Crystal structure of the Bowman-Birk inhibitor from barley seeds in ternary complex with porcine trypsin

The structure and function of Bowman-Birk inhibitors (BBIs) from dicotyledonous plants such as soybean have been studied extensively. In contrast, relatively little is known about the BBIs from monocotyledonous plants such as barley, which differ from dicot BBIs in size and tertiary structure. The BBI from barley seeds (BBBI) consists of 125 amino acid residues with two separate inhibitory loops. Previously we determined the high-resolution structure of a 16 kDa BBBI in the free state. The BBBI folds into two compact domains (N and C domain) with tertiary structures that are similar to that of the 8 kDa BBI from dicots. Here we report the structure of a 1:2 complex between BBBI and porcine pancreatic trypsin (PPT) at 2.2 A resolution. This structure confirms that several regions, including the inhibitory loops in the free BBBI structure, show exceptionally low temperature factors and a distorted conformation due to crystalline packing in the lattice. Extensive analysis of the interaction between BBBI and trypsin, and comparison with other known canonical inhibitor-protease complexes, reveals that the mode of interaction between BBBI and PPT is similar to that of known serine protease inhibitors, as expected; however, several unique features are also identified in the primary binding sites near the inhibitory loops as well as in additional binding sites. The carboxy-terminal tail of the inhibitor extends into the interface between the two trypsin molecules and interacts with both of them simultaneously. The longest distance between the two P1 residues (Arg17 and Arg76) in the complex structure is approximately 34 A, which is shorter than in the free inhibitor, but it is still possible for BBBI to bind and inhibit two trypsin molecules simultaneously and independently.     

Crystal structure of a 16 kDa double-headed Bowman-Birk trypsin inhibitor from barley seeds at 1.9 A resolution.

The Bowman-Birk trypsin inhibitor from barley seeds (BBBI) consists of 125 amino acid residues with two inhibitory loops. Its crystal structure in the free state has been determined by the multiwavelength anomalous diffraction (MAD) method and has been refined to a crystallographic R-value of 19.1 % for 8.0-1.9 A data. This is the first report on the structure of a 16 kDa double-headed Bowman-Birk inhibitor (BBI) from monocotyledonous plants and provides the highest resolution picture of a BBI to date. The BBBI structure consists of 11 beta-strands and the loops connecting these beta-strands but it lacks alpha-helices. BBBI folds into two compact domains of similar tertiary structure. Each domain shares the same overall fold with 8 kDa dicotyledonous BBIs. The five disulfide bridges in each domain are a subset of the seven disulfide bridges in 8 kDa dicotyledonous BBIs. Two buried water molecules form hydrogen bonds to backbone atoms in the core of each domain. One interesting feature of this two-domain inhibitor structure is that the two P1 residues (Arg17 and Arg76) are approximately 40 A apart, allowing the two reactive-site loops to bind to and to inhibit two trypsin molecules simultaneously and independently. The conformations of the reactive-site loops of BBBI are highly similar to those of other substrate-like inhibitors. This structure provides the framework for modeling of the 1:2 complex between BBBI and trypsin.     

Crystal structure of bovine trypsin and wheat germ trypsin inhibitor (I-2b) complex (2:1) at 2.3 a resolution

The Bowman-Birk trypsin inhibitor (BBI) from wheat germ (I-2b) consists of 123 amino acid residues with two inhibitory loops. The crystal structure of a bovine trypsin-wheat germ trypsin inhibitor (I-2b) complex (2:1) has been determined at 2.3 A resolution to a final R-factor of 0.177. A distance of 37.2 A between the contiguous contact loops allows them to bind and inhibit two trypsin molecules simultaneously and independently. Each domain shares the same overall fold with 8 kDa BBIs. The five disulfide bridges in each domain are a subset of seven disulfide bridges in the 8 kDa BBIs. I-2b consists of ten beta-strands and the loops connecting these strands but it lacks alpha-helices. The conformations of the contiguous contact loops of I-2b are in a heart-like structure. The reactive sites in both domains, Arg 17 and Lys 76, are located on the loop connecting anti-parallel beta-strands, beta 1/beta 2 and beta 6/beta 7. Strands beta 1 and beta 6 are in direct contact with trypsin molecules and form stable triple stranded beta-sheet structures via hydrogen bonds.     

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots. S. Selvaraj is on leave from Department of Physics, Bharathidasan University, Tiruchirapalli 620 024, Tamilnadu, India Correspondence to: M.R.N. Murthy     

The structural basis of a conserved P2 threonine in canonical serine proteinase inhibitors

Bowman-Birk inhibitors (BBIs) are a well-studied family of canonical inhibitor proteins of serine proteinases. In nature, the active region of BBIs possesses a highly conserved Thr at the P2 position. The importance of this residue has been reemphasized by synthetic BBI reactive site loop proteinomimetics. In particular, this residue was exclusively identified for active chymotrypsin inhibitors selected from a BBI template-assisted combinatorial peptide library. A further kinetic analysis of 26 P2 variant peptides revealed that Thr provides both optimal binding affinity and optimal resistance against enzymatic turnover by chymotrypsin. Herein, we report the (1)H-NMR spectroscopic study of a 5-membered sub-set of these reactive site loop peptides representing a stepwise elimination of the Thr side-chain functionalities and inversion of its side-chain chirality. The P2 Thr variant adopts a three-dimensional structure that closely mimics the one of the corresponding region of the complete protein. This validates the use of this template for the investigation of structure-function relationships. While the overall backbone geometry is similar in all studied variants, conformational changes induced by the modification of the P2 side chain have now been identified and provide a rational explanation of the kinetically observed functional differences. Eliminating the gamma-methyl group has little structural effect, whereas the elimination of the gamma-oxygen atom or the inversion of the side-chain chirality results in characteristic changes to the intramolecular hydrogen bond network. We conclude that the transannular hydrogen bond between the P2 Thr side-chain hydroxyl and the P5' backbone amide is an important conformational constraint and directs the hydrophobic contact of the P2 Thr side chain with the enzyme surface in a functionally optimal geometry, both in the proteinomimetic and the native protein. In at least four canonical inhibitor protein families similar structural arrangements for a conserved P2 Thr have been observed, which suggests an analogous functional role. Substitutions at P2 of the proteinomimetic also affect the conformational balance between cis and trans isomers at a distant Pro-Pro motif (P3'-P4'). Presented with a mixture of cis/trans isomers chymotrypsin appears to interact preferably with the conformer that retains the cis-P3' Pro-trans-P4' Pro geometry found in the parent BBI protein.     

Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides: creation of a new tri-functional inhibitor

Bowman-Birk inhibitor proteins (BBIs), which are potent inhibitors of chymotrypsin-like proteases, do not inhibit human beta-tryptase despite this protein having a chymotrypsin-like fold. We have reported previously that, in contrast, BBI-derived peptides (whose sequences incorporate the solvent exposed reactive site loop motif) are able to inhibit human beta-tryptase. This is due to their small size, which allows them to access the restricted active site(s) of tryptase, which has an unusual tetrameric arrangement with four active sites flanking a central pore. In this paper, we have examined the possibility of creating additional interactions within this pore by adding extensions to the BBI-peptide motif. We have taken the core disulfide-bridged sequence SCTKSIPPQCY and examined a series of extensions, at both the C- and N-termini, that bear a second positively charged Lys residue at their end. The aim was to construct inhibitors that could make additional interactions in tryptase by spanning the gap between adjacent active sites in the enzyme, producing a double-headed inhibitor; a positively charged group was used as the dominant specificity of this enzyme is for a positively charged P1 residue. Both N- and C-terminal extensions are found to produce inhibitors of much increased potency, with a strong dependence of potency on chain length. Moreover, it was found that the C- and N-terminal extensions were able to synergise, with their combination on the same peptide producing an even better inhibitor with a potency 10(4)-fold greater than the original sequence. We suggest that the C- and N-terminal extensions are picking up interactions with separate additional sites on the tryptase, making the doubly extended BBI peptide a tri-functional tryptase inhibitor.     

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.     

The Structural Basis of a Conserved P2 Threonine in Canonical Serine Proteinase Inhibitors

Abstract

Bowman-Birk inhibitors (BBIs) are a well-studied family of canonical inhibitor proteins of serine proteinases. In nature, the active region of BBIs possesses a highly conserved Thr at the P2 position. The importance of this residue has been reemphasized by synthetic BBI reactive site loop proteinomimetics. In particular, this residue was exclusively identified for active chymotrypsin inhibitors selected from a BBI template-assisted combinatorial peptide library. A further kinetic analysis of 26 P2 variant peptides revealed that Thr provides both optimal binding affinity and optimal resistance against enzymatic turnover by chymotrypsin.

Herein, we report the H-NMR spectroscopic study of a 5-membered sub-set of these reactive site loop peptides representing a stepwise elimination of the Thr side-chain functionalities and inversion of its side-chain chirality. The P2 Thr variant adopts a three-dimensional structure that closely mimics the one of the corresponding region of the complete protein. This validates the use of this template for the investigation of structure-function relationships. While the overall backbone geometry is similar in all studied variants, conformational changes induced by the modification of the P2 side chain have now been identified and provide a rational explanation of the kinetically observed functional differences. Eliminating the γ-methyl group has little structural effect, whereas the elimination of the γ-oxygen atom or the inversion of the side-chain chirality results in characteristic changes to the intramolecular hydrogen bond network. We conclude that the transannular hydrogen bond between the P2 Thr side-chain hydroxyl and the P5′ backbone amide is an important conformational constraint and directs the hydrophobic contact of the P2 Thr side chain with the enzyme surface in a functionally optimal geometry, both in the proteinomimetic and the native protein.

In at least four canonical inhibitor protein families similar structural arrangements for a conserved P2 Thr have been observed, which suggests an analogous functional role. Substitutions at P2 of the proteinomimetic also affect the conformational balance between cis and trans isomers at a distant Pro-Pro motif (P3′-P4′). Presented with a mixture of cis/trans isomers chymotrypsin appears to interact preferably with the conformer that retains the cis-P3′ Pro-trans-P4′ Pro geometry found in the parent BBI protein.     

The absolute structural requirement for a proline in the P3'-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp14 was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro13 and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop.     

Molecular evolution of Bowman-Birk type proteinase inhibitors in flowering plants

The Bowman-Birk family (BBI) of proteinase inhibitors is probably the most studied family of plant inhibitors. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the GenBank database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events.     

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.     

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1′ pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

Studies on simultaneous inhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences. The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.     

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.     

Participation of S-S loops in inhibitory activity of peanut protease inhibitor B-III

A peanut Bowman-Birk (BBI) type protease inhibitors B-III has two regions, 1 and 2, homologous with each other. Each region contains three S-S loops and a reactive site in its outermost loop. The inhibitor was used to investigate the contribution of the S-S loops of BBI-type inhibitors to their inhibitory activity. Two steps of Edman degradation of the native inhibitor cleaved loop III (the innermost S-S loop) of region 1 of B-III, and the antichymotryptic activity of the first reactive site decreased to about 1/4 of that of native B-III. A third step of Edman degradation split loop II and the inhibitory activity at that site became extremely low (about 1/200 of the original value). These results suggest that protease inhibitor B-III maintains its active conformation by means of the three S-S loops and that the conformation is markedly changed by the splitting of loop II.     

Contribution of conserved Asn residues to the inhibitory activities of Kunitz-type protease inhibitors from plants

Plant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Erythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex.     

Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families

By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.