Action of plant proteinase inhibitors on enzymes of physiopathological importance

BIOMEDICAL AND MEDICAL SCIENCES

Action of plant proteinase inhibitors on enzymes of physiopathological importance

Maria Luiza V. Oliva; Misako U. Sampaio

Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina Rua Três de Maio, 100, 04044-020 São Paulo, SP, Brasil

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ABSTRACT

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

Key words: Bowman-Birk, chymotrypsin, Kunitz inhibitors, plasma kallikrein, primary structure, trypsin.

RESUMO

Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.

Palavras-chave: Bowman-Birk, quimotripsina, inibidoresKunitz, calicreína plasmática, estrutura primária, tripsina.

INTRODUCTION

Proteolytic enzymes are abundant in living cells and play important roles in intracellular proteolysis. Many studies have shown that proteinases are targets for the investigation of several diseases.

By cleaving proteins, proteinases are involved in the control of a large number of key physiological processes, such as cell-cycle progression, cell death, cell proliferation, DNA replicatin, haemostasis, immune response, tissue remodeling and wound healing (Turk 2006 - review). For instance, in the case of cysteine proteinases, since the imbalance of their enzymatic activities causes serious diseases, such as osteoporosis (Delaissé et al. 1984) and tumor invasion (Denhardt et al. 1987), the search for inhibitors that can moderately control their activity is desired for drug development. Also, these enzymes have been correlated with the invasion process of many parasites, which demonstrates important interactions with the host immune system (Renslo and McKerrow 2006 - review).

These inhibitors interact reversibly with proteinases forming stoichiometric complexes and competitively influencing the catalytic activity (Radisky et al. 2004).

Serine proteinases activity is blocked through a tight binding of the enzyme active site and the inhibitor, resulting in a complex resistant to proteolysis (Laskowski and Kato 1980, Bode and Huber 1992).

Multiple molecular forms of protein inhibitors have been characterized from animals, microorganisms and plants (Ryan 2000, Birk 2003).

The interest in enzyme inhibitors obtained from plants began in the 1940s, when Kunitz (1946, 1947) isolated and purified from soybean a protein which inhibited trypsin. Inhibitor proteins have been studied as model systems for elucidating proteinase inhibition mechanisms, as well as for studying the protein-protein associations. Being considered anti-nutritional factors, those inhibitors are believed to participate in various physiological functions, such as the regulation of proteolytic cascades and the safe storage of proteins, as well as to act as defense molecules against plant pest and pathogens (Birk 2003, Sumikawa et al. 2008).

The best known groups of inhibitors obtained fromseeds include serine proteinase inhibitors (EC. 3. 4. 21) of chymotrypsin (EC. 3. 4. 21. 3), trypsin (EC. 3. 4. 21. 4)and subtilisin (EC. 3. 4. 21. 62). Numerous examples of inhibitors are also known for aspartyl proteinases (EC. 3. 4. 23), cysteine proteinases (EC. 3. 4. 22) and metallo-proteinases (EC. 3. 4. 12).

Kunitz-type proteinase inhibitors are abundant in seeds from Leguminosae subfamilies, i. e. Mimosoideae, Caesalpinoideae and Papilionoideae. This type of inhibitor normally occurs as a single polypeptide chain; however, some inhibitors have also been shown to be dimeric proteins (Richardson 1991, Krauchenco et al. 2001, 2004).

This review deals with our recent data on the structure and function of plant Kunitz-type inhibitor interactions in biochemical processes involved in some diseases.

PLANT KUNITZ-TYPE INHIBITORS

Plant Kunitz-type inhibitors are easily found in leguminous seeds. As mentioned previously, the first inhibitor from this family (SBTI) was obtained from Glycine max seeds and, over the past three decades, a large number of other inhibitors have been purified and their primary structures determined. This lead s to the conclusion that these inhibitors are not restricted only to the leguminous group, but are also found in other plants (Richardson 1991, Birk 2003).

Information on the structure of plant Kunitz-type inhibitors is helpful to understand the mechanisms underlying their specificity for coagulation factors, inflammation and tumors, and to allow us to investigate which region of the protein is responsible for its biological activity.

The primary sequences of inhibitors may be highly similar within the same family. Several structural features are conserved in most Kunitz-type inhibitors: molecular mass of approximately 20 kDa, four cysteine residues and the sequence neighboring the single reactive site, which in general is Arg-Ser or Arg-Lys situated in a loop closed off by one disulfide bridge, and involved in trypsin inhibition (Richardson 1991, Birk 2003).

Souza-Pinto et al. (1996) purified a Kunitz trypsin inhibitor (LlTI) from Leucaena leucocephala (Fig. 1). Biochemical studies showed that LlTI blocks enzymes involved in blood clotting and fibrinolysis (Table I), has anti-inflammatory effects and decreases bradykinin release.

Inhibitors isolated from different species of Bauhinia seeds inhibit blood clotting enzymes, as well as other serine and cysteine proteinases. The inhibitors BbKI and BbCI, obtained from seeds of Bauhinia bauhinioides, a plant known in Brazil by the popular name of "cow paw" due the shape of its leaves, are 18 kDa proteins that present a high primary structure similarity with other plant Kunitz-type inhibitors (Fig. 2), but differ by the absence of disulfide bridges and in their inhibition specificity (Oliva et al. 1999a, b, 2003, de Oliveira et al. 2001). The description of other inhibitors lacking the four conservative cysteine residues (Macedo et al. 2007) reinforces the establishment of a new group of plant Kunitz family.

BbCI and BbKI recombinants were obtained by heterogonous expression and production in E. coli, and both proteins similar to the wild-type proteins (Araújo et al. 2005) showed potent inhibitory activities towards their target proteinases. What distinguishes BbCI is the inhibition of two different classes of proteinases, e.g., it inhibits the serine proteinases human neutrophil elastase and pancreatic porcine elastase, and the cysteine proteinases cathepsin L and cruzipain from Trypanosoma cruzi. Alanine in the P1 position is essential for these inhibitions. Although BbKI primary structure is highly identical to BbCI (84%), it differs by inhibiting bovine trypsin, human plasmin and plasma kallikrein (Table I). BbKI does not affect the activity of cysteine proteinases, neither BbCI interfere on blood clotting enzyme activities (de Oliveira et al. 2001, Neuhof et al. 2003).

Hansen and co-workers (2007) reported the threedimensional structure of the recombinant BbCI at 1.7Å resolution and, in comparison to the structures of BbKI and other plant Kunitz-type inhibitors, it was shown that they share a common β-trefoil fold. Furthermore, the crystallographic structure of BbCI showed that the maintenance of the canonical conformation of the reactive site loop is important for a proper inhibitory function, and that the protein scaffold plays an important role at this site. The absence of disulfide bridges in the structure of BbCI is compensated for by essential interactions that maintain its structural stability and preserve its biological function.

BrTI, a Kunitz-type proteinase inhibitor purified from Bauhinia rufa seeds, contains the RGD sequence and inhibits human plasma kallikrein and trypsin, but not other related enzymes (Nakahata et al. 2006). A variety of studies have demonstrated that proteinase inhibitors can suppress several stages of carcinogenesis, including tumor initiation, promotion and progression. Although their mechanism of action is not yet clear, in 2006, Nakahata and co-workers reported the inhibitory action of YLEPVARGDGGLA-NH2, a synthetic peptide containing the RGD sequence derived from the structure of BrTI (Fig. 2). This peptide inhibited the adhesion of B16F10 (a high-metastatic B16 murine melanoma cell line) and Tm5 (a murine melanoma cell line derived from a non-tumorigenic lineage of pigmented murine melanocytes, melan-a) to fibronectin. When Asp9 was changed to Glu (YLEPVARGEGGLA-NH₂), cell attachment was not affected. Moreover, this peptide was functional only when the sequence present in the native protein was preserved, since changing Glu3 to Ile (YLIPVARGDGGLA-NH2) did not interfere with B16F10 cell adhesion and was less effective on the adhesion of Tm5 cells. Neither YLEPVARGDGGLA-NH2 nor YLIPVARGDGGLA-NH2 and YLEPVARGEGGLA-NH2 affected the interaction of RAEC (an endothelial cell line from rabbit aorta) with fibronectin. Differently fromother Bauhinia inhibitors, BrTI is the only one that exhibits insecticidal activity on Callosobruchus maculatuslarvae (J. T. Sumikawa et al. , unpublished data).

Purified from Enterolobium contortisiliquum seeds, EcTI (Fig. 1) appears to be an interesting inhibitor sinceit shows a strong capacity for inhibiting trypsin (K_i(app) 0. 88 nM), chymotrypsin (K_i(app) 1. 11 nM), plasma kallikrein (K_i(app) 6. 15 nM), plasmin (K_(iapp 9. 36 nM) and human neutrophil elastase (Ki (app) 55. 00 nM) (Oliva et al. 1987, Batista et al. 1996, 2001) (Table I), but not cysteine proteinases.

The inhibitory capacity of these proteinase inhibitors was investigated on the cell viability of different tumor cell lines, primary human fibroblasts and on the proliferation capacity of human mesenchymal stem cells, in addition to their mechanism of action on blood coagulation, fibrinolysis, inflammation and platelet aggregation.

ACKNOWLEDGMENTS

The authors thank the collaborative work of students in our laboratory and Reinhart Mentele from Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik und Polyklinik, LMU, Munich, Germany, for performing the structure determinations. The skilled technical assistance of Lucimeire A. Santana and Magda Theodoro de Souza is also gratefully acknowledged. This work was partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento e Tecnologia (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Fundo de Auxílio aos Docentes e Alunos/Fundação de Apoio à UNIFESP (FADA/FAP).

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Manuscript received on July 25, 2008; accepted for publication on May 26, 2009; presented by LUIZ R. TRAVASSOS

In commemoration of the 75th anniversary of Escola Paulista de Medicina / Universidade Federal de São Paulo.

Dedicated to the memory of Prof. Dr. Claudio A.M. Sampaio (1943-2005)

Publication Dates

• Publication in this collection
  20 Aug 2009
• Date of issue
  Sept 2009

History

• Received
  25 July 2008
• Accepted
  26 May 2009