Macrocypins, a family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera Jerica Sabotic ˇ 1 , Tatjana Popovic ˇ 1 , Vida Puizdar 2 and Joz ˇ e Brzin 1 1 Department of Biotechnology, Joz ˇ ef Stefan Institute, Jamova 39, Ljubljana, Slovenia 2 Department of Biochemistry and Molecular and Structural Biology, Joz ˇ ef Stefan Institute, Jamova 39, Ljubljana, Slovenia Introduction Papain-like cysteine proteases are widespread in organ- isms ranging from bacteria to humans. They play impor- tant roles in many facets of physiology, and dysregulation of proteolytic activity can lead to a variety of pathologies, including cancer, rheumatoid arthritis, osteoarthritis and neurological disorders [1,2]. The most important regulators of protease activity are specific protease inhibitors. In addition to their considerable potential in diverse medical applications, protease inhib- itors have been studied as tools for analysing proteolytic mechanisms and protein–protein interactions, and as biocidal agents against various organisms. There are several groups of inhibitors, mainly from animal and plant origins, that specifically inhibit papain-like cyste- ine proteases [3,4]. The first cysteine protease inhibitor isolated from higher fungi was clitocypin from the basidiomycete Clitocybe nebularis [5]. Clitocypin is a 16.8-kDa protein lacking cysteine and methionine resi- Keywords basidiomycetes; clitocypin; cysteine protease; mycocypin; protease inhibitor Correspondence J. Sabotic ˇ , Department of Biotechnology, Joz ˇ ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Tel: +386 1 477 3754 Fax: +386 1 477 3594 E-mail: Jerica.Sabotic@ijs.si Note The nucleotide sequences reported in this paper have been deposited in the DDBI ⁄ EMBL ⁄ GenBank databases under accession numbers FJ495239, FJ495240, FJ495241, FJ495242, FJ495243, FJ495244, FJ495245, FJ495246, FJ495247, FJ495248, FJ495249, FJ495250, FJ548751 and FJ548752 (Received 25 March 2009, revised 22 May 2009, accepted 9 June 2009) doi:10.1111/j.1742-4658.2009.07138.x A new family of cysteine protease inhibitors from the basidiomycete Mac- rolepiota procera has been identified and the family members have been termed macrocypins. These macrocypins are encoded by a family of genes that is divided into five groups with more than 90% within-group sequence identity and 75–86% between-group sequence identity. Several differences in the promoter and noncoding sequences suggest regulation of macrocypin expression at different levels. High yields of three different recombinant macrocypins were produced by bacterial expression. The sequence diversity was shown to affect the inhibitory activity of macrocypins, the heterolo- gously expressed macrocypins belonging to different groups showing differ- ences in their inhibitory profiles. Macrocypins are effective inhibitors of papain and cysteine cathepsin endopeptidases, and also inhibit cathepsins B and H, which exhibit both exopeptidase and endopeptidase activities. The cysteine protease legumain is inhibited by macrocypins with the excep- tion of one representative that exhibits, instead, a weak inhibition of serine protease trypsin. Macrocypins exhibit similar basic biochemical characteris- tics, stability against high temperature and extremes of pH, and inhibitory profiles similar to those of clitocypin from Clitocybe nebularis, the sole rep- resentative of the I48 protease inhibitor family in the MEROPS database. This suggests that they belong to the same merops family of cysteine prote- ase inhibitors, the mycocypins, and substantiates the establishment of the I48 protease inhibitor family. Abbreviations AMC, 7-amido-4-methylcoumarin; Clt, clitocypin; Mcp1, 2, 3, 4, 5, macrocypin 1, 2, 3, 4, 5; rMcp1, 3, 4, recombinant macrocypin 1, 3, 4; Z, benzyloxycarbonyl. 4334 FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS dues, which, on account of its unique characteristics, was assigned to a new family of cysteine protease inhibi- tors, I48 in the merops database inhibitor classification, also named mycocypins. Its profile of inhibition differs from those of other then known families of cysteine pro- tease inhibitors. Clitocypin inhibits papain, cathepsins L and K, legumain and bromelain, but is inactive against cathepsin H, trypsin and pepsin. Clitocypin is encoded by a small family of genes that show sequence hetero- geneity, which does not affect its inhibitory activity. In addition to a defensive role, a regulatory role in mushroom endogenous proteolytic systems was pro- posed, based on the specific inhibition of several puta- tive fungal cysteine proteases [5–7]. The proteolytic potential of higher fungi is consid- erable in terms of the number and diversity of prote- ases they contain [8], and basidiomycetes are a rich source of novel proteolytic enzymes and their inhibi- tors [5,8]. As a result of a study carried out to inves- tigate the extent and function of the I48 inhibitor family that to date includes only one characterized member, we report a novel family of cysteine prote- ase inhibitors from basidiocarps, or fruiting bodies, of the basidiomycete Macrolepiota procera, and have characterized some of its natural and recombinant members. Results Isolation of cysteine-protease inhibitors from M. procera Cysteine protease inhibitors were purified from basid- iocarps of M. procera by a method similar to that used to purify clitocypin from basidiocarps of C. nebularis, which included hydrophobic interaction, ion-exchange and papain affinity chromatographies [5,9]. The inhibi- tory proteins were separated on SDS–PAGE under denaturing, nonreducing, conditions into two bands corresponding to apparent molecular masses of 21 and 17 kDa (Fig. 1). The 43-residue N-terminal sequence for the former protein (Fig. 2) was 42% identical and 58% similar to the N-terminal sequence of clitocypin. The new 21-kDa cysteine protease inhibitor has been named macrocypin ( Macrolepiota procera cysteine pro- tease inhibitor, Mcp). The N-terminal sequence for the lower-molecular-mass protein could not be determined, possibly because of a blocked N terminus. The pres- ence of a protein of similar size to that of clitocypin and purified by the same procedure, and with similar biochemical properties, indicated the presence of a clit- ocypin-like cysteine protease inhibitor in M. procera [5,6]. This was confirmed at the genetic level by geno- mic DNA dot-blot analysis and by amplification of partial clitocypin-like gene sequences. The partial sequences of the clitocypin-like genes from M. procera are more than 90% identical to those of clitocypin at the nucleotide level (see Supplementary Data in Doc. S1 and Doc. S2 and Fig. S1 and S2). Cloning and analysis of macrocypin cDNA and gene sequences The N-terminal sequence (H 2 N-GLEDGLYTIRHLVE GQPPNIPGGMYASSKDGKDXPVTAEPPLP) and the sequence of an internal peptide fragment obtained by digestion with cyanogen bromide (H 2 N-YIP- RKVFK) were used to design degenerate primers (Table S1). Degenerate primers and M. procera cDNA synthesized from total RNA as template were used to obtain a specific macrocypin sequence. This was then used to design specific nested primers for use in Genome walking and 3¢ RACE methods. Two fragments were cloned from the two genomic libraries obtained by the Universal Genome Walker kit (Clontech, Heidelberg, Germany), using mcp gene-spe- cific antisense primers – Pr4 (1000 bp) and Pr3 (204 bp). Each corresponds to the mcp 5¢ UTR and promoter regions. Sequences spanning the 5¢ coding region of the mcp gene were not identical (Fig. S3), suggesting that more than one gene encoding macrocy- pin is present in the M. procera genome, each of which has its corresponding promoter. Both promoter sequences have a typical TATA box (TATAAAA) present at position )85, and a putative transcription initiation site (CTAGTCC) at position )55, indicating Fig. 1. SDS–PAGE analysis of the natural cysteine protease inhibi- tors clitocypin (nClt) and macrocypin (nMcp). Clitocypin (from Clito- cybe nebularis) and macrocypin (from Macrolepiota procera) purified from basidiocarps were analyzed under nonreducing, dena- turing conditions and stained with Coomassie Blue. Lane M, protein molecular mass markers. J. Sabotic ˇ et al. Fungal cysteine protease inhibitors FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS 4335 transcriptional activity of macrocypin genes. The loca- tion of the TATA box at a position 30 nucleotides upstream from the putative transcription-initiation site is in accordance with the locations found in several other fungal genes, which are usually within 30–60 nucleotides of the transcription start site [10]. To obtain the coding and 3¢ UTR regions of the mcp mRNA, we used the 3¢ RACE method with mcp-specific sense primers that differentiate between the two sequences obtained by genome walking. Partial sequences corresponding to promoter Pr4 showed two different lengths of the 3¢ UTR (66 nucleotides and 87 nucleotides). Sequences corresponding to promoter Pr3 showed even more variation in 3¢UTR length (64, 69, or 88 nucleotides), indicating the possibility of macrocypin translation being regulated via the 3¢ UTR [11]. There is no typical polyadenylation signal present in the 3¢ UTR. The genomic sequence corresponding to the 3¢ UTR was amplified from the two genomic libraries created using the Universal Genome Walker kit (Clontech), using mcp-specific sense primers. Two fragments, spanning 175 and 151 bp downstream of the stop codon, were obtained that were identical to the overlapping sequence of the 3¢UTR region amplified from M. procera cDNA, corresponding to the promoter fragment Pr4. The full-length gene and cDNA sequences of macro- cypin were obtained, using primers annealing to the 5¢ UTR and 3¢ UTR regions and genomic DNA or first- strand cDNA synthesized from the total RNA of M. procera as templates. Several different gene and cDNA sequences were amplified, all of which, how- ever, shared the same gene structure. The mcp genes were found to be composed of four exons and three short introns with exon–intron boundaries matching the consensus splice sites predicted for eukaryotic genes [10]. Based on a sequence identity matrix of the deduced amino acid sequences, we divided the obtained sequences into five groups, naming them macrocypin 1–5. Sequence identity at the deduced amino acid sequence level between the five macrocypin groups was 75–86%, while sequences within groups exhibited more than 90% sequence identity. The length of the macrocypin 1 (Mcp1) deduced amino acid sequence was 169 residues, with a molecu- lar mass of 19 193 Da, while the length of the repre- sentatives of the other four groups was 167 residues, with molecular masses between 18 770 and 19 031 Da. Single cysteines were present in Mcp1, macrocypin 4 (Mcp4) and macrocypin 5 (Mcp5) (C106), none in macrocypin 2 (Mcp2) and none or one (C75) in macro- Fig. 2. Diversity in the cysteine protease inhibitor macrocypin family. Amino acid sequences deduced from macrocypin (Mcp) genes (pref- aced by g) and from cDNA sequences (prefaced by c) belonging to different groups (macrocypins 1–5) are aligned with the N-terminal sequence determined for the natural macrocypin (nMcp) isolated from basidiocarps of Macrolepiota procera. Identical residues in at least 9 of 11 sequences (80 %) are highlighted in dark gray and similar residues are highlighted in light gray. Sequences corresponding to clones used for the heterologous expression of macrocypins are indicated in bold. Residues subjected to positive evolution, as determined using the Datamonkey rapid detection of positive selection web server [20], are marked with an asterisk. Fungal cysteine protease inhibitors J. Sabotic ˇ et al. 4336 FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS cypin 3 (Mcp3). All the sequences showed high con- tents of proline, of around 9.2% (the average overall protein content is 4.7 %), of tryptophan, of 3.6% (average 1.1 %) and of tyrosine, of 6.0–6.6% (average 2.9%), and low leucine contents of around 3.6% (aver- age 9.7%) [12]. With the exception of Mcp3, macrocy- pin sequences also showed a high glycine content of around 9.8% (average 6.9%). Variability in the sequences of macrocypin genes The diversity observed for the macrocypin coding sequences was greater than that for clitocypins [7]. The coding sequence, composed of four exons, was 507 bp for Mcp1 and 501 bp for the other macrocypins. The deduced amino acid sequence for Mcp1 was thus two amino acids longer, because of an insertion near the N terminus. The lengths of the four exons were 147 or 153, 212, 70 and 75 bp, and of the three introns were 56, 49 or 51, and 50 or 54 bp. The sequence diversity was equally distributed in introns and in exons (Fig. S3). The second intron showed the greatest diver- sity in sequence and was 2 bp shorter in Mcp3 and Mcp5. The third intron was the most highly conserved of the three, with the exception of Mcp3, where it was 4 bp shorter. Diversity in the coding sequence was dis- tributed throughout the sequence (Fig. 2) and this was caused by one, two or three nucleotide substitutions that resulted in 47 variable codons at the level of the deduced amino acid sequences. Some of these codons subjected to positive selection (codons 18, 85, 89, 105, 107 and 116) were identified at the P < 0.05 level (Fig. 2). Codons under negative selection (31 codons identified) appeared to be evenly distributed along the whole mcp genes without particular clustering. The few positively selected sites in the mcp genes may provide insights into the physiological role of macrocypins, as these sites are more likely to be involved in interac- tions with other proteins. Similarity searches for the macrocypin sequences against NCBI databases using blastn [13] revealed no significant similarities (cut-off e < 0.1). tblastn searches, by contrast, found significant sequence simi- larity to the clitocypin cysteine protease inhibitor annotated in the Laccaria bicolor S238N-H82 genome [14]. There was 39–44% sequence similarity and 26–29% sequence identity for different macrocypin sequences, the highest being with that of Mcp5. No significant similarities were found for the macrocypin sequences in the completed and unfinished archaeal or bacterial genomes or in other eukaryotic genomes, which is probably the result of low overall sequence similarity between mycocypins. Alignment of macrocypin deduced amino acid sequences with clitocypin sequences showed 17–21% sequence identity (Fig. 3). The N-terminal halves of the sequences showed more similarity. The higher molecular masses relative to the clitocypins are caused by a few insertions and deletions of two to seven amino acids, distributed along the sequence. Another important difference between the macrocypin and clit- ocypin sequences was the presence of a cysteine residue Fig. 3. Alignment of mycocypin deduced amino acid sequences. Deduced amino acid sequences of macrocypins belonging to each of the five groups are aligned with three representative clitocypin deduced amino acid sequences (GenBank accession numbers: gClt-Kras, AAZ78483.1; cClt-Kras, AAZ78481.1; and cClt-Vrh, AAZ78482.1). Sequences are prefaced by g or c to indicate whether they are deduced from genomic or cDNA sequences. Identical residues in at least seven of the eight sequences (90 %) are highlighted in dark grey and similar residues are highlighted in light grey. J. Sabotic ˇ et al. Fungal cysteine protease inhibitors FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS 4337 in most macrocypins, and of several histidine and methionine residues in all the macrocypin sequences, all of which are absent in clitocypin. High proline and glycine contents were, by contrast, common to both macrocypins and clitocypin. Heterologous expression of macrocypins The cDNA clones that were available for Mcp1, Mcp3 and Mcp4 (marked in bold in Fig. 2), were cloned into expression vectors of the pET System (Novagen, Madi- son, WI, USA) for heterologous expression in Escheri- chia coli (Fig. S4). Macrocypin 1a (Mcp1a) and macrocypin 3a (Mcp3a) cDNA clones were each cloned into two expression vectors (pET3a and pET11a) to assess their expression in the bacterial expression system. Heterologous expression of recom- binant Mcp1 (rMcp1) was higher with the pET11a expression vector construct, while that of recombinant Mcp3 (rMcp3) was higher using pET3a. The amounts of both recombinant proteins were highest 6 h after induction (data not shown). The macrocypin 4a (Mcp4a) cDNA clone was introduced into the pET14b expression vector and expressed in two strains of E. coli. The expression of recombinant Mcp4 (rMcp4) was highest when using the pET14b::Mcp4a construct in combination with the E. coli BL21(DE3) pLysS strain grown for 8 h after induction. Recombinant macrocypins rMcp1 and rMcp3 were expressed mainly as insoluble inclusion bodies, and rMcp4 was expressed as an equal distribution of protein between the soluble form and inclusion bodies. All three rMcps were purified from the inclusion bodies, which were almost completely solubilized in 3 m urea. One-step purification using size-exclusion chromatography yielded purified recombinant macrocypins rMcp1 and rMcp4, while rMcp3 still contained some impurities, which were taken into consideration when calculating the concentration. Characterization of macrocypins Recombinant macrocypins rMcp1, rMcp3 and rMcp4 were resolved on SDS–PAGE under reducing condi- tions as single 19 kDa bands (Fig. 4A). Under non- reducing conditions, however, they all showed an additional band at 38 kDa, corresponding to a dimer, probably formed between the single cysteines present in each protein. The calculated isoelectric point of 5.1 for Mcp4 was confirmed by IEF and was similar to the isoelectric point of the natural macrocypin isolated from basidiocarps of M. procera. For Mcp1 and Mcp3, the calculated isoelectric points were 4.8, which was also confirmed by IEF (Fig. 4B). N-terminal sequences were confirmed for all three recombinant macrocypins. That for rMcp1 [NH 2 - (M)GFEDG] revealed N-terminal cleavage of methio- nine in approximately one-third of the molecules. N-terminal sequences of rMcp3 and rMcp4 (NH 2 - ALEDG) showed complete cleavage of N-terminal methionine. N-terminal cleavage of methionine in E. coli is strongly influenced by the amino acid resi- dues at the P1¢,P2¢ and P3¢ positions (the P1 position being the first methionine) [15]. In the case of macro- cypins, the NH 2 -MALE sequence favours N-terminal methionine cleavage in E. coli, and the NH 2 -MGFE sequence of rMcp1 favours only partial cleavage. The far-UV CD spectra of rMcp1 and rMcp4 con- firmed the expectation from the sequences that the conformations of the inhibitors are very similar (Fig. S5A). The marked tryptophan bands, seen also in clitocypin [9] and ascribed to interaction of buried tryptophan residues, prevent analysis of secondary structure, but underline the similarity in tertiary struc- ture, at least in this region, as well as of secondary structure. Clitocypin has been proven to be a very stable pro- tein [5,9]. The temperature and pH stability of recom- binant macrocypins were determined by following their inhibitory activity (measured after return to native conditions) after heating and after incubation at extremes of pH. The macrocypins rMcp1 and rMcp3 retained their inhibitory activity after heating at 75 °C, or even at 100 °C, for 15 min, whereas rMcp4 partially lost its inhibitory activity after heating at 75 °C for 15 min and completely lost its inhibitory activity after AB Fig. 4. Comparison of natural and recombinant macrocypins by SDS–PAGE (A) and IEF (B). Purified natural (nMcp) and recombinant macrocypins (rMcp) were subjected to SDS–PAGE analysis under reducing denaturing conditions and to IEF. Lane M, protein mole- cular mass markers; lane S, standard protein IEF markers; lane 1, natural macrocypin (nMcp); lane 2, recombinant macrocypin 4 (rMcp4); lane 3, recombinant macrocypin 1 (rMcp1); lane 4, recom- binant macrocypin 3 (rMcp3). Fungal cysteine protease inhibitors J. Sabotic ˇ et al. 4338 FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS heating at 100 °C. Similarly, rMcp1 and rMcp3 retained their inhibitory activities after incubation in acidic (pH 2) or alkaline (pH 11) conditions. While rMcp4 lost its inhibitory activity after incubation at pH 2, incubation at pH 11 had no influence. Stability was also examined by following conforma- tion directly by CD. rMcp1 and rMcp4 were unfolded on thermal denaturation, with closely similar transi- tions, with temperature midpoints of 78 °C (Fig. S5B). The similarity of this value to that for clitocypin shows that the differences in sequence between these three inhibitors are not critical for the stability of the pro- teins. Combined, the above results show that the inhib- itors unfold reversibly. Measurements of CD spectra at pH 2.2 and pH 11 showed only a slight decrease in ellipticity after 24 h of exposure. Specificity of macrocypins The inhibitory specificities of the natural macrocypin isolated from basidiocarps and the three recombinant macrocypins were determined against several cysteine proteases. Association (k a ) and dissociation (k d ) rate constants and equilibrium constants (K i ) were deter- mined by continuous assays for papain, cathepsins L and V, and legumain (Table 1), for which typical biphasic progress curves were obtained. For inhibition of cathepsins S, K, B and H and trypsin by macrocy- pins, only equilibrium constants were determined (Table 2). Macrocypins 1 and 3 were found to be effec- tive inhibitors of papain, cathepsin L and cathepsin V, with a mean K i value of 0.5 nm. Macrocypin 4 was also an effective inhibitor of papain but exhibited weaker inhibition of cathepsin L and V than the other two recombinant macrocypins. The sample of natural macrocypin showed weaker inhibition for these Table 1. Inhibition of cysteine proteases by natural and recombi- nant macrocypins. Kinetic and equilibrium constants for the inhibi- tion of papain, cathepsins L and V, and legumain were determined under pseudo-first-order conditions in continuous kinetic assays at 25 °C and calculated by nonlinear regression analysis according to Morrison [32]. Standard deviation is given where appropriate; ND, not determined; nMcp, natural macrocypin; rMcp1, recombinant macrocypin 1; rMcp3, recombinant macrocypin 3; rMcp4, recombi- nant macrocypin 4. Enzyme 10 )6 k a (M )1 Æs )1 )10 4 k d (s )1 ) K i (nM) nMcp Papain 0.36 ± 0.07 18.3 ± 3.91 5.04 ± 0.98 Cathepsin L 0.33 ± 0.11 1.25 ± 0.26 3.81 ± 1.66 Cathepsin V 0.08 ± 0.01 9.85 ± 1.54 12.6 ± 3.8 Legumain 0.006 ± 0.001 6.49 ± 0.99 110 ± 23 rMcp1 Papain 2.25 ± 0.49 21.4 ± 0.7 0.95 ± 0.33 Cathepsin L 5.52 ± 0.51 35.1 ± 3.2 0.64 ± 0.22 Cathepsin V 1.48 ± 0.01 10.3 ± 0.7 0.69 ± 0.06 Legumain 0.22 ± 0.04 7.66 ± 4.21 3.38 ± 1.44 rMcp3 Papain 4.28 ± 0.95 5.10 ± 0.81 0.12 ± 0.05 Cathepsin L 3.58 ± 0.45 11.1 ± 0.5 0.31 ± 0.06 Cathepsin V 1.88 ± 0.09 8.43 ± 0.76 0.45 ± 0.01 Legumain 0.063 ± 0.020 5.77 ± 1.27 9.17 ± 1.09 rMcp4 Papain 3.41 ± 0.15 6.32 ± 0.75 0.19 ± 0.01 Cathepsin L 1.62 ± 0.55 45.1 ± 5.1 2.76 ± 0.92 Cathepsin V 2.29 ± 0.66 33.3 ± 6.3 1.44 ± 0.11 Legumain ND ND > 1000 Table 2. Inhibition of various proteases by natural and recombinant mycocypins. Equilibrium constants for the inhibition of different prote- ases were determined in continuous or stopped kinetic assays and analyzed according to Morrison [32] or Henderson [33], respectively. Kinetic data for the interaction of clitocypin with papain, cathepsins L, K and H, and legumain were reported previously [6]. Standard devia- tion is given where appropriate. nMcp, natural macrocypin; rMcp1, recombinant macrocypin 1; rMcp3, recombinant macrocypin 3; rMcp4, recombinant macrocypin 4; nClt, natural clitocypin; rClt, recombinant clitocypin; n.i., no inhibition. Enzyme K i (nM) nMcp rMcp1 rMcp3 rMcp4 nClt rClt Papain 5.04 ± 0.98 0.95 ± 0.33 0.12 ± 0.05 0.19 ± 0.01 2.5 ± 0.94 6.2 ± 0.55 Cathepsin L 3.81 ± 1.66 0.64 ± 0.22 0.31 ± 0.06 2.76 ± 0.92 0.03 ± 0.002 0.02 ± 0.001 Cathepsin V 12.6 ± 3.77 0.69 ± 0.06 0.45 ± 0.01 1.44 ± 0.11 0.14 ± 0.01 0.08 ± 0.03 Cathepsin S 47.1 ± 3.1 23.1 ± 1.2 5.1 ± 0.5 6.3 ± 0.6 3.2 ± 0.3 2.2 ± 0.3 Cathepsin K 4.5 ± 0.5 170 ± 20 17.5 ± 1.2 21.8 ± 5.2 0.02 ± 0.005 0.03 ± 0.002 Cathepsin B 515 ± 36 490 ± 18 > 1000 125 ± 10 > 1000 > 1000 Cathepsin H 370 ± 11 100 ± 10 24 ± 5 32 ± 6 n.i. n.i. Legumain 110 ± 23 3.38 ± 1.44 9.17 ± 1.09 > 1000 7.1 ± 1.12 21.5 ± 2.81 Trypsin n.i. n.i. n.i. 160 ± 14 n.i. n.i. Pepsin n.i. n.i. n.i. n.i. n.i. n.i. J. Sabotic ˇ et al. Fungal cysteine protease inhibitors FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS 4339 enzymes than the recombinant macrocypins. Inhibition of cathepsins S and K by recombinant macrocypins was somewhat weaker, with K i values in the range of 5–25 nm, and 10 times higher for inhibition of cathep- sin K by rMcp1. Cathepsin B, which has both endo- peptidase and exopeptidase activities, was inhibited by macrocypins with K i values in the micromolar range, with the exception of rMcp3 (K i >1lm). By contrast, cathepsin H was inhibited by natural macrocypin and rMcp1, with K i values in the micromolar range, while rMcp3 and rMcp4 inhibited with 10-fold lower values. Like clitocypin, recombinant macrocypins inhibited the cysteine protease legumain, a member of the C13 fam- ily, with an average K i value of 6 nm. The natural macrocypin exhibited only very weak inhibition of legumain (K i in the micromolar range), while rMcp4 showed no inhibition at all. The serine protease trypsin was not inhibited by natural macrocypin, rMcp1 or rMcp3, while rMcp4 exhibited weak inhibition with a K i value in the micromolar range. The aspartic prote- ase pepsin was not inhibited by natural or recombinant macrocypins. The ability of natural and recombinant clitocypins [6] to inhibit cathepsins V, S and B was also deter- mined for comparison (Table 2). Clitocypin is an effec- tive inhibitor of cathepsins V and S. The weak inhibition of cathepsin B reported for the natural clito- cypin, with a K i value of 0.48 lm [5], was not con- firmed with either recombinant or natural clitocypin, for which K i values were > 1 lm. Discussion A novel cysteine protease inhibitor, macrocypin, has been isolated from basidiocarps of the parasol mush- room (M. procera), in addition to a putative clito- cypin-like cysteine protease inhibitor. Based on similarities in genetic and biochemical characteristics, macrocypin was assigned to the mycocypin family of fungal cysteine protease inhibitors, family I48 in the merops database, together with clitocypin from clouded agaric (C. nebularis) [6,7]. Based on the partial protein sequence, several macr- ocypin-coding genes and corresponding cDNAs were amplified and sequenced, together with the promoter and 5¢ UTR and 3¢UTR sequences (Fig. S3). The diversity observed was even greater than that observed for clitocypin-coding genes [7], the macrocypin sequences being divided into five groups. The variabil- ity was distributed throughout the coding sequence and comprised one to five consecutive amino acid sub- stitutions (Fig. 2). The variability was not limited to the coding sequence, as two different promoter sequences were cloned. A few 3¢ UTR sequences were found that showed no sequence diversity, but differed in their length. Different promoter and 5¢UTR and 3¢UTR sequences, and their lengths, together with some differences found in intron sequences and their lengths, which could influence transcription, splicing, mRNA transport, stability and translation, all suggest complex regulation of macrocypin expression at diff- erent levels. Macrocypin- and clitocypin deduced amino acid sequences show similarities, despite a low overall sequence identity of 17–21% (Fig. 3). They both have high contents of proline and tyrosine and low contents of leucine. A major difference between clitocypin and macrocypin sequences lies in the presence of sulfur- containing amino acids in macrocypins, but not in clitocypins. In spite of very low overall sequence simi- larity, several amino acid residues are conserved in all macrocypin and clitocypin sequences, mainly in the N-terminal half (Fig. 3). These are probably important for the inhibitory activity and⁄ or structure, and many of them are proline residues. A similarity search using macrocypin deduced amino acid sequences against the translated nucleotide data- base at the NCBI server showed significant similarity to the putative L. bicolor clitocypin-like cysteine prote- ase inhibitor. Alignment of the deduced sequences of the latter with those of macrocypin from M. procera and with clitocypin from C. nebularis revealed con- served amino acid residues (Fig. S6), the majority of which were already present in the alignment of macro- cypin and clitocypin deduced amino acid sequences, confirming their functional and ⁄ or structural imp- ortance. Macrocypins and clitocypins exhibit similar basic biochemical characteristics. They have similar molecu- lar masses of 19 and 16.8 kDa and similar isoelectric points of around 4.8. They both exhibit stability against high temperature and extremes of pH. The CD spectrum in the far-UV region for macrocypin, show- ing the characteristic peak around 232 nm that indi- cates an unusually strong contribution of tryptophan residues (Fig. S5A), indicates further structural similar- ity to clitocypin [6,9]. The high degree of diversity in macrocypin gene sequences indicates a mixture of inhibitors in the natu- ral macrocypin sample isolated from basidiocarps of M. procera. In order therefore to characterize the inhibitors further, recombinant macrocypins were pre- pared. Three different macrocypin cDNA clones were used that belong to different macrocypin groups or isoforms. Heterologous expression in the bacterial expression system proved successful for all three, Fungal cysteine protease inhibitors J. Sabotic ˇ et al. 4340 FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS inhibitory macrocypins (rMcp1, rMcp3, rMcp4) being obtained by one-step purification from inclusion bodies with high yields of 50–100 mgÆL )1 . The inhibitory profiles of macrocypins and clito- cypin for several cysteine proteases are similar, but not identical. Of the cysteine proteases tested, macrocypins inhibit papain and cathepsins L and V most strongly. Compared with clitocypin [6], macrocypins are stron- ger inhibitors of papain, as a result of higher rate constants of association, but weaker inhibitors of cathepsin L, mainly because of increased rate constants of dissociation. Cathepsins S and K are inhibited by macrocypins with K i values in the nanomolar range, while clitocypin inhibits cathepsin K with K i values in the picomolar range. The most notable difference between macrocypin- and clitocypin-inhibition profiles for cysteine proteases is the weak inhibition by macro- cypins of cathepsins B and H, the papain-like prote- ases with endopeptidase and exopeptidase activities. As with clitocypin, legumain, a member of C13 family, is inhibited by Mcp1 and Mcp3 with K i values in the micromolar range. The inhibition profile of rMcp4, with K i >1lm for legumain inhibition and K i values in the nanomolar range for papain-like proteases, con- trasts with those of other macrocypins that inhibit both papain-like proteases and legumain with K i values in the same range. This strongly suggests different binding sites for the inhibition of the two families of cysteine proteases. Similarly, two independent and nonoverlapping binding sites have been reported for family C1 and C13 inhibition by legumain-inhibiting cystatins C, E ⁄ M and F [16]. The ability of macrocypins to inhibit proteases of other catalytic classes was tested. Neither macrocypins nor clitocypin [5] inhibit the aspartic protease pepsin. The serine protease trypsin is weakly inhibited by rMcp4, which is the most significant difference in the inhibitory profiles of macrocypins and clitocypin. The physiological function of the macrocypin cyste- ine protease inhibitor family is proposed to be defence against pathogen infection and ⁄ or predation by insects or other pests, analogously to the phytocystatins that are involved in plant defence by inhibiting exogenous cysteine proteases during herbivory or infection [17]. The sequence diversity includes amino acid sites of positive selection. The variations in inhibitory profile between different members of the macrocypin family reveal different specificities and strengths of inhibition of cysteine proteases of different evolutionary families, and even a serine protease. These findings together suggest an adaptation process and the selection of appropriate inhibitor isoforms providing effective defence. In addition, a regulatory role in intracellular proteolysis may also be considered for mycocypins, because cysteine protease activity is present in basidio- mycetes and its inhibition by clitocypin was shown in a few selected basidiomycete species belonging to dif- ferent orders [8]. In conclusion, we have characterized a novel family of fungal cysteine protease inhibitors – the macrocy- pins – from M. procera, at the genetic and biochemical levels and analyzed their inhibition profiles. Similarity to clitocypin from C. nebularis is evident at all these levels, suggesting that they belong to the same family of cysteine protease inhibitors, the mycocypins, thus substantiating the establishment of the I48 family of protease inhibitors in the MEROPS classification that previously comprised only one member. In addition to the high conformational stability of mycocypins, their other common characteristic is high genetic diversity, with sequence variability influencing the inhibitory activity in macrocypins, but not in clitocypins. Myco- cypins could find use in medical research, as their unique inhibitory profiles could answer the challenge of finding highly selective inhibitors against proteases important in certain stages of diseases, without affect- ing nontarget proteases. Additionally, certain represen- tatives of the macrocypin family, showing inhibition of different classes of proteases, would have applications in plant protection. Double-inhibitory activity against two catalytic classes of proteases in one stable mole- cule could provide more effective protection of plants against insect pests. Experimental procedures Isolation of cysteine protease inhibitors from M. procera Basidiocarps of the basidiomycete M. procera were col- lected from their natural habitat and frozen at )20 °C. Thawed basidiocarps were homogenized in an equal volume of 0.1 m Tris–HCl buffer, pH 7.5, containing 0.5 m NaCl, and centrifuged at 8000 g for 30 min. Ammonium sulfate was added to the supernatant to a final concentration of 1.3 m before application to a column of Phenyl Sepharose (GE Healthcare, Uppsala, Sweden). After thorough wash- ing with 0.1 m Tris–HCl buffer, pH 7.5, containing 1.3 m ammonium sulphate, the bound proteins were eluted by a 1.3–0 m gradient of ammonium sulphate in the same buffer. Inhibitory fractions, measured against papain, were pooled, concentrated by ultrafiltration (Amicon UM-10; Millipore, Vienna, Austria) and dialyzed against 0.02 m Tris–HCl, pH 7.5. The sample was then applied to a column of DEAE– Sephacel (Pharmacia-LKB, Uppsala, Sweden) equilibrated with 0.02 m Tris–HCl, pH 7.5. Bound proteins were eluted J. Sabotic ˇ et al. Fungal cysteine protease inhibitors FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS 4341 with a gradient of 0–0.4 m NaCl in the same buffer. Inhibi- tory fractions were pooled and subjected to an affinity col- umn of carboxymethylpapain–Sepharose, prepared as described previously [5], and equilibrated with 0.02 m Tris– HCl, pH 7.5, containing 0.3 m NaCl. Bound inhibitory fractions were eluted with 0.02 m NaOH, neutralized with dilute HCl, and pooled and concentrated by ultrafiltration (Amicon UM-3). N-terminal sequence analysis Automated amino acid sequencing of purified natural and recombinant macrocypins was performed as described pre- viously [6]. An internal peptide sequence was determined after cleavage with cyanogen bromide in 80 % formic acid at room temperature in the dark for 36 h. The resulting peptide fragments were separated using reverse-phase HPLC, as described previously [6]. Isolation of genomic DNA and total RNA Basidiocarps of the basidiomycete M. procera, harvested from their natural habitat, were frozen in liquid nitrogen, homogenized and stored at )80 °C until use. High- molecular-weight genomic DNA was isolated from frozen powdered tissue as described previously [18] and total RNA was extracted using an RNeasy Kit (Qiagen, Vienna, Austria) according to the manufacturer’s protocol for isolation of total RNA from plant tissues and fila- mentous fungi. Cloning of the genomic and cDNA sequences encoding macrocypins First-strand cDNA was synthesized from the total RNA by RT-PCR using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA) with anchored oli- go(dT)-adapter primer (dT (17) 3¢RACE) (Table S1). Forward and reverse degenerate primers (forward 1N-mar-Clit, nested forward 2N-mar-Clit and reverse C-mar-Clit) were designed based on the N-terminal amino acid sequence and an internal peptide fragment sequence. First-strand cDNA was used for PCR to amplify the partial macrocypin cDNA sequence, which was then used to design specific primers. Forward specific primer (Mp-CliHom-N-uni) was used to amplify the 3¢ end of the cDNA sequence, using the 3¢ RACE method, together with the 3¢RACE adapter primer. The resulting PCR product was used in a secondary PCR with two different nested forward primers (Mp-CliHom-N- A12 and Mp-CliHom-N-A3), together with the 3¢ RACE adapter primer. To amplify the complete macrocypin gene (mcp) with its upstream and downstream regions, Genome Walker libraries were constructed using the Genome WalkerÔ Universal kit (BD Biosciences Clontech) according to the manufacturer’s instructions. High-molecular-weight geno- mic DNA (2.5 lg) was digested separately with two restric- tion enzymes (PvuII and StuI) at 37 °C overnight and after purification by ethanol precipitation, Genome WalkerÔ Adaptors were ligated to the digested DNA at 16 °C over- night. The resulting Genome Walker libraries were used as templates in genome walking PCR amplifications, using nested forward specific primers (Mp-CliHom-ter1A and 1) for downstream amplification and nested reverse specific primers (Mp-CliHom-pro 1 and 2) for upstream amplifica- tion, paired with Adaptor Primer 1 and Nested Adaptor Primer 2 provided by the manufacturer. Advantage 2 Poly- merase Mix (Clontech) was used for amplification under the conditions suggested by the manufacturer. Complete mcp gene and cDNA sequences were obtained using pairs of primers annealing to the 5¢ UTR (Mcp-N- A12-1 and Mcp-N-A3-1 with nested primers Mcp-N-uni-1 or Mp-CliHom-N-uni) and the 3¢ UTR (Mcp-C-uni-1 with nested primers Mp-CliHom-C-uni, Mp-CliHom-C-A12 or Mp-CliHom-C-A3) regions in two-step PCR amplification, using nested primers in the secondary PCR with recombi- nant Taq polymerase (MBI Fermentas, Vilnius, Lithuania). All PCR products were cloned into the pGEM-T Easy Vector System (Promega, Vienna, Austria) for sequencing by the Automated DNA Sequencing Service at MWG Biotech (Ebersberg, Germany). Sequence analyses Sequence analysis and multiple sequence alignments were performed in the BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Promoter analysis was performed using the Transcription Element Search System (TESS, [19]; http://www.cbil.upenn.edu/cgi- bin/tess/tess). Similarity searches were performed using blastn and tblastn algorithms [13] against different data- bases at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST). Evolutionary analysis The Datamonkey web interface [20] (http://www.datamon- key.org) was used to examine selective pressure acting upon individual sites of codon alignments. Three methods were implemented to test for purifying or diversifying selection: the single likelihood ancestor counting (SLAC); fixed effects likelihood (FEL); and random effects likelihood (REL) [21]. A P-value of 0.05 was used for inference of positive and negative selection for individual codons. A hierarchical and information theoretic model selection procedure was applied to choose a model of nucleotide substitution. HKY85 was selected as the optimal time-reversible nucleo- tide substitution model using the implementation in the HyPhy package [22]. Fungal cysteine protease inhibitors J. Sabotic ˇ et al. 4342 FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS Expression and purification of recombinant macrocypins Whole-length cDNA clones of Mcp1a and Mcp3a were used as templates in the PCR amplification with Pfu DNA polymerase (Promega) and primers that introduced NdeI and BamHI restriction sites to the 5¢ and 3¢ ends of the insert, respectively. After resequencing the resulting prod- uct, and digesting the inserts and vectors with NdeI ⁄ BamHI (New England Biolabs, Frankfurt am Main, Germany), the inserts were subcloned into pET3a and pET11a vectors (Novagen) to generate recombinant proteins without tags. Similarly, NcoI and NdeI restriction sites were introduced into the 5¢ and 3¢ ends of the whole-length cDNA of Mcp4a. After digestion of the Mcp4a insert and vector with NcoI ⁄ NdeI (New England Biolabs), the insert was subcl- oned into the pET14b expression vector (Novagen) to gen- erate protein without tags. All constructed expression vectors with inserts were transformed into E. coli BL21(DE3), which was grown in LB (Luria–Bertani) med- ium supplemented with 100 lgÆmL )1 of ampicillin at 37 °C. The construct pET14b::Mcp4a was also transformed into the E. coli BL21(DE3) pLysS strain, which was grown at 37 °C in LB medium supplemented with 100 lgÆmL )1 of ampicillin and 34 lgÆmL )1 of chloramphenicol. When the attenuance (D) at 600 nm reached 1–1.2, the inducer iso- propyl thio-b-d-galactoside was added to a final concentra- tion of 0.4 mm for strains transformed with pET3a or pET14b constructs and to a final concentration of 1 mm for strains transformed with the pET11a construct. Cells were then grown for an additional 6 or 8 h. They were har- vested by centrifugation for 15 min at 6000 g, resuspended in buffer A (50 mm Tris–HCl, pH 7.5, 0.1 % Triton X-100, 2mm EDTA), frozen and thawed once, then sonicated at 4 °C. The insoluble fraction was separated by centrifuga- tion for 15 min at 10000 g, redissolved in the same buffer containing 3 m urea and solubilized by stirring for 4 h at 4 °C. The remaining insoluble material was removed by centrifugation and the supernatant was applied to a Sepha- rose S200 column (4 · 110 cm) equilibrated with Tris–HCl buffer, pH 7.5, containing 0.3 m NaCl. Inhibitory fractions were pooled and concentrated by ultrafiltration (Amicon UM-3) to approximately 1 mgÆmL )1 . SDS–PAGE and IEF Proteins were separated by 12.5% SDS–PAGE under dena- turing and reducing or non-reducing conditions, as appro- priate, and stained using Coomassie Brilliant Blue. The molecular mass values of the separated proteins were estimated using low-molecular-mass standard proteins of 14.4–97 kDa (LMW Calibration Kit; GE Healthcare). The Phast System (Pharmacia) was used to perform SDS– PAGE (precast 8–25 % gradient gels) and IEF (precast pH 3–9 gradient gels), as described previously [6]. Theoretical molecular mass and pI values were determined from sequences using the protparam tool available at the Exp- aSy server of the Swiss Institute of Bioinformatics (http:// www.expasy.org/tools/protparam.html). Inhibition assay Inhibitory activities against papain were determined as described previously [5] using Bz-DL-Arg-2-naphthylamide (Sigma, Taufkirchen, Germany) as the substrate [23]. Enzymes and determination of kinetic constants Human cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) were purified as described previously [24]. Papain (2· crystallized) (EC 3.4.22.2) was further purified by affin- ity chromatography [25]. Beta-trypsin (EC 3.4.21.4) was prepared from type IX trypsin (Sigma), as described previ- ously [26]. Legumain (EC 3.4.22.34) was isolated from pig kidney cortex following a previously described procedure [27]. Recombinant human cathepsin K (EC 3.4.22.38) [28], cathepsin S (EC 3.4.22.27) [29] and cathepsin B (EC 3.4.22.1) [30], all expressed in E. coli, were provided by Prof. Boris Turk, and recombinant human cathepsin V (EC 3.4.22.43) [31] was provided by Professor Dus ˇ an Turk (both from the Department of Biochemistry and Molecular and Structural Biology, Jozˇ ef Stefan Institute, Ljubljana, Slovenia). Inhibition kinetics for natural and recombinant macro- cypins and clitocypins were measured under pseudo-first- order conditions, as previously described [6]. Continuous kinetic assays were performed for the cysteine proteases papain, cathepsin L and cathepsin V using benzyloxycar- bonyl (Z)-Phe-Arg-7-amido-4-methylcoumarin (AMC) as substrate, and for legumain with Z-Ala-Ala-Asn-AMC as the substrate, while stopped assays were performed for cathepsins K, S and B using Z-Phe-Arg-AMC as the sub- strate and for cathepsin H with Arg-AMC as the substrate [6]. Trypsin was assayed using the stopped kinetic assay with fluorogenic substrate Z-Phe-Arg-AMC in 0.1 m Tris– HCl buffer, pH 8.0, containing 1.5 mm EDTA and 0.02 m CaCl 2 . Data for continuous assays were analyzed by non- linear regression analysis according to Morrison [32], while kinetic constants for cathepsins K, S, B and H, and trypsin, were determined according to Henderson [33]. Porcine pepsin (3.4.32.1) from Sigma was assayed in 0.1 m acetate buffer, pH 3.5, using the fluorogenic substrate fluorescein isothiocarbamoyl–hemoglobin, as described for fluorescein isothiocarbamoyl–casein [34]. CD spectroscopy CD spectra measurements and thermal-unfolding transi- tions were performed on an Aviv model 60 spectropolarim- J. Sabotic ˇ et al. Fungal cysteine protease inhibitors FEBS Journal 276 (2009) 4334–4345 ª 2009 The Authors Journal compilation ª 2009 FEBS 4343 [...]... D190–D195 13 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 25, 3389–3402 14 Martin F, Aerts A, Ahren D, Brun A, Danchin EG, Duchaussoy F, Gibon J, Kohler A, Lindquist E, Pereda V et al (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis Nature... proteinases Anal Biochem 47, 280–293 24 Popovic T, Puizdar V, Ritonja A & Brzin J (1996) Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation J Chromatogr B: Biomed Appl 681, 251–262 25 Blumberg S, Schechter I & Berger A (1970) The purification of papain by affinity chromatography Eur J Biochem 15, 97–102 26 Strop P & Cechova D (1981) Separation of alpha-trypsin and beta-trypsin... ClustalW alignment of partial clitocypin-like gene sequences from M procera (MpClt) with that of clitocypin from C nebularis (CnClt) Fig S3 ClustalW alignment of the full-length macrocypin gene with promoter and cDNA sequences Fig S4 Expression and purification of recombinant macrocypins rMcp1 (A) , rMcp3 (B) and rMcp4 (C) Fig S5 Far UV CD spectra and thermal unfolding transitions of recombinant macrocypins... measurements, discussion of the results and critical reading of the manuscript Dr Dusˇ an Kordisˇ is gratefully acknowledged for assistance in the evolutionary analysis of sequences We thank Adrijana Leonardi for determining N-terminal protein sequences References 1 Turk B, Turk D & Turk V (2000) Lysosomal cysteine proteases: more than scavengers Biochim Biophys Acta 1477, 98–111 2 Turk B (2006) Targeting... enzymes and subcellular particles Fungal cysteine protease inhibitors interacting with tightly bound inhibitors Biochem J 127, 321–333 34 Twining SS (1984) Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes Anal Biochem 143, 30–34 Supporting information The following supplementary material is available: Fig S1 Clitocypin-encoding genes are present in the Macrolepiota procera genome... for 2 h at room temperature in 0.1 m citrate buffer, pH 2, in 0.1 m phosphate buffer, pH 6.5, or in 0.1 m Tris–HCl buffer, pH 11, and neutralized afterwards Residual activities were then determined against papain with Z-Phe-Arg-para-nitroanilide as the substrate [5] Acknowledgements This work was supported by the Slovenian Research Agency Grant No P4-0127 (J.K.) We are very grateful to Dr Roger Pain for... Dobersek A, Guncar G & Turk D (2008) Inhibitory fragment from the p41 form of invariant chain can regulate activity of cysteine cathepsins in antigen presentation J Biol Chem 283, 14453–14460 32 Morrison JF (1982) The slow-binding and slow, tightbinding inhibition of enzyme-catalyzed reactions Trends Biochem Sci 7, 102–105 33 Henderson PJF (1972) Linear equation that describes steady-state kinetics of enzymes... rMcp1 and rMcp4 Fig S6 ClustalW alignment of macrocypin and clitocypin sequences with deduced clitocypin-like sequences from Laccaria bicolor genome Table S1 List of degenerate and specific primers used in PCR reactions Doc S1: Supplementary results Doc S2: Supplementary experimental procedures This supplementary material can be found in the online article Please note: As a service to our authors and readers,...ˇ J Sabotic et al Fungal cysteine protease inhibitors eter (Aviv, Lakewood, NJ, USA), as described previously [9] Temperature and pH stability Recombinant macrocypins (0.1 mgÆmL)1) were incubated for 15 min in 0.1 m phosphate buffer, pH 6.5, containing 1.5 mm EDTA and 5 mm dithiothreitol, at 37, 75 or 100 °C, then incubated for 1 h at room temperature Macrocypins (0.1 mgÆmL)1) were also incubated for... Purif 15, 213–220 29 Kopitar G, Dolinar M, Strukelj B, Pungercar J & Turk V (1996) Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli Eur J Biochem 236, 558–562 30 Kuhelj R, Dolinar M, Pungercar J & Turk V (1995) The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies Eur . Macrocypins, a family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera Jerica Sabotic ˇ 1 , Tatjana Popovic ˇ 1 , Vida Puizdar 2 and. report a novel family of cysteine prote- ase inhibitors from basidiocarps, or fruiting bodies, of the basidiomycete Macrolepiota procera, and have characterized