María A. Chávez

Purification and characterization of two hemolysins from Stichodactyla helianthus

Two hemolysins, Sticholysin I (St I) and Sticholysin II (St II) were purified from the sea anemone Stichodactyla helianthus combining gel filtration and ion exchange chromatography. The amino acid composition of both cytolysins was determined revealing a high proportion of glycine, lysine, tyrosine and non-polar amino acids (alanine, leucine and valine). Cysteine was not found in either polypeptide. Molecular masses of St I and St II were 19401 and 19290 Da, respectively. N-terminal sequence analysis of St I and St II showed a high homology between them suggesting they are isoforms of the same cytolysin. Compared with other sea anemone cytolysins, St I and St II contain a 22 amino acid insertion fragment also present in Eq T II/Tn C and probably in CaT I and Hm T and absent in C III, the major hemolysin previously reported in this anemone.

Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla helianthus

Isolation of proteinase inhibitors from the sea anemone Stichodactyla helianthus was achieved by trichloroacetic acid treatment of the aqueous extract followed by affinity chromatography on trypsin-Sepharose and ion-exchange chromatography on CM-cellulose. The average molecular mass of the major inhibitor (ShPI-I) obtained by fast atom bombardment mass spectrometry (FAB-MS) was 6110.6 Da. The amino acid sequence was determined by FAB-MS combined with manual Edman degradation, digestions with endopeptidases and exopeptidases and automatic sequencing. The sequence of ShPI-I (55 amino acids) was compared with those reported in the SwissProt database for several proteinase inhibitors and significant similarity to inhibitors belonging to the Kunitz family was observed. ShPI-I exhibits a broad specificity for serine, cysteine and aspartic proteinases. The dissociation constants of the complexes formed with different enzymes were determined. The affinity-purified fraction (PI) was immobilized on Sepharose and used in the purification of different classes of proteinases.

Generation of an affinity matrix useful in the purification of natural inhibitors of plasmepsin II, an antimalarial-drug target

An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) aspartic proteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N‐hydroxysuccinimide)‐activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 °C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic‐proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea‐fan coral) Plexaura homomalla (black sea rod), with increases of 10.2‐ and 5.9‐fold in the specific inhibitory activity respectively. The preliminary Ki values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight‐binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates.Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSF proteins were chosen for the design of a CSF-specific depletion setup. Affibody molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower-abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.

Characterization and comparative 3D modeling of CmPI-II, a novel ‘non-classical’ Kazal-type inhibitor from the marine snail Cenchritis muricatus (Mollusca)

Abstract The complete amino acid sequence obtained by electrospray ionization tandem mass spectrometry of the proteinase inhibitor CmPI-II isolated from Cenchritis muricatus is described. CmPI-II is a 5480-Da protein with three disulfide bridges that inhibits human neutrophil elastase (HNE) (K i 2.6±0.2 nM), trypsin (K i 1.1±0.9 nM), and other serine proteinases such as subtilisin A (K i 30.8±1.2 nM) and pancreatic elastase (K i 145.0±4.4 nM); chymotrypsin, pancreatic and plasma kallikreins, thrombin and papain are not inhibited. CmPI-II shares homology with the Kazal-type domain and may define a new group of ‘non-classical’ Kazal inhibitors according to its CysI-CysV disulfide bridge position. The 3D model of CmPI-II exhibits similar secondary structure characteristics to Kazal-type inhibitors and concurs with circular dichroism experiments. A 3D model of the CmPI-II/HNE complex provides a structural framework for the interpretation of its experimentally determined K i value. The model shows both similar and different contacts at the primary binding sites in comparison with the structure of turkey ovomucoid third domain (OMTKY3)/HNE used as template. Additional contacts calculated at the protease-inhibitor interface could also contribute to the association energy of the complex. This inhibitor represents an exception in terms of specificity owing to its ability to strongly inhibit elastases and trypsin.

Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata In vivo effects in rodent brain

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica

Background: Several protein inhibitors of metallocarboxypeptidases have already been described. Results: We have characterized of a tri-domain inhibitor from Sabellastarte magnifica, recombinant forms and truncated variants. Conclusion: The whole tri-domain is required for full inhibition of metallocarboxypeptidases A. Monodomains are designed to inhibit serine proteases. Significance: The first reported multidomain protein inhibitor of metallocarboxypeptidases is also able to act on another mechanistic class of proteases (serine-type). This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature.

Crystal Structure of Novel Metallocarboxypeptidase Inhibitor from Marine Mollusk Nerita versicolor in Complex with Human Carboxypeptidase A4

Background: Only a few proteinaceous inhibitors of metallocarboxypeptidases have been characterized structurally in depth. Results: The structure of human carboxypeptidase A4 in complex with a Nerita versicolor inhibitor (NvCI) was derived at 1.7 Å. Conclusion: NvCI displays a different protein fold that inhibits carboxypeptidases in a substrate-like manner. Significance: We deciphered the structural determinants for picomolar inhibition constants for A-type carboxypeptidases, the most potent by now. NvCI is a novel exogenous proteinaceous inhibitor of metallocarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 Å resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris

Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.

The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels.

The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the N- and C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the N- and C-terminal ends.