Expression of LEKTI domains 6–9′ in the baculovirus expression system: recombinant LEKTI domains 6–9′ inhibit trypsin and subtilisin A

Jayakumar, Arumugam; Kang, Ya’an; Mitsudo, Kenji; Henderson, Ying C.; Frederick, Mitchell J.; Wang, Mary; El‐Naggar, Adel K.; Marx, Ute C.; Briggs, Katrina; Clayman, Gary L.

doi:10.1016/j.pep.2003.12.004

Cited by 43 publications

(37 citation statements)

References 27 publications

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“…First, processed amino-terminal fragments of rEPI10 containing one or two Kazal domains might be functional in interacting with P69. Indeed, there are many examples of active shorter peptides derived from a multidomain inhibitor and some Kazal inhibitors are naturally processed into single-domain peptides (Scott et al, 1987;Magert et al, 1999;Mitsudo et al, 2003;Jayakumar et al, 2004;Mende et al, 2004). Second, rEPI10 was not totally processed, and some intact molecules could still be detected in the eluates (Fig.…”

Section: Discussionmentioning

confidence: 99%

A Second Kazal-Like Protease Inhibitor from Phytophthora infestans Inhibits and Interacts with the Apoplastic Pathogenesis-Related Protease P69B of Tomato

2005

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The plant apoplast forms a protease-rich environment in which proteases are integral components of the plant defense response. Plant pathogenic oomycetes, such as the potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) pathogen Phytophthora infestans, secrete a diverse family of serine protease inhibitors of the Kazal family. Among these, the two-domain EPI1 protein was shown to inhibit and interact with the pathogenesis-related protein P69B subtilase of tomato and was implicated in counter-defense. Here, we describe and functionally characterize a second extracellular protease inhibitor, EPI10, from P. infestans. EPI10 contains three Kazal-like domains, one of which was predicted to be an efficient inhibitor of subtilisin A by an additivity-based sequence to reactivity algorithm (Laskowski algorithm). The epi10 gene was up-regulated during infection of tomato, suggesting a potential role during pathogenesis. Recombinant EPI10 specifically inhibited subtilisin A among the major serine proteases, and inhibited and interacted with P69B subtilase of tomato. The finding that P. infestans evolved two distinct and structurally divergent protease inhibitors to target the same plant protease suggests that inhibition of P69B could be an important infection mechanism for this pathogen.The plant apoplast forms a protease-rich environment in which proteases are integral components of the plant defense response (Tornero et al., 1997;Jorda et al., 1999;van Loon and van Strien, 1999;Kruger et al., 2002;Xia et al., 2004). For example, the subtilisin-like Ser protease P69B, an apoplastic pathogenesis-related (PR) protein of tomato (Lycopersicon esculentum), has long been tied to plant defense (Tornero et al., 1997;Zhao et al., 2003;Tian et al., 2004). P69B is induced by multiple plant pathogens, including the oomycete Phytophthora infestans, citrus exocortis viroid, and the bacterium Pseudomonas syringae (Tornero et al., 1997;Zhao et al., 2003;Tian et al., 2004), and appears to account for a significant portion of the increase in total endoprotease activity observed in defense-activated tomato (Tornero et al., 1997;Zhao et al., 2003;Tian et al., 2004). Rcr3, an apoplastic papain-like Cys protease from tomato, is required for specific resistance to the plant pathogenic fungus Cladosporium fulvum (Kruger et al., 2002). In Arabidopsis (Arabidopsis thaliana), the extracellular aspartic protease CDR1 functions in disease resistance signaling as a positive regulator of cell death (Xia et al., 2004). These findings suggest that suppression of plant protease-mediated host defenses could be one of the diverse strategies that plant pathogens have evolved to survive in the plant intercellular space and colonize plant tissue. Indeed, our laboratory recently reported that plant pathogenic oomycetes secrete a diverse family of Kazal-like Ser protease inhibitors with at least 35 members identified from P. infestans, Phytophthora sojae, Phytophthora ramorum, Phytophthora brassicae, and the downy mildew Plasmopara halstedii (Tian et ...

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Section: Discussionmentioning

confidence: 99%

A Second Kazal-Like Protease Inhibitor from Phytophthora infestans Inhibits and Interacts with the Apoplastic Pathogenesis-Related Protease P69B of Tomato

2005

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“…Recombinant pro-KLC1 was produced in Chinese hamster ovary cells, and recombinant pro-KLK1 was produced in the human embryonic kidney cell line, HEK293, as described previously (32,33). LEKTI fragments containing intact domains 1-6, LEKTI(1-6), domains 6 -8 and partial domain 9, LEKTI(6 -9Ј), domains 9 -12, LEKTI (9 -12), and domains 12-15, LEKTI (12)(13)(14)(15), were produced in a baculovirus/insect system as reported previously (34,35). Recombinant SLPI, neutrophil elastase, and elafin were purchased from R & D Systems Inc. (Minneapolis, MN), Calbiochem, and Sigma, respectively, and diluted to a final concentration of 0.5g/liter and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade

Eissa

Amodeo

Smith

et al. 2011

Journal of Biological Chemistry

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Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg 164 . Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1-P2. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.

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“…Purified proteins were analyzed by SDS-PAGE after Coomassie Blue staining. HisD9 -D15 was purified following the experimental procedures as previously described (Jayakumar et al, 2004). Proteins were dialyzed against a solution of HEPES 10 mM, pH 7.4, for inhibitory activity assay and Biacore analysis.…”

Section: Cloning Expression and Purification Of Lekti Domainsmentioning

confidence: 99%

“…The full-length LEKTI recombinant protein has been shown to inhibit trypsin, subtilisin A, plasmin, cathepsin G, and neutrophil elastase, but not chymotrypsin (Mitsudo et al, 2003). A partial recombinant form of LEKTI containing domains 6 -9 (rLEKTI6 -9) has been shown to inhibit trypsin, subtilisin A, chymotrypsin, kallikrein 5 (KLK5, stratum corneum tryptic enzyme [SCTE]), and kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme [SCCE]) but not plasmin, cathepsin G, or elastase (Jayakumar et al, 2004;Schechter et al, 2005). In addition, the single domain D6 was shown to be a potent inhibitor of trypsin, KLK5, and KLK7, whereas D15 was not effective against these two kallikreins .…”

Section: Introductionmentioning

confidence: 99%

LEKTI Fragments Specifically Inhibit KLK5, KLK7, and KLK14 and Control Desquamation through a pH-dependent Interaction

Deraison

Bonnart

Lopez

et al. 2007

MBoC

Self Cite

287

266

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LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8 -D11, and D9 -D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8 -D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.

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Expression of LEKTI domains 6–9′ in the baculovirus expression system: recombinant LEKTI domains 6–9′ inhibit trypsin and subtilisin A

Cited by 43 publications

References 27 publications

A Second Kazal-Like Protease Inhibitor from Phytophthora infestans Inhibits and Interacts with the Apoplastic Pathogenesis-Related Protease P69B of Tomato

A Second Kazal-Like Protease Inhibitor from Phytophthora infestans Inhibits and Interacts with the Apoplastic Pathogenesis-Related Protease P69B of Tomato

Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade

LEKTI Fragments Specifically Inhibit KLK5, KLK7, and KLK14 and Control Desquamation through a pH-dependent Interaction

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