Mohamed Azarkan

Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism.

Abstract. In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.

Fractionation and purification of the enzymes stored in the latex of Carica papaya

The latex of the tropical species Carica papaya is well known for being a rich source of the four cysteine endopeptidases papain, chymopapain, glycyl endopeptidase and caricain. Altogether, these enzymes are present in the laticifers at a concentration higher than 1 mM. The proteinases are synthesized as inactive precursors that convert into mature enzymes within 2 min after wounding the plant when the latex is abruptly expelled. Papaya latex also contains other enzymes as minor constituents. Several of these enzymes namely a class-II and a class-III chitinase, an inhibitor of serine proteinases and a glutaminyl cyclotransferase have already been purified up to apparent homogeneity and characterized. The presence of a beta-1,3-glucanase and of a cystatin is also suspected but they have not yet been isolated. Purification of these papaya enzymes calls on the use of ion-exchange supports (such as SP-Sepharose Fast Flow) and hydrophobic supports [such as Fractogel TSK Butyl 650(M), Fractogel EMD Propyl 650(S) or Thiophilic gels]. The use of covalent or affinity gels is recommended to provide preparations of cysteine endopeptidases with a high free thiol content (ideally 1 mol of essential free thiol function per mol of enzyme). The selective grafting of activated methoxypoly(ethylene glycol) chains (with M(r) of 5000) on the free thiol functions of the proteinases provides an interesting alternative to the use of covalent and affinity chromatographies especially in the case of enzymes such as chymopapain that contains, in its native state, two thiol functions.

X-ray Structure of Papaya Chitinase Reveals the Substrate Binding Mode of Glycosyl Hydrolase Family 19 Chitinases

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation

Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a lambdamax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.

Interplay between predicted inner-rod and gatekeeper in controlling substrate specificity of the type III secretion system.

The type III secretion apparatus (T3SA) is a multi‐protein complex central to the virulence of many Gram‐negative pathogens. Currently, the mechanisms controlling the hierarchical addressing of needle subunits, translocators and effectors to the T3SA are still poorly understood. In Shigella, MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle. However, molecules involved in linking the needle and MxiC are unknown. Here, we demonstrate a molecular interaction between MxiC and the predicted inner‐rod component MxiI suggesting that this complex plugs the T3SA entry gate. Our results suggest that MxiI–MxiC complex dissociation facilitates the switch in secretion from translocators to effectors. We identified MxiCF206S variant, unable to interact with MxiI, which exhibits a constitutive secretion phenotype although it remains responsive to induction. Moreover, we identified the mxiIQ67A mutant that only secretes translocators, a phenotype that was suppressed by coexpression of the MxiCF206S variant. We demonstrated the interaction between MxiI and MxiC homologues in Yersinia and Salmonella. Lastly, we identified an interaction between MxiC and chaperone IpgC which contributes to understanding how translocators secretion is regulated. In summary, this study suggests the existence of a widely conserved T3S mechanism that regulates effectors secretion.

Thiol pegylation facilitates purification of chymopapain leading to diffraction studies at 1.4 Å resolution

Abstract Thiol pegylation of a protein profoundly affect its chromatographic behavior on ion-exchange supports as a results of charge shielding effects induced by the presence of the polyethylene glycol (PEG) chain(s) at the surface of the polypeptide. When PEG chain(s) is(are) covalently bound via disulfide bonds, thiol pegylation is reversible and may be used in the context of purifying enzymes such as chymopapain, the dithiol proteinase from papaya latex, investigated here. Reaction of chymopapain with a dithiopyridyl poly(ethylene glycol) (PEG) reagent, possessing an extended spacer arm, followed by cation-exchange chromatography on S-Sepharose Fast Flow, afforded for the first time an homogeneous preparation of the native form of this proteinase. This constituted the key for obtaining highly diffracting crystals for chymopapain (as the protected S,S′-dimethylthio derivative) exhibiting diffraction spots visible up to a resolution of 1.4 A.

Reversible modification of thiol-containing polypeptides with poly (ethylene glycol) through formation of mixed disulfide bonds. The case of papaya proteinase III.

Papaya proteinase III (PPIII) was purified, as the S-methylthio derivative from the latex ofCarica papaya L., by ion-exchange chromatography. Separation of reactivable PPIII from the irreversibly oxidized molecular species of this enzyme was readily achieved after a selective conversion of the reactivated proteinase into the S-monomethoxypoly-(ethylene glycol) thio derivative (S-mPEG thio PPIII). From this derivative, a PPIII preparation titrating 1 mol of thiol/mol of enzyme was regenerated. From the physicochemical properties of S-mPEG thio PPIII that were investigated, it is concluded that interactions between the mPEG and the PPIII chains occur only to a limited extent. In addition to its usefulness for purifying thiol-containing enzymes, the mPEG modification resulting from mixed disulfide bond formation may find other practical applications.

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex.

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.

The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

Background: The Kunitz-STI family is a paradigm of protease-inhibitor interaction in particular and protein-protein recognition in general. Results: PPI is a versatile protease inhibitor that targets several subfamilies of serine proteases. Conclusion: The β-trefoil fold constitutes an evolutionary platform for protease inhibition and molecular recognition. Significance: Fold plasticity influences protein evolution toward multiple function and binding promiscuity. Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.

Evaluation of the polyethylene glycol–KF–water system in the context of purifying PEG–protein adducts

Abstract Covalent binding of PEG to proteins leads to conjugates widely investigated in several biotechnological processes. Their use as pharmaceuticals requires both careful purification and proper characterization. In this context, this paper examines the potentialities offered by hydrophobic interaction chromatography and compares aqueous potassium fluoride and ammonium sulfate as the eluents. Relative contribution of the various forces which dictate the chromatographic behaviour of PEG–protein adducts on Fractogel TSK–Butyl 650 is discussed.