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1.
Sci Adv ; 7(24)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34108208

RESUMO

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

2.
Blood ; 138(13): 1182-1193, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33945603

RESUMO

Events mediated by the P-selectin/PSGL-1 pathway play a critical role in the initiation and propagation of venous thrombosis by facilitating the accumulation of leukocytes and platelets within the growing thrombus. Activated platelets and endothelium express P-selectin, which binds P-selectin glycoprotein ligand-1 (PSGL-1) that is expressed on the surface of all leukocytes. We developed a pegylated glycomimetic of the N terminus of PSGL-1, PEG40-GSnP-6 (P-G6), which proved to be a highly potent P-selectin inhibitor with a favorable pharmacokinetic profile for clinical translation. P-G6 inhibits human and mouse platelet-monocyte and platelet-neutrophil aggregation in vitro and blocks microcirculatory platelet-leukocyte interactions in vivo. Administration of P-G6 reduces thrombus formation in a nonocclusive model of deep vein thrombosis with a commensurate reduction in leukocyte accumulation, but without disruption of hemostasis. P-G6 potently inhibits the P-selectin/PSGL-1 pathway and represents a promising drug candidate for the prevention of venous thrombosis without increased bleeding risk.

3.
Cell Chem Biol ; 27(9): 1207-1219.e9, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32610041

RESUMO

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.


Assuntos
Glicopeptídeos/química , Análise em Microsséries/métodos , Anticorpos/imunologia , Cromatografia Líquida de Alta Pressão , Polarização de Fluorescência , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Biblioteca de Peptídeos , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Glycobiology ; 30(5): 282-300, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-31742337

RESUMO

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.


Assuntos
Antígenos Glicosídicos Associados a Tumores/análise , Biomarcadores Tumorais/análise , Carcinoma/diagnóstico , Glicoproteínas/análise , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma/genética , Carcinoma/imunologia , Feminino , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Lactente , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Células Tumorais Cultivadas , Adulto Jovem
6.
Cell Chem Biol ; 26(4): 535-547.e4, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30745240

RESUMO

The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine (Asn)-linked N-glycans, we synthesized a library of isomeric multiantennary N-glycans that vary in terminal non-reducing sialic acid, galactose, and N-acetylglucosamine residues, as well as core fucose. We identified specific recognition of N-glycans by several plant lectins, human galectins, influenza viruses, and Siglecs, and explored the influence of sialic acid linkages and branching of the N-glycans. These results show the unique recognition of complex-type N-glycans by a wide variety of glycan-binding proteins and their abilities to distinguish isomeric structures, which provides new insights into the biological roles of these proteins and the uses of lectins in biological applications to identify glycans.


Assuntos
Asparagina/metabolismo , Polissacarídeos/metabolismo , Proteínas/metabolismo , Animais , Asparagina/análogos & derivados , Sítios de Ligação , Galectinas/metabolismo , Humanos , Isomerismo , Orthomyxoviridae/metabolismo , Lectinas de Plantas/metabolismo , Plantas/metabolismo , Polissacarídeos/química , Ligação Proteica , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo
7.
PLoS One ; 12(6): e0180242, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28665962

RESUMO

Cosmc is an endoplasmic reticulum chaperone necessary for normal protein O-GalNAc glycosylation through regulation of T-synthase, its single client. Loss-of-function of Cosmc results in expression of the Tn antigen, which is associated with multiple human diseases including cancer. Despite intense interest in dysregulated expression of the Tn antigen, little is known about the structure and function of Cosmc, including domain organization, secondary structure, oligomerization, and co-factors. Limited proteolysis experiments show that Cosmc contains a structured N-terminal domain (CosmcΔ256), and biochemical characterization of CosmcΔ256 reveals wild type chaperone activity. Interestingly, CosmcE152K, which shows loss of function in vivo, exhibits wild type-like activity in vitro. Cosmc and CosmcE152K heterogeneously oligomerize and form monomeric, dimeric, trimeric, and tetrameric species, while CosmcΔ256 is predominantly monomeric as characterized by chemical crosslinking and blue native page electrophoresis. Additionally, Cosmc selectively binds divalent cations in thermal shift assays and metal binding is abrogated by the CosmcΔ256 truncation, and perturbed by the E152K mutation. Therefore, the N-terminal domain of Cosmc mediates T-synthase binding and chaperone function, whereas the C-terminal domain is necessary for oligomerization and metal binding. Our results provide new structure-function insight to Cosmc, indicate that Cosmc behaves as a modular protein and suggests points of modulation or regulation of in vivo chaperone function.


Assuntos
Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Cátions Bivalentes , Células HEK293 , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
8.
J Biol Chem ; 290(37): 22385-97, 2015 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-26216880

RESUMO

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have Kd values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3.


Assuntos
Quimiocina CXCL12/química , Modelos Moleculares , Biblioteca de Peptídeos , Receptores CXCR4/química , Receptores CXCR/química , Animais , Quimiocina CXCL12/genética , Quimiocina CXCL12/farmacologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Células Jurkat , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Engenharia de Proteínas , Receptores CXCR/genética , Receptores CXCR4/genética
9.
Proteins ; 79(4): 1267-76, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21294157

RESUMO

Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/ß-lactamase inhibitor protein (BLIP) and SHV-1/BLIP are homologous ß-lactamase/ß-lactamase inhibitor protein complexes with disparate K(d) values (3 nM and 2 µM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network.


Assuntos
Proteínas de Bactérias/química , Domínios e Motivos de Interação entre Proteínas , beta-Lactamases/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Termodinâmica , beta-Lactamases/metabolismo
10.
Protein Sci ; 19(10): 1996-2000, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20669180

RESUMO

Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the ß-lactamase TEM-1 and various ß-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies.


Assuntos
Proteínas de Bactérias/metabolismo , Complexos Multiproteicos/química , Proteínas Mutantes/química , beta-Lactamases/química , Algoritmos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Cinética , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Ligação Proteica , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Ureia/química , Ureia/farmacologia , beta-Lactamases/metabolismo
11.
Biochemistry ; 48(39): 9185-93, 2009 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-19731932

RESUMO

KPC beta-lactamases hydrolyze the "last resort" beta-lactam antibiotics (carbapenems) used to treat multidrug resistant infections and are compromising efforts to combat life-threatening Gram-negative bacterial infections in hospitals worldwide. Consequently, the development of novel inhibitors is essential for restoring the effectiveness of existing antibiotics. The beta-lactamase inhibitor protein (BLIP) is a competitive inhibitor of a number of class A beta-lactamases. In this study, we characterize the previously unreported interaction between KPC-2 beta-lactamase and BLIP. Biochemical results show that BLIP is an extremely potent inhibitor of KPC enzymes, binding KPC-2 and KPC-3 with subnanomolar affinity. To understand the basis of affinity and specificity in the beta-lactamase-BLIP system, the crystallographic structure of the KPC-2-BLIP complex was determined to 1.9 A resolution. Computational alanine scanning was also conducted to identify putative hot spots in the KPC-2-BLIP interface. Interestingly, the two complexes making up the KPC-2-BLIP asymmetric unit are distinct, and in one structure, the BLIP F142 loop is absent, in contrast to homologous structures in which it occupies the active site. This finding and other sources of structural plasticity appear to contribute to BLIP's promiscuity, enabling it to respond to mutations at the beta-lactamase interface. Given the continuing emergence of antibiotic resistance, the high-resolution KPC-2-BLIP structure will facilitate its use as a template for the rational design of new inhibitors of this problematic enzyme.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Inibidores de beta-Lactamases , beta-Lactamases/química , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Klebsiella pneumoniae/enzimologia , Dados de Sequência Molecular , Conformação Proteica , beta-Lactamases/fisiologia
12.
J Mol Biol ; 382(5): 1265-75, 2008 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-18775544

RESUMO

Beta-lactamases are enzymes that catalyze the hydrolysis of beta-lactam antibiotics. beta-lactamase/beta-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A beta-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 beta-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 A resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.


Assuntos
Inibidores Enzimáticos/química , Inibidores de beta-Lactamases , beta-Lactamases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Cinética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Modelos Moleculares , Complexos Multiproteicos , Mutagênese , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , beta-Lactamases/genética
13.
J Comput Chem ; 28(14): 2378-88, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17471459

RESUMO

Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.


Assuntos
Algoritmos , Biblioteca de Peptídeos , Proteínas/química , Software , Modelos Moleculares , Conformação Proteica , Termodinâmica
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