Stabilization of adalimumab Fab through the introduction of disulfide bonds between the variable and constant domains.

Fab is a promising format for antibody drug. Therefore, efforts have been made to improve its thermal stability for therapeutic and commercial use. So far, we have attempted to introduce a disulfide bond into the Fab fragment to improve its thermal stability and demonstrated that it is possible to do this without sacrificing its biochemical function. In this study, to develop a novel stabilization strategy for Fab, we attempted to introduce a disulfide bond between the variable and constant domains and prepared three variants of Fab; H:G10C + H:P210C, L:P40C + L:E165C, and H:G10C + H:P210C + L:P40C + L:E165C. Differential scanning calorimetry measurements showed that each of these variants had improved thermal stability. In addition, the variants with two disulfide bonds demonstrated a 6.5 °C increase in their denaturation temperatures compared to wild-type Fab. The introduction of disulfide bonds was confirmed by X-ray crystallography, and the variants retained their antigen-binding activity. The variants were also found to be less aggregative than the wild type. Our results demonstrate that the introduction of a disulfide bond between the variable and constant domains significantly improves the thermal stability of Fab.

C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond.

Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1 : C177-CL : C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH1 and CL domains was deleted by substitution of Cys with Ala (FabΔSS). DSC measurements showed that the Tm values of FabΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of FabΔSS. The resulting Fab (mutSS FabΔSS) had the mutations H:V177C and L:Q160C in FabΔSS, confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS FabΔSS was approximately 5 °C higher than that of FabΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of FabΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.

Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development.

A role for Ras in inhibiting circular foraging behavior as revealed by a new method for time and cell-specific RNAi.

BACKGROUND: The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. In particular, RNAi is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible RNAi or cell-specific RNAi. However, conventional RNAi is insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions. RESULTS: Here, we investigated the Ras gene for its role in exploratory behavior in C. elegans. We found that, under poor environmental conditions, mutations in the Ras-MAPK signaling pathway lead to circular locomotion instead of normal exploratory foraging. Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion. We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons. To investigate the temporal- and cell-specific profiles of the functions of Ras, we developed a new RNAi method that enables simultaneous time- and cell-specific knockdown. In this method, one RNA strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded RNA in the target cell when heat shock is induced. This technique revealed that control of GLR-1 localization in RMD neurons requires Ras at the adult stage. Further, we demonstrated the application of this method to other genes. CONCLUSIONS: We have established a new RNAi method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the Ras-MAPK pathway in the control of exploratory behavior under poor environmental conditions.

Retinal hemorrhages following fingolimod treatment for multiple sclerosis; a case report.

BACKGROUND: Fingolimod is the first oral agent used for treatment of relapsing-remitting multiple sclerosis. Macular edema, but not retinal hemorrhage, is a well-known adverse effect of fingolimod treatment. To the best of our knowledge, this is the first case report of extensive retinal hemorrhages following fingolimod treatment. CASE PRESENTATION: A 31-year-old male with relapsing-remitting multiple sclerosis developed macular edema and retinal hemorrhages in his left eye, 1 month after starting fingolimod treatment; treatment was then discontinued. The hemorrhages were flame-shaped, and were extensive along retinal arteries and veins. The hemorrhages started to decrease at 4 weeks and disappeared completely at 24 weeks after cessation of fingolimod treatment. CONCLUSIONS: Occurrence of retinal hemorrhage warrants careful follow-up for multiple sclerosis patients treated with fingolimod.

Supramolecular Hybrids of Proteins from Habu Snake Venom with Discrete [Pt(CN)4]2- Complex.

The venom of the Habu snake Protobothrops flavoviridis (P. flavoviridis) is known to contain a diverse array of proteins and peptides, with a notable presence of phospholipase A2 (PLA2) enzymes. These PLA2 enzymes have been extensively studied for their function and molecular evolution. Nevertheless, several aspects, such as the physical properties and the self-assembly mechanism of hierarchical structure from the nanoscale to the microscale with different chemical compounds, remain poorly understood. This study aims to fill this knowledge gap by investigating the behavior of enzyme components purified from P. flavoviridis venom in the presence of anionic [Pt(CN)4]2- complexes, which have the potential for soft metallophilic interactions and interesting optical properties. The purified PLA2 isozymes were diluted in Dulbecco's phosphate buffered saline (D-PBS (-)) and combined with the anionic metal complex, resulting in the formation of microstructures several micrometers in size, which further grew to form fibrous structures. This novel approach of combining PLA2 enzymes with discrete functional metal complexes opens up exciting possibilities for designing flexible and functional supramolecular and biomolecular hybrid systems in aqueous environments. These findings shed light on the potential applications of snake venom enzymes in nanotechnology and bioengineering.

Structural characteristics and evolution of the Protobothrops elegans pancreatic phospholipase A2 gene in contrast with those of Protobothrops genus venom phospholipase A2 genes.

The nucleotide sequence of the gene encoding Protobothrops elegans (Crotalinae) pancreatic phospholipase A(2) (PLA(2)), abbreviated PePancPLA(2), was determined by means of inverted PCR techniques. Since its deduced amino acid sequence contains a pancreatic loop and shows high similarity to that of Laticauda semifasciata (Elapinae) group IB pancreatic PLA(2), PePancPLA(2) is classified into group IB PLA(2). The nucleotide sequences of the PePancPLA(2) gene, the L. semifasciata group IB pancreatic PLA(2) gene, and the L. semifasciata group IA venom PLA(2) gene are similar to one another but greatly dissimilar to those of Protobothrops genus (Crotalinae) group II venom PLA(2) genes, suggesting that the Elapinae group IB PLA(2) gene and the group IA PLA(2) gene appeared after Elapinae was established, and that the Crotalinae group II venom PLA(2) genes came into existence before Elapinae and Crotalinae diverged. A phylogenetic analysis of their amino acid sequences confirms this.

[Four-year changes in ganglion cell complex thickness in homonymous hemianopia].

PURPOSE: Aquired transsynaptic retrograde degeneration of the human visual system can be identified using magnetic resonance imaging (MRI) and optical coherence tomography (OCT), which can reveal optic tract atrophy, retinal nerve fiber layer loss, and ganglion cell complex (GCC) thinning. We investigated GCC changes in the first 4 years following post-geniculate lesions. METHODS: Nine patients with congruous homonymous hemianopia were scanned with OCT. A 6-mm circle centered on the macula was divided vertically into hemianopia (H) and unaffected (U) sides. GCC and retinal thicknesses were calculated using an average for both eyes and compared with the hemianopia/unaffected side (H/U) ratio. The relationship between the H/U ratio of GCC and time elapsed since occipital damage was evaluated. RESULTS: The average GCC thicknesses was 89.1 and 96.6 microm, and of the retina 295.7 microm and 292.7 microm in the H and U sides, respectively. The H/U ratio of GCC at 0.92 was significantly lower than of the retina at 0.99 (p=0.042). Regression analysis revealed a negative linear relationship (linear regression r= -0.868, p= 0.012) described by the equation: H/U ratio of GCC = 0.99 - 0.004 x elapsed time (months). CONCLUSION: For 4 years following post-geniculate damage, GCC on the hemianopia side decreased approximately 5% per year while the retinal thickness was almost unchanged.

Peritoneal dialysis-related peritonitis caused by Rhodococcus corynebacterioides.

A 57-year-old Japanese man on peritoneal dialysis developed peritoneal dialysis-associated peritonitis caused by Rhodococcus corynebacterioides. After the introduction of peritoneal dialysis, he had experienced four episodes of peritonitis, but the causative organism was not identified in any of episode. When he was hospitalized for the fifth episode of peritonitis, Rhodococcus corynebacterioides was detected in the ascitic fluid. He improved after an intraperitoneal administration of vancomycin (VCM) that was used based on the treatment of peritonitis caused by Corynebacterium spp. However, he then had repeated flare-ups and eventually required the removal of the peritoneal dialysis catheter due to recurrent peritonitis. 16S rRNA gene sequencing is generally needed to positively identify Rhodococcus corynebacterioides. In this case, we were able to rapidly identify the organism by using mass spectrometry and then apply this knowledge to the patient's treatment. To the best of our knowledge, this is the first reported case of peritoneal dialysis-associated peritonitis caused by Rhodococcus corynebacterioides.

Analysis of thermostability for seven Phe to Ala and six Pro to Gly mutants in the Fab constant region of adalimumab.

To identify amino acids that play important roles in the structural stability of Fab, seven phenylalanine residues in the Fab constant region of the therapeutic antibody adalimumab were subjected to alanine mutagenesis. Six Fab mutants, H:F130A, H:F154A, H:F174A, L:F118A, L:F139A and L:F209A, showed decreased thermostability compared with wild-type Fab. In contrast, the Tm for the L:F116A mutant was 1.7°C higher than that of wild-type Fab, indicating that the F116 residue was unfavorable for Fab thermostability. Six proline mutants, H:P131G, H:P155G, H:P175G, L:P119G, L:P120G and L:P141G, were also prepared to investigate the effect of proline residues adjacent to mutated phenylalanine residues. The thermostability of the H:P155G and L:P141G mutants in particular was significantly reduced, with decreases in Tm of 5.0 and 3.0°C, respectively, compared with wild-type Fab. The H:P155 and L:P141 residues have a cis conformation, whereas the other mutated proline residues have a trans conformation. H:P155 and L:P141 had stacking interactions with the H:F154 and L:Y140, respectively, at the interface between the variable and constant regions. It is suggested that the interactions of the aromatic ring with a cis-form proline at the interface between the variable and constant regions is important for stability of Fab.

Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine): a highly potent, selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8 g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8 g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S(2) extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8 g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8 g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Structural characteristics and evolution of a novel venom phospholipase A2 gene from Protobothrops flavoviridis.

A novel phospholipase A(2) (PLA(2)) gene, named PfPLA 6, was found in a 6,328-bp NIS-1(5')-a segment in the Protobothrops flavoviridis (Habu, Crotalinae) genome. A comparison of the aligned nucleotide sequences of Viperidae (Viperinae and Crotalinae) venom PLA(2) genes, including PfPLA 6, revealed the deletion of a 12-bp segment called S1EX 1 and a 55-bp segment called S2EX 1 in exon 1 and the interposition of a 219-bp segment called SINT 2 (SINE) in intron 2. A classification of Viperidae PLA(2) genes based on these structural modes indicated that the A-type genes (without SINE), including PfPLA 6, are evolutionarily ancestral to the B-type (Viperinae) and C-type (Crotalinae) PLA(2) genes (both with SINE). Since PfPLA 6 is a pseudogene, an active prototype of PfPLA 6 can be assumed to be the ancestral PLA(2) gene. Putative evolutionary processes from this A-type prototype PLA(2) gene to descendent PLA(2) genes are discussed.

Application of machine learning in the diagnosis of vestibular disease.

Machine learning is considered a potential aid to support human decision making in disease prediction. In this study, we determined the utility of various machine learning algorithms in classifying peripheral vestibular (PV) and non-PV diseases based on the results of equilibrium function tests. A total of 1009 patients who had undergone our standardized neuro-otological examinations were recruited. We applied five supervised machine learning algorithms (random forest, adaboost, gradient boosting, support vector machine, and logistic regression). After preprocessing the data, optimizing the hyperparameters using GridSearchCV, and performing a final evaluation on the test set using scikit-learn, we evaluated the predictive capability using various performance metrics, namely, accuracy, F1-score, area under the receiver operating characteristic curve, precision, recall, and Matthews correlation coefficient (MCC). All five machine learning algorithms yielded satisfactory results; the accuracy of the algorithms ranged from 76 to 79%, with the support vector machine classifier having the highest accuracy. In cases where the predictions of the five models were consistent, the accuracy of the PV diagnostic results was improved to 83%, whereas it increased to 85% for the non-PV diagnostic results. Future research should increase the number of patients and optimize the classification methods to obtain the highest diagnostic accuracy.

Efficiency of a novel middle ear pressure device for intractable definite Meniere's disease and delayed endolymphatic hydrops after certification by the public health insurance system in Japan.

BACKGROUND: Middle ear pressure therapy (MEPT) is effective in treating intractable vertigo in patients with definite Meniere's disease (MD) and delayed endolymphatic hydrops (DEH) refractory to conservative treatment. A novel middle ear pressure device, the EFET01®, which requires no transtympanic ventilation tubes, was developed in Japan, approved by the Japanese Ministry of Health, Labour and Welfare, and has been used under Japanese national health insurance since September 2018. OBJECTIVES: To examine short-term therapeutic effect of MEPT using the ETET01® compared with previous clinical trial results. METHODS: Patients selected according to Japan Society for Equilibrium Research (JSER) guidelines underwent MEPT using the EFET01 from September 2018 to July 2021, and 44 patients were enrolled in this retrospective study. Clinical data analysed at 4 months after the start of MEPT were compared with those of the previous clinical trial for the EFET01. RESULTS: MEPT using the EFET01 showed the same therapeutic efficacy as that of the previous clinical trial, i.e. improvement in the intensity and frequency of vertigo with no effect on hearing, even under JSER guidelines for proper use of MEPT. CONCLUSION: MEPT using the EFET01 provided an effective treatment option for intractable vertigo in patients with definite MD and DEH.

12-month effect of middle ear pressure therapy with the EFET01 device for intractable definite Meniere's disease and delayed endolymphatic hydrops after certification by the public health insurance system in Japan.

BACKGROUND: Middle ear pressure therapy (MEPT) is effective for intractable vertigo in patients with definite Meniere's disease (MD) and treatment-refractory delayed endolymphatic hydrops (DEH). Four-month MEPT with the EFET01®, an MEPT device developed in Japan and covered by national health insurance since September 2018, has shown efficacy. However, efficacy and safety after 12 months of treatment, which is appropriate for determining the therapeutic effect of MEPT devices, is unclear. OBJECTIVES: Examine the therapeutic effect of 12-month MEPT using the ETET01®. MATERIAL AND METHODS: Patients underwent MEPT using the EFET01® from September 2018 to July 2021. Thirty-three patients followed for >12 months were enrolled in this retrospective study. Clinical data were evaluated in the first and second 6-month treatment periods. Data from the second 6-month period were compared with data from an MEPT study using a different device. RESULTS: MEPT with the EFET01® significantly improved vertigo in the first period, with further improvement in the second period. The efficacy and safety were comparable to MEPT with other devices. CONCLUSIONS: MEPT with the EFET01® is effective for intractable vertigo in patients with definite MD and DEH, and 12-month follow-up is recommended. SIGNIFICANCE: The efficacy of 12-month MEPT with the EFET01® was demonstrated.

Effect of an intermolecular disulfide bond introduced into the first loop of CH1 domain of Adalimumab Fab on thermal stability and antigen-binding activity.

The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135-141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the KD value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.

Zinc suppresses Th17 development via inhibition of STAT3 activation.

Zinc (Zn) is an essential trace metal required by many enzymes and transcription factors for their activity or the maintenance of their structure. Zn has a variety of effects in the immune responses and inflammation, although it has not been well known how Zn affects these reactions on the molecular basis. We here showed that Zn suppresses T(h)17-mediated autoimmune diseases at lest in part by inhibiting the development of T(h)17 cells via attenuating STAT3 activation. In mice injected with type II collagen to induce arthritis, Zn treatment inhibited T(h)17 cell development. IL-6-mediated activation of STAT3 and in vitro T(h)17 cell development were all suppressed by Zn. Importantly, Zn binding changed the alpha-helical secondary structure of STAT3, disrupting the association of STAT3 with JAK2 kinase and with a phospho-peptide that included a STAT3-binding motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn suppresses STAT3 activation, which is a critical step for T(h)17 development.

A [Lys49]phospholipase A2 from Protobothrops flavoviridis venom induces caspase-independent apoptotic cell death accompanied by rapid plasma-membrane rupture in human leukemia cells.

Protobothrops flavoviridis venom contains plural phospholipase A(2) (PLA(2)) isozymes. A [Lys(49)]PLA(2) called BPII induced cell death in human leukemia cells. PLA2, an [Asp(49)]PLA(2) that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA(2) lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.

Identification and evolution of venom phospholipase A2 inhibitors from Protobothrops elegans serum.

The cDNAs encoding venom phospholipase A(2) (PLA(2)) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A(a) and PeαPLI-A(b), were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA(2)s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA(2) isozymes.