RESUMO
Recent advances in immunotherapeutic approaches to the treatment of Alzheimer's disease (AD) have increased the importance of understanding the exact binding preference of each amyloid-beta (Aß) antibody employed, since this determines both efficacy and risk for potentially serious adverse events known as amyloid-related imaging abnormalities. Lecanemab is a humanized IgG1 antibody that was developed to target the soluble Aß protofibril conformation. The present study prepared extracts of post mortem brain samples from AD patients and non-demented elderly controls, characterized the forms of Aß present, and investigated their interactions with lecanemab. Brain tissue samples were homogenized and extracted using tris-buffered saline. Aß levels and aggregation states in soluble and insoluble extracts, and in fractions prepared using size-exclusion chromatography or density gradient ultracentrifugation, were analyzed using combinations of immunoassay, immunoprecipitation (IP), and mass spectrometry. Lecanemab immunohistochemistry was also conducted in temporal cortex. The majority of temporal cortex Aß (98 %) was in the insoluble extract. Aß42 was the most abundant form present, particularly in AD subjects, and most soluble Aß42 was in soluble aggregated protofibrillar structures. Aß protofibril levels were much higher in AD subjects than in controls. Protofibrils captured by lecanemab-IP contained high levels of Aß42 and lecanemab bound to large, medium, and small Aß42 protofibrils in a concentration-dependent manner. Competitive IP showed that neither Aß40 monomers nor Aß40-enriched fibrils isolated from cerebral amyloid angiopathy reduced lecanemab's binding to Aß42 protofibrils. Immunohistochemistry showed that lecanemab bound readily to Aß plaques (diffuse and compact) and to intraneuronal Aß in AD temporal cortex. Taken together, these findings indicate that while lecanemab binds to Aß plaques, it preferentially targets soluble aggregated Aß protofibrils. These are largely composed of Aß42, and lecanemab binds less readily to the Aß40-enriched fibrils found in the cerebral vasculature. This is a promising binding profile because Aß42 protofibrils represent a key therapeutic target in AD, while a lack of binding to monomeric Aß and cerebral amyloid deposits should reduce peripheral antibody sequestration and minimize risk for adverse events.
RESUMO
Amyloid beta (Aß) peptides, which aggregate to form neocortical plaques in Alzheimer's disease, exist in states that range from soluble monomers and oligomers/protofibrils to insoluble fibrillar amyloid. The present study evaluated the effects of mAb158, a mouse monoclonal antibody version of lecanemab that preferentially binds to soluble Aß protofibrils, in aged transgenic mice (Tg2576) with Aß pathology. Female Tg2576 mice (12 months old) received weekly intraperitoneal mAb158 (35 mg/kg) or vehicle for 4 weeks or for 18 weeks, with or without a subsequent 12-week off-treatment period. Aß protofibril levels were significantly lower in mAb158-treated animals at both 4 and 18 weeks, while longer treatment duration (18 weeks) was required to observe significantly lower Aß42 levels in insoluble brain fractions and lower Aß plaque load. Following the off-treatment period, comparison of the vehicle- and mAb158-treated mice demonstrated that the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load remained significantly lower in mAb158-treated animals, as compared with age-matched controls. However, there was a significant increase of brain accumulation of both the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load after treatment cessation. Thus, repeated mAb158 treatment of aged Tg2576 mice first reduced Aß protofibril levels within 4 weeks of treatment, which then was followed by a reduction of amyloid plaque pathology within 18 weeks of treatment. These effects were maintained 12 weeks after the final dose, indicating that mAb158 had a disease-modifying effect on the Aß pathology in this mouse model. In addition, brain accumulation of both Aß protofibril levels and amyloid pathology progressed after discontinuation of the treatment which supports the importance of continued treatment with mAb158 to maintain the effects on Aß pathology.
RESUMO
Therapeutic antibodies have been developed to target amyloid-beta (Aß), and some of these slow the progression of Alzheimer's disease (AD). However, they can also cause adverse events known as amyloid-related imaging abnormalities with edema (ARIA-E). We investigated therapeutic Aß antibody binding to cerebral amyloid angiopathy (CAA) fibrils isolated from human leptomeningeal tissue to study whether this related to the ARIA-E frequencies previously reported by clinical trials. The binding of Aß antibodies to CAA Aß fibrils was evaluated in vitro using immunoprecipitation, surface plasmon resonance, and direct binding assay. Marked differences in Aß antibody binding to CAA fibrils were observed. Solanezumab and crenezumab showed negligible CAA fibril binding and these antibodies have no reported ARIA-E cases. Lecanemab showed a low binding to CAA fibrils, consistent with its relatively low ARIA-E frequency of 12.6%, while aducanumab, bapineuzumab, and gantenerumab all showed higher binding to CAA fibrils and substantially higher ARIA-E frequencies (25-35%). An ARIA-E frequency of 24% was reported for donanemab, and its binding to CAA fibrils correlated with the amount of pyroglutamate-modified Aß present. The findings of this study support the proposal that Aß antibody-CAA interactions may relate to the ARIA-E frequency observed in patients treated with Aß-based immunotherapies.
Assuntos
Peptídeos beta-Amiloides , Angiopatia Amiloide Cerebral , Humanos , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloide/imunologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Angiopatia Amiloide Cerebral/diagnóstico por imagem , Angiopatia Amiloide Cerebral/imunologia , Angiopatia Amiloide Cerebral/patologia , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Despite years of utilizing the transferrin receptor 1 (TfR1) to transport large biomolecules into the brain, there is no consensus on how to optimally measure affinity to it. The aim of this study was to compare different methods for measuring the affinities of anti-TfR1 antibodies. Antibodies 15G11, OX26 and 8D3 are known to successfully carry large biologics across the blood-brain barrier in humans, rats, and mice, respectively. The affinity to their respective species of TfR1 was measured with different surface plasmon resonance setups in Biacore and an on-cell assay. When the antibody was captured and TfR1 was the analyte, the dissociation in Biacore was very slow. The dissociation was faster when the antibody was the analyte and TfR1 was the ligand. The Biacore setup with capture of N-terminal FLAG-tag TfR1 yielded the most similar apparent affinities as the cell assay. In conclusion, it is important to evaluate assay parameters including assay orientation, surface capture method, and antibody-format when comparing binding kinetics for TfR1 antibodies. Although it seems possible to determine relative affinities of TfR1 antibodies using the methods described here, both the FLAG-tag TfR1 capture setup and cell assays likely yield apparent affinities that are most translatable in vivo.
Assuntos
Anticorpos , Ressonância de Plasmônio de Superfície , Ratos , Camundongos , Humanos , Animais , Ressonância de Plasmônio de Superfície/métodos , Anticorpos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Receptores da Transferrina/metabolismoRESUMO
Immunotherapy against amyloid-beta (Aß) is a promising option for the treatment of Alzheimer's disease (AD). Aß exists as various species, including monomers, oligomers, protofibrils, and insoluble fibrils in plaques. Oligomers and protofibrils have been shown to be toxic, and removal of these aggregates might represent an effective treatment for AD. We have characterized the binding properties of lecanemab, aducanumab, and gantenerumab to different Aß species with inhibition ELISA, immunodepletion, and surface plasmon resonance. All three antibodies bound monomers with low affinity. However, lecanemab and aducanumab had very weak binding to monomers, and gantenerumab somewhat stronger binding. Lecanemab was distinctive as it had tenfold stronger binding to protofibrils compared to fibrils. Aducanumab and gantenerumab preferred binding to fibrils over protofibrils. Our results show different binding profiles of lecanemab, aducanumab, and gantenerumab that may explain clinical results observed for these antibodies regarding both efficacy and side effects.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismoRESUMO
A growing body of evidence suggests that aggregated α-synuclein, the major constituent of Lewy bodies, plays a key role in the pathogenesis of Parkinson's disease and related α-synucleinopathies. Immunotherapies, both active and passive, against α-synuclein have been developed and are promising novel treatment strategies for such disorders. Here, we report on the humanization and pharmacological characteristics of ABBV-0805, a monoclonal antibody that exhibits a high selectivity for human aggregated α-synuclein and very low affinity for monomers. ABBV-0805 binds to a broad spectrum of soluble aggregated α-synuclein, including small and large aggregates of different conformations. Binding of ABBV-0805 to pathological α-synuclein was demonstrated in Lewy body-positive post mortem brains of Parkinson's disease patients. The functional potency of ABBV-0805 was demonstrated in several cellular assays, including Fcγ-receptor mediated uptake of soluble aggregated α-synuclein in microglia and inhibition of neurotoxicity in primary neurons. In vivo, the murine version of ABBV-0805 (mAb47) displayed significant dose-dependent decrease of α-synuclein aggregates in brain in several mouse models, both in prophylactic and therapeutic settings. In addition, mAb47 treatment of α-synuclein transgenic mice resulted in a significantly prolonged survival. ABBV-0805 selectively targets soluble toxic α-synuclein aggregates with a picomolar affinity and demonstrates excellent in vivo efficacy. Based on the strong preclinical findings described herein, ABBV-0805 has been progressed into clinical development as a potential disease-modifying treatment for Parkinson's disease.
Assuntos
Anticorpos Monoclonais , Doença de Parkinson , Sinucleinopatias , Animais , Anticorpos Monoclonais/uso terapêutico , Humanos , Longevidade , Camundongos , Camundongos Transgênicos , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Sinucleinopatias/terapia , alfa-Sinucleína/metabolismoRESUMO
Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin αL /ß2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic αL domain and a yellow fluorescent protein-tagged ß2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and ß2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and ß2 subunits (Kd = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin αM , αX , and ß3 subunits (Kd = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of αL and ß2 show that LtxA binds membrane-proximal domain of αL and intermediate domain of ß2 .
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Exotoxinas/metabolismo , Interações Hospedeiro-Patógeno , Imunossupressores/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Células Jurkat , Microscopia de Fluorescência , Ligação ProteicaRESUMO
Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1ß [IL-1ß] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Colesterol/metabolismo , Infecções por Pasteurellaceae/microbiologia , Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/genética , Motivos de Aminoácidos , Toxinas Bacterianas/genética , Humanos , Interleucina-1beta/imunologia , Macrófagos/imunologia , Infecções por Pasteurellaceae/imunologia , Infecções por Pasteurellaceae/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the ß3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the ß3 tail binding site. Accordingly, we have re-examined c-Src binding to ß3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and ß3. Following platelet activation, however, c-Src was co-immunoprecipitated with ß3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal ß3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the ß1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the ß3 peptide to the RT- loop. Under these conditions, the ß3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the ß3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with ß3 are weak and insensitive to ß3 tail mutations.
Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Plaquetas/química , Plaquetas/citologia , Proteína Tirosina Quinase CSK , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Peptídeos/química , Peptídeos/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Domínios de Homologia de src , Quinases da Família src/química , Quinases da Família src/genéticaRESUMO
During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen - a region encompassing the groove of Ranvier - caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium's phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier.
Assuntos
Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Cartilagem/patologia , Exostose Múltipla Hereditária/patologia , Heparitina Sulfato/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Osso e Ossos/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/embriologia , Condrogênese/efeitos dos fármacos , Coristoma/patologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Exostose Múltipla Hereditária/embriologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Cinética , Camundongos , Modelos Biológicos , N-Acetilglucosaminiltransferases/deficiência , Fenótipo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
We demonstrated that ß-(1-azulenyl)-L-alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.
Assuntos
Proteínas/química , Triptofano/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Metionina/química , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Triptofano/químicaRESUMO
Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the ß3 cytosolic tail causes activation of the integrin αIIbß3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the ß3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the ß3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the ß3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the ß3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.
Assuntos
Citosol/metabolismo , Integrina beta3/química , Integrina beta3/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Talina/metabolismo , Proteínas do Citoesqueleto/química , Dextranos/metabolismo , Proteínas Imobilizadas/metabolismo , Cinética , Proteínas de Membrana/química , Proteínas dos Microfilamentos/química , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Propriedades de SuperfícieRESUMO
We show that it is possible to induce a defined secondary structure in de novo designed peptides upon electrostatic attachment to negatively charged lipid bilayer vesicles without partitioning of the peptides into the membrane, and that the secondary structure can be varied via small changes in the primary amino acid sequence of the peptides. The peptides have a random-coil conformation in solution, and results from far-UV circular dichroism spectroscopy demonstrate that the structure induced by the interaction with silica nanoparticles is solely alpha-helical and also strongly pH-dependent. The present study shows that negatively charged vesicles, to which the peptides are electrostatically adsorbed via cationic amino acid residues, induce either alpha-helices or beta-sheets and that the conformation is dependent on both lipid composition and variations in peptide primary structure. The pH-dependence of the vesicle-induced peptide secondary structure is weak, which correlates well with small differences in the vesicles' electrophoretic mobility, and thus the surface charge, as the pH is varied.
Assuntos
Membrana Celular/metabolismo , Desenho de Fármacos , Bicamadas Lipídicas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Eletricidade Estática , Sequência de Aminoácidos , Membrana Celular/química , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Cinética , Luz , Bicamadas Lipídicas/química , Dados de Sequência Molecular , Nanopartículas/química , Peptídeos/síntese química , Fosfatidilgliceróis/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Espalhamento de Radiação , Dióxido de Silício/química , Propriedades de Superfície , TermodinâmicaRESUMO
We have shown that it is possible to design a peptide that has a very low helical content when free in solution but that adopts a well-defined helix when interacting with silica nanoparticles. From a systematic variation of the amino acid composition and distribution in designed peptides, it has been shown that the ability to form helical structure upon binding to the silica surface is dominated by two factors. First, the helical content is strongly correlated with the net positive charge on the side of the helix that interacts with the silica, and arginine residues are strongly favored over lysine residues in these positions. The second important factor is to have a high net negative charge on the side of the helix that faces the solution. Apparently, both attractive and repulsive electrostatic forces dominate the induction and stabilization of a bound helix. It is also evident that using amino acids that have high propensity to form helix in solution are also advantageous for the formation of helix on surfaces.
Assuntos
Nanopartículas/química , Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Secundária de Proteína , Dióxido de Silício/química , TitulometriaRESUMO
Chemotaxis is the stimulated directional migration of cells in response to chemotactic factors, manifested for instance during leukocyte interaction with chemoattractants in inflammation. The N-formyl-Met-Leu-Phe (fMLF) bacterial peptide family is particularly potent in attracting and activating neutrophilic granulocytes. To accomplish defined circumstances for recruitment and activation of cells, we fabricated semitransparent gold-coated glass coverslips functionalized with chemoattractant fMLF receptor peptide agonist analogues. Peptides based on a common leading four-amino-acid sequence Gly-Gly-Gly-Cys were thus coupled to two potent fMLF receptor agonists, N-formyl-Tyr-Nle-Phe-Leu-Nle-Gly-Gly-Gly-Cys and N-formyl-Met-Leu-Phe-Gly-Gly-Gly-Cys, and a formylated control peptide, N-formyl-Gly-Gly-Gly-Cys. They were anchored via the SH group of Cys either directly to the gold surface or a mixed self-assembled monolayer composed of maleimide- and hydroxyl-terminated oligo(ethylene glycol) alkyldisulfides. The overall peptide immobilization procedure was characterized with ellipsometry, contact angle measurement, and infrared spectroscopy. When exposed to granulocytes, the agonist surface rapidly recruited neutrophils and the cells responded with extensive spreading and intracellular calcium transients within minutes. The reference peptide generated no such activation, and the cells maintained a more spherical morphology, suggesting that we have been able to immobilize chemoattractant receptor agonist peptides with retained bioactivity. This is a crucial step in designing surfaces with specific effects on cellular behavior.