RESUMEN
Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence for Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse Wise protein.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Endopeptidasas/metabolismo , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Endopeptidasas/química , Endopeptidasas/genética , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Escherichia coli/genética , Femenino , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Péptidos/genética , Péptidos/inmunología , Unión Proteica , Dominios Proteicos , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Semaforinas/química , Semaforinas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Células HEK293 , Humanos , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Semaforinas/genética , Relación Estructura-ActividadRESUMEN
Enpp1 is a membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing extracellular nucleotide triphosphates to produce pyrophosphate. Enpp1 dysfunction causes human diseases characterized by ectopic calcification. Enpp1 also inhibits insulin signaling, and an Enpp1 polymorphism is associated with insulin resistance. However, the precise mechanism by which Enpp1 functions in these cellular processes remains elusive. Here, we report the crystal structures of the extracellular region of mouse Enpp1 in complex with four different nucleotide monophosphates, at resolutions of 2.7-3.2 Å. The nucleotides are accommodated in a pocket formed by an insertion loop in the catalytic domain, explaining the preference of Enpp1 for an ATP substrate. Structural mapping of disease-associated mutations indicated the functional importance of the interdomain interactions. A structural comparison of Enpp1 with Enpp2, a lysophospholipase D, revealed marked differences in the domain arrangements and active-site architectures. Notably, the Enpp1 mutant lacking the insertion loop lost the nucleotide-hydrolyzing activity but instead gained the lysophospholipid-hydrolyzing activity of Enpp2. Our findings provide structural insights into how the Enpp family proteins evolved to exert their diverse cellular functions.
Asunto(s)
Calcificación Fisiológica/genética , Glicoproteínas/química , Insulina/metabolismo , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Conformación Proteica , Pirofosfatasas/química , Transducción de Señal/genética , Animales , Cristalografía por Rayos X , Glicoproteínas/metabolismo , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Ribonucleótidos/metabolismo , Difracción de Rayos XRESUMEN
Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is associated with a poor prognosis, making it an important therapeutic target. Here, we establish a novel cancer-specific anti-HER2 antibody, H2Mab-214. H2Mab-214 reacts with HER2 on cancer cells, but unlike the therapeutic antibody trastuzumab, it does not react with HER2 on normal cells in flow cytometry measurements. A crystal structure suggests that H2Mab-214 recognizes a structurally disrupted region in the HER2 domain IV, which normally forms a ß-sheet. We show that this misfolding is inducible by site-directed mutagenesis mimicking the disulfide bond defects that also may occur in cancer cells, indicating that the local misfolding in the Cys-rich domain IV governs the cancer-specificity of H2Mab-214. Furthermore, we show that H2Mab-214 effectively suppresses tumor growth in xenograft mouse models. Our findings offer a potential strategy for developing cancer-specific therapeutic antibodies that target partially misfolded cell surface receptors.
Asunto(s)
Pliegue de Proteína , Receptor ErbB-2 , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor ErbB-2/metabolismo , Receptor ErbB-2/química , Humanos , Animales , Ratones , Línea Celular Tumoral , Modelos Moleculares , Femenino , Cristalografía por Rayos X , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/química , Trastuzumab/farmacología , Trastuzumab/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Semaphorin-Plexin signaling plays a major role in the tumor microenvironment (TME). In particular, Semaphorin 4D (SEMA4D) has been shown to promote tumor growth and metastasis; however, the role of its high-affinity receptor Plexin-B1 (PLXNB1), which is expressed in the TME, is poorly understood. In this study, we directly targeted PLXNB1 in the TME of triple-negative murine breast carcinoma to elucidate its relevance in cancer progression. We found that primary tumor growth, and metastatic dissemination were strongly reduced in PLXNB1-deficient mice, which showed longer survival. PLXNB1-loss in the TME induced a switch in the polarization of tumor-associated macrophages (TAMs) towards a pro-inflammatory M1 phenotype and enhanced the infiltration of CD8+ T lymphocytes both in primary tumors and in distant metastases. Moreover, PLXNB1-deficiency promoted a shift in the Th1/Th2 balance of the T-cell population and an antitumor gene signature, with the up-regulation of Icos, Perforin-1, Stat3 and Ccl5 in tumor infiltrating lymphocytes (TILs). We thus tested the translational relevance of TME re-programming driven by PLXNB1 inactivation for responsiveness to immunotherapy. Indeed, in the absence of PLXNB1, the efficacy of anti-PD-1 blockade was strongly enhanced, efficiently reducing tumor growth and distant metastasis. Consistent with this, pharmacological PLXNB1 blockade by systemic treatment with a specific inhibitor significantly hampered breast cancer growth and enhanced the antitumor activity of the anti-PD1 treatment in a preclinical model. Altogether, these data indicate that PLXNB1 signaling controls the antitumor immune response in the TME and highlight this receptor as a promising immune therapeutic target for metastatic breast cancers.
RESUMEN
The regenerative functions associated with cytokines and growth factors have immense therapeutic potential; however, their poor pharmacokinetics, resulting from structural features, hinder their effectiveness. In this study, we aimed to enhance the pharmacokinetics of growth factors by designing receptor-binding macrocyclic peptides through in vitro mRNA display and grafting them into loops of immunoglobulin's crystallizable region (Fc). As a model, we developed peptide-grafted Fc proteins with hepatocyte growth factor (HGF) functionality that exhibited a prolonged circulation half-life and could be administered subcutaneously. The Fc-based HGF mimetic alleviated liver fibrosis in a mouse model fed a choline-deficient high-fat diet, which induces hepatic features of non-alcoholic steatohepatitis, including fibrosis, showcasing its potential as a therapeutic intervention. This study provides a basis for developing growth factor and cytokine mimetics with improved pharmacokinetics, expanding their therapeutic applications.
RESUMEN
Short half-lives in circulation and poor transport across the blood-brain barrier limit the utility of cytokines and growth factors acting as receptor agonists. Here we show that surrogate receptor agonists with longer half-lives in circulation and enhanced transport rates across the blood-brain barrier can be generated by genetically inserting macrocyclic peptide pharmacophores into the structural loops of the fragment crystallizable (Fc) region of a human immunoglobulin. We used such 'lasso-grafting' approach, which preserves the expression levels of the Fc region and its affinity for the neonatal Fc receptor, to generate Fc-based protein scaffolds with macrocyclic peptides binding to the receptor tyrosine protein kinase Met. The Met agonists dimerized Met, inducing biological responses that were similar to those induced by its natural ligand. Moreover, lasso-grafting of the Fc region of the mouse anti-transferrin-receptor antibody with Met-binding macrocyclic peptides enhanced the accumulation of the resulting Met agonists in brain parenchyma in mice. Lasso-grafting may allow for designer protein therapeutics with enhanced stability and pharmacokinetics.
Asunto(s)
Barrera Hematoencefálica , Péptidos , Humanos , Animales , Ratones , Encéfalo , Citocinas , SemividaRESUMEN
Enpp1 is an extracellular membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing ATP to generate pyrophosphate. The extracellular region of mouse Enpp1 was expressed in HEK293S GnT1(-) cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 3.0â Å resolution. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 105.3, c = 173.7â Å. A single-wavelength anomalous dispersion (SAD) data set was also collected to 2.7â Å resolution using a selenomethionine-labelled crystal. The experimental phases determined by the SAD method produced an interpretable electron-density map.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , Pirofosfatasas/química , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Pirofosfatasas/genética , Pirofosfatasas/aislamiento & purificaciónRESUMEN
The Wnt signaling pathway plays a critical role in the developmental and physiological processes of metazoans. We previously reported that the Frizzled4 (FZD4) linker domain plays an important role in Norrin binding and signaling. However, the question remains whether the FZD linker contributes to Wnt signaling in general. Here, we show that the FZD linker is involved in Wnt binding and affects downstream Wnt signaling. A FZD4 chimera, in which the linker was swapped with that of the non-canonical receptor FZD6, impairs the binding with WNT3A and suppresses the recruitment of LRP6 and Disheveled, resulting in reduced canonical signaling. A similar effect was observed for non-canonical signaling. A FZD6 chimera containing the FZD1 linker showed reduced WNT5A binding and impaired signaling in ERK, JNK, and AKT mediated pathways. Altogether, our results suggest that the FZD linker plays an important role in specific Wnt binding and intracellular Wnt signaling.
Asunto(s)
Receptores Frizzled , Vía de Señalización Wnt , Proteínas Portadoras/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Dominios Proteicos , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Semaphorins constitute a large family of secreted and membrane-bound proteins that signal through cell-surface receptors, plexins. Semaphorins generally use low-affinity protein-protein interactions to bind with their specific plexin(s) and regulate distinct cellular processes such as neurogenesis, immune response, and organogenesis. Sema6D is a membrane-bound semaphorin that interacts with class A plexins. Sema6D exhibited differential binding affinities to class A plexins in prior cell-based assays, but the molecular mechanism underlying this selectivity is not well understood. Therefore, we performed hybrid in vitro/in silico analysis to examine the binding mode of Sema6D to class A plexins and to identify residues that give rise to the differential affinities and thus contribute to the selectivity within the same class of semaphorins. Our biophysical binding analysis indeed confirmed that Sema6D has a higher affinity for Plexin-A1 than for other class A plexins, consistent with the binding selectivity observed in the previous cell-based assays. Unexpectedly, our present crystallographic analysis of the Sema6D-Plexin-A1 complex showed that the pattern of polar interactions is not interaction-specific because it matches the pattern in the prior structure of the Sema6A-Plexin-A2 complex. Thus, we performed in silico alanine scanning analysis and discovered hotspot residues that selectively stabilized the Sema6D-Plexin-A1 pair via Van der Waals interactions. We then validated the contribution of these hotspot residues to the variation in binding affinity with biophysical binding analysis and molecular dynamics simulations on the mutants. Ultimately, our present results suggest that shape complementarity in the binding interfaces is a determinant for binding selectivity.
Asunto(s)
Semaforinas , Semaforinas/genética , Semaforinas/química , Semaforinas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Comunicación CelularRESUMEN
Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.
Asunto(s)
Anticuerpos Monoclonales , Mieloma Múltiple , Anticuerpos Monoclonales/uso terapéutico , HumanosRESUMEN
Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6ß1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin ß1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the ß-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.
Asunto(s)
Células Epiteliales/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6beta1/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Secuencia de Aminoácidos , Membrana Basal/metabolismo , Sitios de Unión/fisiología , Adhesión Celular/fisiología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Conformación Proteica , Dominios Proteicos/fisiología , Electricidad EstáticaRESUMEN
Lasso-grafting (LG) technology is a method for generating de novo biologics (neobiologics) by genetically implanting macrocyclic peptide pharmacophores, which are selected in vitro against a protein of interest, into loops of arbitrary protein scaffolds. In this study, we have generated a neo-capsid that potently binds the hepatocyte growth factor receptor MET by LG of anti-MET peptide pharmacophores into a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS), a self-assembling protein nanocapsule. By virtue of displaying multiple-pharmacophores on its surface, the neo-capsid can induce dimerization (or multimerization) of MET, resulting in phosphorylation and endosomal internalization of the MET-capsid complex. This work demonstrates the potential of the LG technology as a synthetic biology approach for generating capsid-based neobiologics capable of activating signaling receptors.
RESUMEN
Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.
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Péptidos/química , Péptidos/farmacología , Ingeniería de Proteínas/métodos , Proteínas de la Cápside/química , Proteínas Portadoras/química , Línea Celular , Dependovirus , Humanos , Inmunoglobulina G/química , Modelos Moleculares , Albúmina Sérica/química , Bibliotecas de Moléculas PequeñasRESUMEN
SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/farmacología , Tratamiento Farmacológico de COVID-19 , Mutación , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/virología , Células Cultivadas , Cricetinae , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Masculino , Simulación de Dinámica Molecular , Unión Proteica , Ingeniería de Proteínas/métodos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismoRESUMEN
Here we report de novo macrocyclic peptide binders to Wnt3a, a member of the Wnt protein family. By means of the Random non-standard Peptides Integrated Discovery (RaPID) system, we have performed in vitro selection against the complex of mouse Wnt3a (mWnt3a) with human afamin (hAFM) to discover macrocyclic peptides that bind mWnt3a with K D values as tight as 110 nM. One of these peptides, WAp-D04 (Wnt-AFM-peptide-D04), was able to inhibit the receptor-mediated signaling process, which was demonstrated in a Wnt3a-dependent reporter cell-line. Based on this initial hit, we applied a block-mutagenesis scanning display to identify a mutant inhibitor, WAp-D04-W10P, with 5-fold greater potency in a reporter assay. This work represents the first instance of molecules capable of inhibiting Wnt signaling through direct interaction with a Wnt protein, a molecular class for which targeting has been challenging due its highly hydrophobic nature.
RESUMEN
Wnt signaling plays fundamental roles in organogenesis, tissue regeneration and cancer, but high-resolution structural information of mammalian Wnt proteins is lacking. We solved a 2.8-Å resolution crystal structure of human Wnt3 in complex with mouse Frizzled 8 Cys-rich domain (CRD). Wnt3 grabs the receptor in a manner very similar to that found in Xenopus Wnt8 complexed with the same receptor. Unlike Xenopus Wnt8-bound CRD, however, Wnt3-bound CRD formed a symmetrical dimer in the crystal by exchanging the tip of the unsaturated acyl chain attached to each Wnt3, confirming the ability of Wnt and Frizzled CRD to form a 2:2 complex. The hypervariable 'linker' region of Wnt3 formed a ß-hairpin protrusion opposite from the Frizzled binding interface, consistent with its proposed role in the coreceptor recognition. Direct binding between this segment and the Wnt coreceptor LRP6 was confirmed, enabling us to build a structural model of the Wnt-Frizzled-LRP6 ternary complex.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Proteína Wnt3/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3/metabolismo , XenopusRESUMEN
N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a ß1-6-linked N-acetylglucosamine branch. ß1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GnT-V a promising target for drug development. However, the molecular basis of GnT-V's catalytic mechanism and substrate specificity are not fully understood. Here, we report crystal structures of human GnT-V luminal domain with a substrate analog. GnT-V luminal domain is composed of a GT-B fold and two accessary domains. Interestingly, two aromatic rings sandwich the α1-6 branch of the acceptor N-glycan and restrain the global conformation, partly explaining the fine branch specificity of GnT-V. In addition, interaction of the substrate N-glycoprotein with GnT-V likely contributes to protein-selective and site-specific glycan modification. In summary, the acceptor-GnT-V complex structure suggests a catalytic mechanism, explains the previously observed inhibition of GnT-V by branching enzyme GnT-III, and provides a basis for the rational design of drugs targeting N-glycan branching.
Asunto(s)
Dominio Catalítico/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , Neoplasias/patología , Polisacáridos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biocatálisis , Cristalografía por Rayos X , Diseño de Fármacos , Pruebas de Enzimas , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Neoplasias/tratamiento farmacológico , Polisacáridos/química , Especificidad por SustratoRESUMEN
Plexins are type I membrane proteins that function as receptors for semaphorins. All of the known plexins contain a large globular domain, termed the sema domain, in the N-terminal extracellular region, which interacts with semaphorins during signal transduction. Here, we describe procedures for protein production and purification that we utilized in the crystallographic study of the mouse Plexin A2 (mPlxnA2) extracellular fragment, including the sema domain. A mutant mammalian cell line, HEK293S GnTI-, was used as an expression host for the production of a crystallizable-quality mPlxnA2 fragment, which contains several N-glycosylation sites and disulfide bonds.
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Cromatografía de Afinidad/métodos , Proteínas del Tejido Nervioso/aislamiento & purificación , Receptores de Superficie Celular/aislamiento & purificación , Animales , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Glicosilación , Células HEK293 , Humanos , Ratones , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation.