Lysyl Endopeptidase Research Articles

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

Extracellular Proteins Isolated from L. acidophilus as an Osteomicrobiological Therapeutic Agent to Reduce Pathogenic Biofilm Formation, Regulate Chronic Inflammation, and Augment Bone Formation In Vitro.

Periprosthetic joint infection (PJI) is a challenging complication that can occur following joint replacement surgery. Efficacious strategies to prevent and treat PJI and its recurrence remain elusive. Commensal bacteria within the gut convey beneficial effects through a defense strategy named "colonization resistance" thereby preventing pathogenic infection along the intestinal surface. This blueprint may be applicable to PJI. The aim is to investigate Lactobacillus acidophilus spp. and their isolated extracellular-derived proteins (LaEPs) on PJI-relevant Staphylococcus aureus, methicillin-resistant S. aureus, and Escherichia coli planktonic growth and biofilm formation in vitro. The effect of LaEPs on cultured macrophages and osteogenic, and adipogenic human bone marrow-derived mesenchymal stem cell differentiation is analyzed. Data show electrostatically-induced probiotic-pathogen species co-aggregation and pathogenic growth inhibition together with LaEP-induced biofilm prevention. LaEPs primemacrophages for enhanced microbial phagocytosis via cathepsin K, reduce lipopolysaccharide-induced DNA damage and receptor activator nuclear factor-kappa B ligand expression, and promote a reparative M2 macrophage morphology under chronic inflammatory conditions. LaEPs also significantly augment bone deposition while abating adipogenesis thus holding promise as a potential multimodal therapeutic strategy. Proteomic analyses highlight high abundance of lysyl endopeptidase, and urocanate reductase. Further, in vivo analyses are warranted to elucidate their role in the prevention and treatment of PJIs.

Development of an LC-MS/MS method for absolute quantification of IgG4 by evaluating dependence on the digestion efficiency using a non-cleavable/dually-cleavable internal calibrator set

The measurement of serum IgG4 levels is mandatory for the diagnosis of IgG4-related disease, but no widely accepted reference material exists due to a lack of consensus on the standard assay. Therefore, we developed here an LC-MS/MS method for absolute quantification of IgG4 in a purified IgG sample, addressing a concern over the reliability depending on the proteolytic digestion efficiency. Our method uses internal calibrator sets containing unique amino acid sequences within IgG4, each of which comprises non-cleavable and dually-cleavable peptides labeled with different numbers of isotopes for mass separation, to determine digestion efficiency. Surrogate peptides generated by trypsin or lysyl endopeptidase digestion were selected based on selectivity, stability, and identifiability. IgG4 quantification using synthetic calibrator peptides showed high precision across the two conditions with different peptidases (relative differences ≤6.1%), even with low digestion efficiencies (<20%), which was within the interday precision under an established condition (% coefficient of variation ≤6.9%, digestion efficiencies >90%, n = 5). These results indicate that the LC-MS/MS method for quantifying IgG4 is robust against digestion efficiency variations and is applicable to validating an IgG4 reference material.

Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies.

Peptide mapping is an important tool used to confirm that the correct sequence has been expressed for a protein and to evaluate protein post-translational modifications (PTMs) that may arise during the production, processing, or storage of protein drugs. Our new orally administered drug (Ab-1), a single-domain antibody, is highly stable and resistant to proteolysis. Analysis via the commonly used tryptic mapping method did not generate sufficient sequence coverage. Alternative methods were needed to study the Ab-1 drug substance (75 mg/mL) and drug product (3 mg/mL). To meet these analytical needs, we developed two new peptide mapping methods using lysyl endopeptidase (Lys-C) digestion. These newly developed protein digestion protocols do not require desalting/buffer-exchange steps, thereby reducing sample preparation time and improving method robustness. Additionally, the protein digestion is performed under neutral pH with methionine acting as a scavenger to minimize artifacts, such as deamidation and oxidation, which are induced during sample preparation. Further, the method for low-concentration samples performs comparably to the method for high-concentration samples. Both methods provide 100% sequence coverage for Ab-1, and, therefore, enable comprehensive characterization for its product quality attribute (PQA) assessment. Both methods can be used to study other antibody formats.

A Mass Spectrometry-Based Strategy for Mapping Modification Sites for the Ubiquitin-Like Modifier NEDD8.

The identification of modification sites for ubiquitin and ubiquitin-like modifiers is an essential step in the elucidation of controlled processes. The ubiquitin-like modifier NEDD8 is an important regulator of plethora of biological processes both under homeostatic and proteotoxic stress conditions. Here, we describe a detailed protocol for proteome-wide identification of NEDDylation sites. The approach is based on the use of cell lines stably expressing the NEDD8R74K mutant. Digestion of samples with Lysyl endopeptidase generates peptides with a di-glycine remnant only from proteins modified with NEDD8R74K but not with ubiquitin or ISG15. The isolation of these peptides with anti-di-glycine antibodies (K-ε-GG) allows the identification of NEDDylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Paper‐Based Bacterial Lysis Enables Sample‐to‐Answer Home‐based DNA Testing

AbstractNucleic acid amplification testing (NAAT) is the gold standard for infectious disease diagnostics. Currently NAATs are mainly limited to centralized laboratories, while paper‐based antigen tests are used for rapid home‐based diagnostics. DNA extraction, the initial sample preparation step in NAATs, remains a bottleneck that hinders its development toward home‐based kits. This step requires the use of compounds detrimental to the enzymes in downstream DNA amplification. Here, this work overcomes this bottleneck by immobilizing the enzyme achromopeptidase (ACP) on nitrocellulose, to both store and enable the separation of the enzymes from the other steps. This work provides proof‐of‐concept that immobilized ACP is effective at lysis and release of amplifiable DNA from gram‐positive Staphylococcus epidermidis and enables the use of the lysate directly for DNA amplification, without the need for heat deactivation of the enzyme. This sample preparation method requires only incubation at 37 °C and mild agitation, which allows to implement it with fully disposable and affordable equipment. Consequently, this work enables to combine the paper‐based DNA extraction method with the isothermal recombinase polymerase amplification (RPA) followed by lateral flow detection to demonstrate a sample‐to‐answer NAAT packaged as an instrument free self‐test kit expanding the capabilities of home‐testing beyond antigen tests.

Analytical Performance Evaluation of Identity, Quality-Attribute Monitoring and new Peak Detection in a Platform Multi-Attribute Method Using Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

The use of multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Phase-appropriate method validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed a MAM workflow for therapeutic monoclonal antibodies (mAbs) with optimized sample preparation using lysyl endopeptidase (Lys-C) digestion. In this study, we evaluated the assay performances of this platform MAM workflow for identity, product quality attributes (PQAs) monitoring and new peak detection (NPD) for single and coformulated mAbs. An IgG4 mAb-1 and its coformulations were used as model molecules in this study. The assay performance evaluation demonstrated the full potential of the platform MAM approach for its intended use for characterization and quality control of single mAb-1 and mAb-1 in its coformulations. To the best of our knowledge, this is the first performance evaluation of MAM for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first performance evaluation of MAM for PQA monitoring using Lys-C digestion with a high-resolution MS, 2) a new approach for mAb identity testing capable of distinguishing single mAb from coformulations using MAM, and 3) the performance evaluation of NPD for MAM with Lys-C digestion. The developed platform MAM workflow and the MAM performance evaluation paved the way for its GMP qualification and enabled clinical release of mAb-1 in GMP environment with MAM.

Characterization of Leishmania (L.) amazonensis oligopeptidase B and its role in macrophage infection.

Leishmania spp. are parasitic protozoa that cause leishmaniasis, a disease endemic in 98 countries. Leishmania promastigotes are transmitted by the vector and differentiate into amastigotes within phagocytic cells of the vertebrate host. To survive in multiple and hostile environments, the parasite has several virulence factors. Oligopeptidase B (OPB) is a serine peptidase present in prokaryotes, some eukaryotes and some higher plants. It has been considered a virulence factor in trypanosomatids, but only a few studies, performed with Old World species, analysed its role in Leishmania virulence or infectivity.L. (L.) amazonensis is an important agent of cutaneous leishmaniasis in Brazil. The L. (L.) amazonensis OPB encoding gene has been sequenced and analysed in silico but has never been expressed. In this work, we produced recombinant L. (L.) amazonensis OPB and showed that its pH preferences, Km and inhibition patterns are similar to those reported for L. (L.) major and L. (L.) donovani OPBs. Since Leishmania is known to secrete OPB, we performed in vitro infection assays using the recombinant enzyme. Our results showed that active OPB increased in vitro infection by L. (L.) amazonensis when present before and throughout infection. Our findings suggest that OPB is relevant to L. (L.) amazonensis infection, and that potential drugs acting through OPB will probably be effective for Old and New World Leishmania species. OPB inhibitors may eventually be explored for leishmaniasis chemotherapy.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30s.The Lys C gene from M14-150s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

Improvements on sample preparation and peptide separation for reduced peptide mapping based multi-attribute method analysis of therapeutic monoclonal antibodies using lysyl endopeptidase digestion

The mass spectrometry based multi-attribute method (MAM) has gained popularity in the field of biopharmaceutical analysis as it promises a single method for comprehensive monitoring of multiple product quality attributes (PQAs) and product purity. Sample preparation for protein digestion and peptide separation are critical considerations for a reduced peptide mapping-based MAM. To avoid desalting steps required in most tryptic protein digestion and in order to improve peptide separation for hydrophilic peptides, we developed an improved robust sample preparation using lysyl endopeptidase (Lys-C) for high-throughput MAM testing. Additionally, this method optimizes the peptide retention and separation of a stability-indicating VSNK peptide using a HSS T3 column for comprehensive PQA monitoring. A fully automated sample preparation had similar assay variations for PQAs monitoring compared to manual sample preparation. To the best of our knowledge, this is the first report of a high-resolution MS-based MAM using a streamlined Lys-C digestion without desalting with enhanced PQA monitoring for hydrophilic peptides. The improved, robust MAM workflow for protein digestion and peptide separation will pave the way for broader MAM qualification and its applications for the characterization and quality control of therapeutic monoclonal antibodies.

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data.

Improved stability and controlled release of lycopene via self-assembled nanomicelles encapsulation

Lycopene with many potential health-promoting attributes is chemically unstable and prone to degradation. This work aimed to develop nanomicelles based on amphiphilic peptides self-assembled for improving the water solubility and physicochemical stability of lycpoene. The amphiphilic peptides were obtained by enzymatic hydrolysis of β-lactoglobulin using recombinant lysyl endopeptidase. Subsequently, the lycopene was docked with amphiphilic peptides to study their binding potential. Furthermore, lycopene loaded nanomicelles were prepared and characterized, and their stability and in vitro simulated release were studied. As a result, lycopene could be embedded in nanomicelles via hydrophobic interaction and lycopene loaded nanomicelles showed spherical with a size approximately 20.00 nm. Circular dichroism results suggested that the secondary structure was changed in nanomicelles after its combination with lycopene. The stability of the encapsulated lycopene was significantly (P < 0.05) higher than that of the free lycopene under UV irradiation and 60 °C heating. The nanomicelles were designed as a new delivery vehicle for lycopene with embedded amount (354.00 mg/g). And lycopene exhibited controlled release behavior from the nanomicelles and its cumulative release amount was 30.29% after 24 h incubated in pH 7.00. The study demonstrated the β-lactoglobulin nanomicelles could be a potential strategy for the delivery of hydrophobic bioactive compounds.

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine.

Simple SummaryOligopeptidase B is a two-domain, trypsin-like peptidase from parasitic protozoa and bacteria which belongs to the least studied group of prolyloligopeptidases. In this study, we describe for the first time a crystal structure of bacterial oligopeptidase B and compare it with those of protozoan oligopeptidases B and related prolyloligopeptidases. The enzyme was crystallized in the presence of spermine and contained a modified sequence of the interdomain linker. Both factors were key for crystallization. The structure showed an uncommon intermediate conformation with a domain arrangement intermediate between open and closed conformations found in the crystals of ligand-free and inhibitor-bound prolyloligopeptidases, respectively. To evaluate the impact of the modification and spermine in the obtained conformation, small-angle X-ray scattering was applied, which showed that in solution wild-type enzymes adopt the open conformation and spermine causes a transition to the intermediate state, while the modification is associated with a partial transition. We suggest that spermine-dependent conformational transition replicates the behavior of the enzyme in bacterial cells and the intermediate state, which is rarely detected in vitro, and might be widely distributed in vivo, and so should be considered during computational studies, including those aimed wanting to develop the small molecule inhibitors targeting prolyloligopeptidases.Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests

Enzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

Crystallographic Study of Mutants and Complexes of Oligopeptidase B from Serratia proteamaculans

The results of the structural studies of bacterial oligopeptidase B (OpB) belonging to the least well-studied prolyl oligopeptidase family are described. The screening of crystallization conditions for mutants of the enzyme, its complexes with peptides, which mimic substrates and catalytic reaction products, and a complex with a transition state analog as an inhibitor was performed in order to determine the three-dimensional structures of OpB from Serratia proteamaculans (PSP) acting at different steps of the catalytic cycle. Crystals suitable for X-ray diffraction were grown. The X-ray diffraction data sets were collected, processed, and subjected to preliminary analysis. These X-ray diffraction data sets are suitable for obtaining the structural data necessary for the description of the catalytic cycle of bacterial ОpBs.

Analysis and characterization of a promising therapeutic target identified in Leishmania spp

Leishmaniasis is a neglected disease caused by protozoa of the genus Leishmania spp., which affects about 1.6 million individuals each year and 500,000 present themselves in the visceral form. In Brazil there are about 30,000 new cases each year. In addition, the country is responsible for 90% of reported cases of Visceral Leishmaniasis, and this is more severe form of the disease. Allied to these facts, the current treatment is ineffective, contributing to the establishment of resistant strains. Currently, the treatment has several side effects and permanent damage to the health of patients, this fact has contributed to the search for new drugs against leishmaniasis. The enzyme oligopeptidase B (OPB) has been studied as a possible therapeutic target in the development of antiparasitic agents. Thus, the objective of this work is to construct the three-dimensional model of the enzyme Oligopeptidase B of different species of Leishmania spp. and compare them to each other. For this purpose, the comparative modeling method was used. In this method, the models of the species L. brasiliensis, L. donovani, L. infantum, L. mexicana and L. panamensis were constructed using the MODELLER program. Once the models were ready, the validation process was carried out and subsequently characterized, which was possible to verify a promising degree of similarity between the models. Finally, these models were submitted to the method of analysis by normal modes, which obtained a similar movement pattern, thus it was possible to verify a movement in a specific region of an alpha-helix, consequently leading to the triad of the enzyme being exposed, which may be indicative of a mechanism of action. Finally, it is expected to use the models built to assist in the development of a promising new therapy for the treatment of leishmaniasis.

Real-Time Measurement of Phosphatase Activity During Proteolytic Digestion by LYSYL Endopeptidase using High Pressure Optical Spectroscopy

Real-Time Measurement of Phosphatase Activity During Proteolytic Digestion by LYSYL Endopeptidase using High Pressure Optical Spectroscopy

Peptidyl-Lys metalloendopeptidase (Lys-N) purified from dry fruit of Grifola frondosa demonstrates "mirror"digestion property with lysyl endopeptidase (Lys-C).

Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)-based de novo sequencing of proteins. Here we present a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification. The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity. The novel production process provides for the first time the extraction of Lys-N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS-based proteomics and other biological studies.

Amyloid Formation under Complicated Conditions in Which β2-Microglobulin Coexists with Its Proteolytic Fragments.

Amyloid formation in vivo occurs under complicated conditions in which various amyloidogenic and non-amyloidogenic components coexist, often under crowding. Controversy surrounds the role of additional components under complicated conditions. They have been suggested to accelerate amyloid formation because molecular crowding or interactions with additives increase effective concentrations and, thus, break the supersaturation of amyloidogenic proteins. On the other hand, cellular crowding conditions with various heterogeneous components may retard or prevent amyloid formation because they impede homologous amyloidogenic associations. To elucidate the roles of these additional components, we examined the amyloid formation of β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, with a simplified model system in which intact β2m and its proteolytic peptides coexist. Among the nine proteolytic peptides of β2m produced in vitro with lysyl endopeptidase, the 22-residue K3 peptide is highly amyloidogenic. The amyloid formation of the K3 peptide, which occurred with a lag time of 1 h at pH 2 and 37 °C, was significantly retarded by the coexistence of β2m or a mixture of the proteolytic digests. To identify the sites of inhibitory interactions, we performed paramagnetic relaxation enhancement measurements using spin-labeled K3 and uniformly 15N-labeled β2m with nuclear magnetic resonance detection. The results revealed that K3 interacted weakly with a broad cluster of the hydrophobic residues of β2m, which accommodated the residues located in some distant sequence, leading to competitive inhibition. The results showed that relatively weak and broad interactions formed a nonproductive complex, implying a role for heterogeneous interactions under complicated conditions.