from the flowers of the this with the of synthetic have been recently as inhibitors of the aspartic proteinase from human immunodeficiency virus (8Wlodawer A. Vondrasek J. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 249-284Crossref PubMed Scopus (636) Google Scholar). have been shown to have potencies the human aspartic proteinases A. J. J. C. A. D. B. 1995; PubMed Scopus Google Scholar, E. S. C.A. L. A. L. T. A. S. A. 1995; PubMed Scopus Google Scholar, D. Kay J. A. J.S. S. Science. PubMed Scopus Google Scholar, D. A. A. Dunn B.M. Kay J. Eur. J. Biochem. 1996; 241: PubMed Scopus Google Scholar). these were for recombinant which is a a A. J. J. C. A. D. B. 1995; PubMed Scopus Google Scholar), was found to as an of cyprosin the plant enzyme HIV proteinase or some of the human for which subnanomolar values were a and has been shown to have values of and for human and E. S. C.A. L. A. L. T. A. S. A. 1995; PubMed Scopus Google Scholar), is not specific for HIV proteinase was with recombinant cyprosin against which was a In contrast, in has been D. Kay J. A. J.S. S. Science. PubMed Scopus Google Scholar) to specific for the aspartic proteinases from and and to have aspartic those from D. A. A. Dunn B.M. Kay J. Eur. J. Biochem. 1996; 241: PubMed Scopus Google Scholar). was found to recombinant at In contrast, in which a not cyprosin to of HIV proteinase inhibitors with recombinant cyprosin in a these it is that the active of cyprosin synthesized inhibitors of mammalian and aspartic proteinases the plant enzyme is not to inhibitors of plant it that the of the plant aspartic proteinase is the plant-specific insert. has been to direct the newly plant into appropriate cytomorphological the in the recombinant cyprosin was by the into the medium that it not to appropriate that were in in to into the significance of the plant-specific it to encoding of the plant from the the plant-specific insert to for the of the recombinant precursor in such a form that it to the mature enzyme. the by M. of and in and C-terminal the of J. B. J. L. H. J. A. D. J. and D. F. for of the inhibitors