RESUMEN
Bifunctional antibody (BfAb) therapeutics offer the potential for novel functionalities beyond those of the individual monospecific entities. However, combining these entities into a single molecule can have unpredictable effects, including changes in pharmacokinetics that limit the compound's therapeutic profile. A better understanding of how molecular modifications affect in vivo tissue interactions could help inform BfAb design. The present studies were predicated on the observation that a BfAb designed to have minimal off-target interactions cleared from the circulation twice as fast as the monoclonal antibody (mAb) from which it was derived. The present study leverages the spatial and temporal resolution of intravital microscopy (IVM) to identify cellular interactions that may explain the different pharmacokinetics of the two compounds. Disposition studies of mice demonstrated that radiolabeled compounds distributed similarly over the first 24 hours, except that BfAb accumulated approximately two- to -three times more than mAb in the liver. IVM studies of mice demonstrated that both distributed to endosomes of liver endothelia but with different kinetics. Whereas mAb accumulated rapidly within the first hour of administration, BfAb accumulated only modestly during the first hour but continued to accumulate over 24 hours, ultimately reaching levels similar to those of the mAb. Although neither compound was freely filtered by the mouse or rat kidney, BfAb, but not mAb, was found to accumulate over 24 hours in endosomes of proximal tubule cells. These studies demonstrate how IVM can be used as a tool in drug design, revealing unpredicted cellular interactions that are undetectable by conventional analyses. SIGNIFICANCE STATEMENT: Bifunctional antibodies offer novel therapeutic functionalities beyond those of the individual monospecific entities. However, combining these entities into a single molecule can have unpredictable effects, including undesirable changes in pharmacokinetics. Studies of the dynamic distribution of a bifunctional antibody and its parent monoclonal antibody presented here demonstrate how intravital microscopy can expand our understanding of the in vivo disposition of therapeutics, detecting off-target interactions that could not be detected by conventional pharmacokinetics approaches or predicted by conventional physicochemical analyses.
Asunto(s)
Anticuerpos Monoclonales , Hígado , Ratas , Ratones , Animales , Distribución Tisular , Anticuerpos Monoclonales/farmacocinética , Hígado/metabolismo , RiñónRESUMEN
Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.
Asunto(s)
Anticuerpos Biespecíficos/fisiología , Anticuerpos Biespecíficos/farmacocinética , Fenómenos Biofísicos/fisiología , Animales , Anticuerpos Biespecíficos/química , Fenómenos Biofísicos/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/fisiología , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.
Asunto(s)
Anticuerpos Biespecíficos/farmacocinética , Capilares/metabolismo , Hígado/metabolismo , Animales , Semivida , Antígenos de Histocompatibilidad Clase I , Humanos , Macaca fascicularis , Tasa de Depuración Metabólica , Receptores FcRESUMEN
Follistatin 315 heparan sulfate-binding deficient mutant human IgG4 Fc fusion (FST-ΔHBS-Fc) is a follistatin (FST) based Fc fusion protein currently being developed as a novel therapy for several potential indications, including muscle wasting. Previous assessments of the pharmacokinetics and therapeutic activity of FST-ΔHBS-Fc have shown a close association of the exposure-response relationship. The current work builds upon these initial studies by investigating the glycosylation characteristics of FST-ΔHBS-Fc after recombinant expression and its impact on the pharmacokinetics in mice and Cynomolgus monkeys. The data presented indicate that FST-ΔHBS-Fc is heterogeneously glycosylated at the three putative sites in FST when recombinantly expressed in stably transfected Chinese hamster ovary cells. Such carbohydrate heterogeneity, especially with regards to sialic acid incorporation, directly results in sugar-dependent clearance in both mice and Cynomolgus monkeys. Examination of the pharmacokinetics of FST-ΔHBS-Fc molecules containing variable sialic acid content in asialoglycoprotein receptor 1 (ASPGR-1) knockout mice supports the receptor's role as part of the clearance mechanism of the molecules. Based on the evaluation of several variably sialylated lots of material in pharmacokinetic assessments, we define specifications for average sialic acid incorporation into FST-ΔHBS-Fc that result in limited sugar-mediated clearance. Taken together, these studies highlight the importance of establishing an early understanding of the glycosylation/pharmacokinetic relationships of FST-ΔHBS-Fc, which will provide a basis for future application toward optimal systemic drug delivery and dosing strategies.
Asunto(s)
Terapia Biológica/tendencias , Folistatina/química , Folistatina/farmacocinética , Animales , Células CHO , Cricetinae , Cricetulus , Glicosilación , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Ratones SCIDRESUMEN
Activin A, a member of the transforming growth factor-ß superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.
Asunto(s)
Activinas/antagonistas & inhibidores , Contracción Muscular/fisiología , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Animales , Electroporación , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunohistoquímica , Ratones Endogámicos C57BL , Regeneración/fisiología , TranscriptomaRESUMEN
Follistatin (FST) is a member of the tissue growth factor ß family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.
Asunto(s)
Folistatina/farmacología , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Músculo Esquelético/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Regeneración , Animales , Folistatina/genética , Ratones , Músculo Esquelético/fisiología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Human follistatin is a regulatory glycoprotein with widespread biologic functions, including antiinflammatory activities, wound-healing properties, and muscle-stimulating effects. The role of follistatin in a wide range of biologic activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential disease-modifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic/pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315 (FST315). Our data indicate that the intrinsic PK/PD properties of native FST315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Here, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing FST315 to a murine IgG(1) Fc and removing the intrinsic heparan sulfate-binding activity of follistatin. The engineered variant molecule had ~100- and ~1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native FST315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy toward the development of a follistatin therapeutic with potential use in patients affected with skeletal muscle diseases.
Asunto(s)
Folistatina/farmacología , Folistatina/farmacocinética , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/farmacocinética , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Folistatina/genética , Células HEK293 , Semivida , Heparina/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Unión Proteica , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Sefarosa/metabolismoRESUMEN
Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of the regulation of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres 1), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of Schizosaccharomyces pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of â¼20 000 compounds yielded a single inhibitor, which we found interacted tightly with sub-micromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red (CR), was able to competitively inhibit hPOT1 binding to telomeric DNA. Isothermal titration calorimetry and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption.
Asunto(s)
Rojo Congo/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Proteínas de Unión a Telómeros/antagonistas & inhibidores , Telómero/metabolismo , Secuencias de Aminoácidos , Dominio Catalítico , Técnicas Químicas Combinatorias , Rojo Congo/metabolismo , Rojo Congo/farmacología , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Fragmentos de Péptidos , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Complejo Shelterina , Bibliotecas de Moléculas PequeñasRESUMEN
Clusters of complement-type ligand binding repeats in the LDL receptor family are thought to mediate the interactions between these receptors and their various ligands. Apolipoprotein E, a key ligand for cholesterol homeostasis, has been shown to interact with LDLR, LRP, and VLDLR, through these clusters. LDLR and VLDLR each contain a single ligand binding repeat cluster, whereas LRP contains three large clusters of ligand binding repeats, each with ligand binding functions. We show that within sLRP3 the three-repeat subcluster CR16-18 recapitulated ligand binding to the isolated receptor binding portion of ApoE (residues 130-149). Binding experiments with LA3-5 of LDLR and CR16-18 showed that a conserved W25/D30 pair appears to be critical for high-affinity binding to ApoE(130-149). The triple repeat LA3-5 showed the expected interaction with ApoE(1-191).DMPC, but surprisingly CR16-18 did not interact with this form of ApoE. To understand these differences in ApoE binding affinity, we introduced mutations of conserved residues from LA5 into CR18 and produced a CR16-18 variant capable of binding ApoE(1-191).DMPC. This change cannot fully be accounted for by the interaction with the proposed ApoE receptor binding region; therefore, we speculate that LA5 is recognizing a distinct epitope on ApoE that may only exist in the lipid-bound form. The combination of avidity effects with this distinct recognition process likely governs the ApoE-LDL receptor interaction.
Asunto(s)
Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores de LDL/metabolismo , Secuencia de Aminoácidos , Apolipoproteínas E/metabolismo , Humanos , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores de LDL/química , Alineación de SecuenciaRESUMEN
The Schizosaccharomyces pombe protection of telomeres 1 (SpPot1) protein recognizes the 3' single-stranded ends of telomeres and provides essential protective and regulatory functions. The ssDNA-binding activity of SpPot1 is conferred by its ssDNA-binding domain, Pot1-DBD (residues 1-389), which can be further separated into two distinct domains, Pot1pN (residues 1-187) and Pot1pC (residues 188-389). Here we show that Pot1pC, like Pot1pN, can function independently of Pot1-DBD and binds specifically to a minimal nonameric oligonucleotide, d(GGTTACGGT), with a K(D) of 400 +/- 70 nM (specifically recognized nucleotides in bold). NMR chemical shift perturbation analysis indicates that the overall structures of the isolated Pot1pN and Pot1pC domains remain intact in Pot1-DBD. Furthermore, alanine scanning reveals modest differences in the ssDNA-binding contacts provided by isolated Pot1pN and within Pot1-DBD. Although the global character of both Pot1pN and Pot1pC is maintained in Pot1-DBD, chemical shift perturbation analysis highlights localized structural differences within the G1/G2 and T3/T4 binding pockets of Pot1pN in Pot1-DBD, which correlate with its distinct ssDNA-binding activity. Furthermore, we find evidence for a putative interdomain interface on Pot1pN that mediates interactions with Pot1pC that ultimately result in the altered ssDNA-binding activity of Pot1-DBD. Together, these data provide insight into the mechanisms underlying the activity and regulation of SpPot1 at the telomere.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Telómero , Ensayo de Cambio de Movilidad Electroforética , Modelos Moleculares , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificaciónRESUMEN
The N-terminal oligonucleotide/oligosaccharide-binding fold domain of the Schizosaccharomyces pombe protection of telomeres 1 (Pot1) protein, Pot1pN (residues 1-187 of full-length Pot1), specifically recognizes telomeric single-stranded DNA (ssDNA) via a complex series of molecular interactions that are punctuated by unusual internucleotide hydrogen bonds. While the structure of ssDNA-bound Pot1pN provides an initial model for understanding how the Pot1pN-ssDNA complex is assembled and how specific nucleotide recognition occurs, further refinement requires knowledge of the ssDNA-free state of Pot1pN and the dynamic changes that accompany the binding of ssDNA. Using NMR strategies, we found that ssDNA-free Pot1pN adopts a similar overall protein backbone topology as ssDNA-bound Pot1pN does. Although the backbone structure remained relatively unchanged, we observed unexpected differential dynamic changes within the ssDNA-binding pockets of Pot1pN upon binding of cognate ssDNA. These studies support a model in which conformational selection and induced fit play important roles in the recognition of ssDNA by Pot1pN. Furthermore, the studies presented here provide a more comprehensive understanding of how specific nucleotide recognition is achieved by the telomere-end protection family of essential proteins.
Asunto(s)
ADN de Hongos/genética , ADN de Cadena Simple/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Oligodesoxirribonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Espectrofotometría , Proteínas de Unión a Telómeros/genética , TermodinámicaRESUMEN
Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3' single-strand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. In Schizosaccharomyces pombe, this protective function is mediated by the Pot1 protein, which binds specifically and with high affinity to telomeric ssDNA. We have characterized the thermodynamics and accommodation of both cognate and noncognate telomeric single-stranded DNA (ssDNA) sequences by Pot1pN, an autonomous ssDNA-binding domain (residues 1-187) found in full-length S. pombe Pot1. Direct calorimetric measurements of cognate telomeric ssDNA binding to Pot1pN show favorable enthalpy, unfavorable entropy, and a negative heat-capacity change. Thermodynamic analysis of the binding of noncognate telomeric ssDNA to Pot1pN resulted in unexpected changes in free energy, enthalpy, and entropy. Chemical-shift perturbation and structural analysis of these bound noncognate sequences show that these thermodynamic changes result from the structural rearrangement of both Pot1pN and the bound oligonucleotide. These data suggest that the ssDNA-binding interface is highly dynamic and, in addition to the conformation observed in the crystal structure of the Pot1pN/d(GGTTAC) complex, capable of adopting alternative thermodynamically equivalent conformations.
Asunto(s)
ADN de Cadena Simple/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Unión a Telómeros/química , Telómero/química , Termodinámica , Secuencia de Bases , Carbohidratos/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , Unión Proteica , Estructura Terciaria de Proteína , Protones , Complejo ShelterinaRESUMEN
The ends of eukaryotic linear chromosomes are unique structures that require special management by the cell. If left unattended, the ends are inappropriately processed, leading to genomic instability and problems with proliferation. Telomeres are specialized nucleoprotein structures that restore chromosome stability by protecting and maintaining chromosome ends. Proper telomere function is facilitated, in part, by the telomere-end protection (TEP) family of proteins, which targets the 3' single-stranded (ss) overhang region of the telomere via a specialized ssDNA-binding domain (DBD). With the recent availability of the structures of these DBDs, the ssDNA-binding characteristics of TEP proteins can be compared and the common underlying mechanisms of ssDNA recognition identified, thus providing insights into telomere function.
Asunto(s)
ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Telómero/química , Animales , Sitios de Unión , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Telómero/metabolismoRESUMEN
The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Genéticos , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/genéticaRESUMEN
LRP1 is a cell surface receptor responsible for clearing some 30 known ligands. We have previously shown that each of the three complete LDL receptor-homology domains of the LRP1 extracellular domain (sLRPs) binds apoE-enriched beta-VLDL particles. Here we show that two peptides from the N-terminal receptor binding domain of apoE, which are known to elicit a number of different cellular responses, bind to LRP1. Solution binding assays show that the two peptides, apoE(130-149) and apoE(141-155)(2), interact with each of the sLRPs (2, 3, and 4). Each peptide was found to exhibit the same solution binding characteristics as apoE-enriched beta-VLDL particles. Surface plasmon resonance analyses of the sLRP-apoE peptide interaction show that both peptides bind the sLRPs with K(D) values in the 100 nM range, a value similar to the effective concentration required for observation of the cellular responses. Consistent with results from mutagenesis studies of binding of apoE to LDLR, apoE(130-149,Arg142Glu) bound with a K(D) similar to that of the wild-type sequence, while apoE(130-149,Lys143Glu) showed a 10-fold decrease in K(D). Each of the peptides bound heparin, and heparin competed for sLRP binding.
Asunto(s)
Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Imitación Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Apolipoproteínas E/síntesis química , Apolipoproteínas E/genética , Unión Competitiva , Línea Celular , Dicroismo Circular , Heparina/metabolismo , Humanos , Cinética , Lipoproteínas VLDL/metabolismo , Liposomas/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Pruebas de Precipitina , Unión Proteica , Soluciones , Resonancia por Plasmón de SuperficieRESUMEN
The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP). Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography. Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter. Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands. Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments. As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451). All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor. All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes. Only sLRP4 bound thrombin-antithrombin III complexes. The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P. pastoris and readily purified. Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP.