Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin
2021; Elsevier BV; Volume: 297; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2021.100879
ISSN1083-351X
AutoresSeandean L. Harwood, Nadia Sukusu Nielsen, Khang Diep, Kathrine Tejlgård Jensen, Peter Kresten Nielsen, Kazuhiro Yamamoto, Jan J. Enghild,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoHuman α2-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer. Human α2-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer. Proteases constitute the largest enzyme family in humans with 641 currently identified members (1Bond J.S. Proteases: History, discovery, and roles in health and disease.J. Biol. Chem. 2019; 294: 1643-1651Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In addition, they are common virulence factors produced by viruses, bacteria, and parasites. As they are involved in virtually every type of biological event and pathogenic in many diseases, they represent potential drug targets (1Bond J.S. Proteases: History, discovery, and roles in health and disease.J. Biol. Chem. 2019; 294: 1643-1651Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, the therapeutic targeting of proteases, such as matrix metalloproteases (MMPs) in cancer, has proven difficult because of broad protease inhibition by small-molecule therapeutics (2Overall C.M. Kleifeld O. Tumour microenvironment - opinion: Validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy.Nat. Rev. Cancer. 2006; 6: 227-239Crossref PubMed Scopus (989) Google Scholar, 3Coussens L.M. Fingleton B. Matrisian L.M. Matrix metalloproteinase inhibitors and cancer: Trials and tribulations.Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2292) Google Scholar, 4Laronha H. Carpinteiro I. Portugal J. Azul A. Polido M. Petrova K.T. 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This conformational collapse is triggered by peptide bond hydrolysis within the bait region, a 39-residue sequence that is unstructured and poorly evolutionarily conserved, but is rapidly and preferentially cleaved by most proteases regardless of their catalytic class (6Gettins P. Cunningham L.W. Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine.Biochemistry. 1986; 25: 5011-5017Crossref PubMed Scopus (26) Google Scholar, 7Sottrup-Jensen L. Sand O. Kristensen L. Fey G.H. The alpha-macroglobulin bait region. Sequence diversity and localization of cleavage sites for proteinases in five mammalian alpha-macroglobulins.J. Biol. Chem. 1989; 264: 15781-15789Abstract Full Text PDF PubMed Google Scholar). 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Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (275) Google Scholar, 36Mortensen S. Kidmose R.T. Petersen S.V. Szilagyi A. Prohaszka Z. Andersen G.R. Structural basis for the function of complement component C4 within the classical and lectin pathways of complement.J. Immunol. 2015; 194: 5488-5496Crossref PubMed Scopus (52) Google Scholar, 37Aleshin A.E. DiScipio R.G. Stec B. Liddington R.C. Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex.J. Biol. Chem. 2012; 287: 19642-19652Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), but not for A2M. A2M can be made to collapse into a conformation that is highly similar to its protease-cleaved conformation by aminolysis of its thiol ester (e.g., using methylamine (MA)) (5Barrett A.J. Brown M.A. Sayers C.A. The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.Biochem. J. 1979; 181: 401-418Crossref PubMed Scopus (408) Google Scholar, 38Bretaudiere J.P. Tapon-Bretaudiere J. Stoops J.K. Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1437-1441Crossref PubMed Scopus (33) Google Scholar, 39Boisset N. Penczek P. Pochon F. Frank J. Lamy J. Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.J. Mol. Biol. 1993; 232: 522-529Crossref PubMed Scopus (41) Google Scholar), and the crystal structure of A2M-MA has been determined as a surrogate for its protease-cleaved conformation (40Marrero A. Duquerroy S. Trapani S. Goulas T. Guevara T. Andersen G.R. Navaza J. Sottrup-Jensen L. Gomis-Ruth F.X. The crystal structure of human alpha2-macroglobulin reveals a unique molecular cage.Angew. Chem. Int. Ed. Engl. 2012; 51: 3340-3344Crossref PubMed Scopus (75) Google Scholar). We have recently developed a low-resolution model of native A2M using negative stain electron microscopy, small-angle X-ray scattering, and cross-linking mass spectrometry (41Harwood S.L. Lyngsø J. Zarantonello A. Kjøge K. Nielsen P.K. Andersen G.R. Pedersen J.S. Enghild J.J. Structural investigations of human A2M identify a hollow native conformation that underlies its distinctive protease-trapping mechanism.Mol. Cell. Proteomics. 2021; 20: 100090Abstract Full Text Full Text PDF PubMed Google Scholar). This model indicates that proteases must enter the interior of native A2M to access and cleave the bait regions, at which point A2M collapses and closes off its entrances, thereby trapping the intruding protease (Fig. 1D). The bait regions occupy the interior space of native A2M and may interact with each other, as suggested by the formation of intersubunit disulfides upon the introduction of cysteine residues into the bait region (42Bowen M.E. Gettins P.G. Bait region involvement in the dimer-dimer interface of human alpha 2-macroglobulin and in mediating gross conformational change. Evidence from cysteine variants that form interdimer disulfides.J. Biol. Chem. 1998; 273: 1825-1831Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The precise molecular mechanism by which bait region cleavage triggers A2M's conformational collapse is not known. In the αM complement factors, the anaphylactic domain corresponds to the bait region of αM protease inhibitors, and its removal by proteolysis is thought to perturb the adjacent MG3 and MG8 domains, thereby initiating a conformational change (36Mortensen S. Kidmose R.T. Petersen S.V. Szilagyi A. Prohaszka Z. Andersen G.R. Structural basis for the function of complement component C4 within the classical and lectin pathways of complement.J. Immunol. 2015; 194: 5488-5496Crossref PubMed Scopus (52) Google Scholar). However, while the anaphylactic domain has a rigid α-helix structure (32Janssen B.J. Huizinga E.G. Raaijmakers H.C. Roos A. Daha M.R. Nilsson-Ekdahl K. Nilsson B. Gros P. Structures of complement component C3 provide insights into the function and evolution of immunity.Nature. 2005; 437: 505-511Crossref PubMed Scopus (391) Google Scholar) and is evolutionarily conserved across species, the bait region is unstructured and poorly conserved (Fig. S1), and it is unclear how it could participate in an equivalent triggering mechanism. While the reaction of A2M's thiol ester with nucleophiles (e.g., lysine side chain ε-amino groups on the surface of proteases) takes place during or after its conformational change (43Sottrup-Jensen L. Hansen H.F. Christensen U. Generation and reactivity of "nascent" alpha 2-macroglobulin: Localization of cross-links in alpha 2-macroglobulin-trypsin complex.Ann. N. Y. Acad. Sci. 1983; 421: 188-208Crossref PubMed Scopus (15) Google Scholar), its amino- or hydrolysis is not required for the conformational change to take place, as A2M lacking a thiol ester is still induced to change its conformation by proteolysis (44Harwood S.L. Nielsen N.S. Pedersen H. Kjoge K. Nielsen P.K. Andersen G.R. Enghild J.J. Substituting the thiol ester of human A2M or C3 with a disulfide produces native proteins with altered proteolysis-induced conformational changes.Biochemistry. 2020; 59: 4799-4809Crossref PubMed Scopus (1) Google Scholar), as are the thiol-ester-lacking αM proteins C5 and chicken ovostatin (10Nagase H. Harris Jr., E.D. Ovostatin: A novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin.J. Biol. Chem. 1983; 258: 7490-7498Abstract Full Text PDF PubMed Google Scholar, 37Aleshin A.E. DiScipio R.G. Stec B. Liddington R.C. Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex.J. Biol. Chem. 2012; 287: 19642-19652Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this study, we have produced and characterized recombinant A2M proteins with modified bait regions, which investigate the bait region's functional role and demonstrate the design of new protease inhibitors. We found that A2M's bait region can be replaced in its entirety with 13 Gly-Gly-Ser triplets without preventing A2M from assembling into its usual homotetrameric structure or assuming its native conformation. This tabula rasa bait region was not initially cleaved by any of 12 tested proteases, but could be cleaved by trypsin, LysC, or MMP2 upon introducing an appropriate cleavage site into its sequence. Bait region cleavage of tabula rasa A2Ms resulted in protease conjugation through A2M's thiol ester, the induction of A2M's conformational collapse, and protease inhibition, as demonstrated for trypsin and MMP2. An MMP2 substrate bait region that was selectively cleaved by MMPs was identified by screening using ten human proteases. Further optimization of the bait region yielded a tabula rasa A2M, which inhibited MMP2 as efficiently as wild-type A2M. Altogether, these results demonstrate an approach to developing more specific A2M-based protease inhibitors through bait region engineering and have identified several factors that are important during bait region design, including the cleavage site position and bait region length. The bait region is both ground zero for A2M's proteolytically induced conformational change and the major determiner of which proteases are inhibited by A2M. However, the low conservation of the bait region sequence across species (Fig. S1) suggests that it has no essential structure or sequence motifs. In order to remove essentially all protease cleavage sites or putative structural features from the bait region and determine the extent to which it tolerates modification, we entirely replaced the 39-residue bait region sequence with 13 Gly-Gly-Ser repeats, chosen for their solubility and low susceptibility to proteolysis (Fig. 2A). The resulting tabula rasa bait region could be incorporated into recombinant A2M, resulting in a tabula rasa A2M protein that was predominantly tetrameric and in its native conformation, with an intact thiol ester apparent from the formation of characteristic heat-induced autolysis products in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2, B and C). Upon aminolysis of its thiol ester with methylamine, tabula rasa A2M underwent a conformational collapse indistinguishable from that of wild-type A2M, as determined by pore-limited native PAGE; however, tabula rasa A2M was not cleaved in its bait region by either trypsin or LysC and remained in its native conformation despite proteolysis by these proteases outside of its bait region (Fig. 2, B and C). While the majority of tabula rasa A2M assembled into the normal homotetramer, dimeric tabula rasa A2M that had failed to tetramerize was slightly more abundant than wild-type A2M; dimers were removed during purification prior to all characterization (Fig. S2). Similarly, while the majority of tabula rasa A2M was in a native conformation, both fully collapsed A2M and conformations with an intermediate electrophoretic mobility were apparent in pore-limited native PAGE and more abundant than in wild-type A2M (Fig. 2B). Before using these proteins in quantitative protease inhibition assays, nonnative A2M was depleted using LRP1-conjugated resin (Fig. S3). The TR K704 bait region added a lysine residue at position 704 and was cleaved by trypsin and LysC, which caused A2M to undergo a conformational collapse (Fig. 2, A–C); as the wild-type bait region and the initial tabula rasa bait region contain no lysine residues, LysC cleaved these A2Ms in locations other than the bait region without inducing a conformational change, as has been previously reported (45Van Leuven F. Marynen P. Cassiman J.J. Van den Berghe H. Proteolysis of human alpha 2-macroglobulin without hydrolysis of the internal thiolesters or expression of the receptor recognition site.J. Biol. Chem. 1988; 263: 468-471Abstract Full Text PDF PubMed Google Scholar). The protease-induced conformational change of A2M TR K704 activated its thiol ester and resulted in the formation of covalent conjugation products that were apparent as high-MW bands in reducing SDS-PAGE (Fig. 2C). Altogether, these results showed that while the wild-type bait region may make minor contributions toward A2M's tetrameric assembly and the assumption of its native conformation, no part of its sequence is structurally or functionally essential. The majority of tabula rasa A2M is tetrameric and in a native conformation and can undergo a methylamine- or proteolysis-induced conformational change (if protease substrate sites are incorporated into the bait region). Bait region cleavage of A2M TR K704 by both trypsin and LysC produced an intense band migrating as ∼250 kDa in reducing SDS-PAGE that was not produced by trypsin-cleaved wild-type A2M (Fig. 2C). The bait region is spatially close to the TE domain when A2M is proteolytically activated (Fig. 1, B–D), and we hypothesized that the nucleophilic ε-amine group of Lys704's side chain might attack the thiol ester following proteolysis, conjugating the N- and C-terminal bait region cleavage fragments together into an aberrantly migrating ∼180 kDa product. This proposed product of Lys704 autoconjugation disappeared when 3-aminopropanenitrile (BAPN), a small nucleophile that reacts with the thiol ester after it is proteolytically exposed and out-competes conjugation to other nucleophiles (9Salvesen G.S. Sayers C.A. Barrett A.J. Further characterization of the covalent linking reaction of alpha 2-macroglobulin.Biochem. J. 1981; 195: 453-461Crossref PubMed Scopus (135) Google Scholar), was included alongside trypsin, demonstrating that it is formed through thiol-ester-mediated conjugation (Fig. 3A). After cleavage of A2M TR K704 with Cy5-labeled trypsin, this band was not fluorescent and therefore does not contain trypsin (Fig. 3A). In fact, the product band became more intense upon cleaving A2M TR K704 with acetylated trypsin, which is not efficiently conjugated as it lacks primary amine groups (Fig. 3A). LC-MS/MS analysis of the pepsin-digested product band identified a cross-linked peptide containing an isopeptide cross-link between the thiol ester Gln975 and the bait region Lys704 (Fig. 3B). Furthermore, the band was not produced upon tryptic cleavage of tabula rasa A2M with a bait region arginine residue instead of a lysine residue (Fig. S4, A and B). These results conclusively show that this band represents an autoconjugation product of the thiol ester and a bait region lysine residue. We proceeded to investigate the trypsin-inhibiting ability of tabula rasa-based A2Ms using a fluorescently labeled gelatin substrate. In addition to A2M TR K704, bait regions incorporating an Arg704, Lys710, or Arg710 residue were produced (Fig. 4A). These A2M proteins appeared similar to A2M TR K704 during their initial characterization by electrophoresis (Fig. S4, A and B). In order to quantitatively determine the inhibitory capacity of the native tabula rasa A2Ms, nonnative A2M was depleted from these proteins using LRP1-conjugated resin before all inhibitory assays (Fig. S3). A2M with the tabula rasa bait regio
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