Asia1型口蹄疫病毒受体结合位点多样性

【目的】口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)通过结构蛋白VP1 G-H环上高度保守的精氨酸-甘氨酸-天门冬氨酸(Arg-Gly-Asp,RGD)基序与整联蛋白结合起始病毒的感染,但FMDV是RNA病毒,在环境条件变化时,FMDV能够以非RGD的途径起始病毒的感染。为了研究FMDV Asia1/JS/China/05田间舌皮毒经两种不同的途径短期传代后细胞受体结合基序RGD的变异。【方法】采用RT-PCR方法扩增FMDV Asia1/JS/China/05田间毒、田间毒的乳鼠适应毒第四代(MF4)和接种田间毒的牛同居感染的猪水泡病料适应细胞的第八代毒(PBF8)结构蛋白VP1基因,并对不同病毒VP1基因的PCR产物测序和cDNA文库测序。【结果】以含RGD受体结合基序为优势的田间毒在乳鼠上短期传代后出现了含精氨酸-丝氨酸-天门冬氨酸(Arg-Ser-Asp,RSD)和RGD受体结合基序的混合种群,而同居感染后的细胞传代病毒种群则以含精氨酸-天门冬氨酸-天门冬氨酸(Arg-Asp-Asp,RDD)受体结合基序为优势种群。【结论】发现了含RGD受体结合位点为优势的FMDV种群,经不同的宿主短期传代后产了含RSD或RDD受体结合基序的优势种群,该发现不仅增加了保守基序RGD发生替换的FMDV变异株的数量,而且为FMDV的细胞识别和宿主嗜性的改变等进一步研究奠定了物质基础。     

VP3 G–H环中氨基酸突变对O型口蹄疫病毒生物学特性的影响

【目的】为了研究O型口蹄疫病毒VP3G–H环中氨基酸突变对其生物学特性的影响。【方法】借助口蹄疫病毒反向遗传操作技术平台拯救出2株定点突变体rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)。进行蚀斑形成试验、一步生长曲线的绘制、TCID_(50)和LD_(50)的测定、间接免疫荧光与激光共聚焦显微镜检测。【结果】结果显示,与骨架病毒rHN相比,虽然rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对BHK-21细胞的感染性及其蚀斑表型和复制动力学无显著性差异;但rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对乳鼠的致病力明显减弱,且均获得了小窝蛋白介导侵染CHO-K1细胞的能力。【结论】VP3上第3174位特征性氨基酸突变影响O型口蹄疫病毒感染宿主细胞的毒力及其内吞作用路径,这有助于我们认知VP3 G–H环在口蹄疫病毒粒子立体空间构象中潜在的作用。     

C-glyco“RGD” as αIIbβ3 and αvβ integrin ligands for imaging applications: Synthesis,in vitro evaluation and molecular modeling

The design of conjugates displaying simultaneously high selectivity and high affinity for different subtypes of integrins is a current challenge. The arginine-glycine-aspartic acid amino acid sequence (RGD) is one of the most efficient short peptides targeting these receptors. We report herein the development of linear and cyclic fluoro-C-glycoside“RGD” conjugates, taking advantage of the robustness and hydrophilicity of C-glycosides. As attested by in vitro evaluation, the design of these C-glyco“RGD” with a flexible three-carbon triazolyl linker allows distinct profiles towards α_IIbβ₃ and α_vβ₃ integrins. Molecular-dynamics simulations confirm the suitability of cyclic C-glyco-c(RGDfC) to target α_vβ₃ integrin. These C-glyco”RGD” could become promising biological tools in particular for Positron Emission Tomography imaging.     

Design and synthesis of novel dual-cyclic RGD peptides for αvβ3 integrin targeting

The specific binding of RGD cyclic peptide with integrin α_vβ₃ attracts great research interest for tumor-targeting drug delivery. Herein, we designed and synthesized a series of dual-ring RGD-peptide derivatives as a drug carrier for α_vβ₃ targeting. Three novel peptides showed excellent cell adhesion inhibition effect, in which, P3 exhibited 7-fold enhancement in IC₅₀ compared with cyclo(RGDfK). Drug-loaded cytotoxicity experiment and imaging experiment indicated that such dual-cyclic RGD peptides have good tumor targeting effects. This work provides a new strategy for the design of novel RGD peptides.     

Preclinical evaluation of a dual sstr2 and integrin αvβ3-targeted heterodimer [68Ga]-NOTA-3PEG4-TATE-RGD

PurposeMultiple receptors are co-expressed in many types of cancers. Octreotate (TATE) and Arg-Gly-Asp (RGD) peptides target somatostatin receptor 2 (sstr2) and integrin α_vβ₃, respectively. We developed and synthesized a heterodimer NOTA-3PEG₄-TATE-RGD (3PTATE-RGD) and aimed to investigate its characteristics for dual-targeting sstr2 and integrin α_vβ₃.MethodsTATE and RGD peptides and 1,4,7-triazacylononane-N’,N’’,N’’’-triacetic acid (NOTA) were linked through a glutamate and polyethylene glycol (PEG) linker, then 3PTATE-RGD was labeled with ⁶⁸Ga ion. Receptor-binding characteristics and tumor-targeting efficacy were tested in vitro and in vivo using H69 and A549 lung cancer cell lines and tumor-bearing mice models.Results[⁶⁸Ga]-3PTATE-RGD had comparable sstr2 and integrin α_vβ₃-binding affinity with monomeric TATE and RGD in cell uptake and PET imaging study, respectively. In the competition study, H69 and A549 tumor uptake of [⁶⁸Ga]-3PTATE-RGD was completed inhibited in the presence of an excess amount of unlabeled TATE or RGD, respectively. The blocked level didn’t grow when both of TATE and RGD mixture was co-injected with [⁶⁸Ga]-3PTATE-RGD. The pharmacokinetics of [⁶⁸Ga]-3PTATE-RGD is comparable with [⁶⁸Ga]-TATE and [⁶⁸Ga]-RGD, resulting in a larger application.Conclusion[⁶⁸Ga]-3PTATE-RGD showed improved and wider tumor-targeting efficacy compared with monomeric TATE and RGD peptides, which warrants its further investigation in detection both of sstr2 and integrin α_vβ₃-related carcinomas.     

Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging

One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y₂O₃). Cyclic RGD peptide binds specifically to integrin α_vβ₃ which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y₂O₃ was observed for U87MG cancer cell (high integrin α_vβ₃ expression), but not for MCF-7 cancer cell (low integrin α_vβ₃ expression), this UC imaging is considered to be integrin α_vβ₃ specific. The non-invasive imaging of integrin α_vβ₃ expression using UCP-based probes will have great potential in cancer imaging in general in living subjects.     

Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha(v)beta3 in vitro.

The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular alpha(v)beta3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of alpha(v)beta3 was exquisitely sensitive to competition by short RGD-containing peptides (50% inhibition at < 10(-8) M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10(4) fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to alpha(v)beta3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for alpha(v)beta3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.     

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α_vβ₃ is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α_vβ₃ binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α_vβ₃ dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.     

RGD-containing ankyrin externalized onto the cell surface triggers αVβ3 integrin-mediated erythrophagocytosis

The RGD motif on the extracellular matrix or cell surface, together with its integrin receptors, constitutes a major recognition system for cell adhesion. There are several erythrocyte major membrane skeletal proteins, e.g., α spectrin, ankyrin, and protein 4.2, that bear an RGD motif. However, it is not known whether the RGD/integrin recognition system is utilized in the erythrocyte-macrophage adhesion during erythrophagocytosis. Here we report that the RGD motif of ankyrin, but not others, is recognized by the α_vβ₃ integrin receptor. In addition, the RGD motif of ankyrin, a peripheral membrane protein, can be externalized onto the cell surface when erythrocytes are incubated with calcium and sheared both at physiological levels. Furthermore, the erythrocyte-macrophage adhesion can be specifically inhibited by ankyrin and/or α_vβ₃. Thus, externalization of ankyrin followed by RGD/integrin recognition may be a novel mechanism by which erythrocytes adhere to macrophages preceding phagocytosis.     

Cyclic RGD peptidomimetics containing 4- and 5-amino-cyclopropane pipecolic acid (CPA) templates as dual αVβ3 and α5β1 integrin ligands

4-Amino- and 5-amino-cyclopropane pipecolic acids (CPAs) with cis relative stereochemistry between the carboxylic and amino groups were used as templates to prepare cyclic peptidomimetics containing the RGD sequence as possible integrin binders. The peptidomimetic c(RGD8) built on the 5-amino-CPA displayed an inhibition activity (IC₅₀ = 2.4 nM) toward the α_vβ₃ integrin receptor (expressed in M21 human melanoma cell line) comparable to that of the most potent antagonists reported so far and it was ten times more active than the corresponding antagonist c(RGD7) derived from the isomeric 4-amino-CPA. Both compounds were also nanomolar ligands of the α₅β₁ integrin (expressed in human erythroleukemia cell line K562). These results suggest that the CPA-derived templates are suitable for the preparation of dual α_vβ₃ and α₅β₁ ligands to suppress integrin-mediated events as well as for targeted drug delivery in cancer therapy.     

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.     

A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Lability and Confer Resistance to Acid-Dependent Uncoating Inhibition

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH₄Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH₄Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH₄Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus in the family Picornaviridae. FMDV displays epithelial tropism and is responsible for a highly contagious disease of cloven-hoofed animals (23, 60). FMDV populations are quasispecies and exhibit a high potential for variation and adaptation, one consequence of which is the extensive antigenic diversity of this virus, reflected in the existence of seven serotypes and multiple antigenic variants (reviewed in references 17 and 60). Different cellular receptors, including α_vβ integrins and heparan sulfate (HS) glycosaminoglycans, have been described for natural isolates and tissue culture-adapted FMDVs (3, 4, 6, 28-31, 56). However, viruses that are infectious in vivo use integrins as receptors (28). The interaction between FMDV and the integrin molecule is mediated by an Arg-Gly-Asp (RGD) triplet located at the G-H loop of capsid protein VP1 (9, 47). FMDV isolates interacting with integrins gain entry into the cell following clathrin-mediated endocytosis (8, 39, 52). On the other hand, it has been described that a genetically engineered HS-binding mutant uses caveolae to enter into cultured cells (51). After internalization, FMDV must release its genomic RNA molecule of positive polarity into the host cell cytoplasm to establish a productive infection. Early work showed that a variety of lysosomotropic agents, such as weak bases and ionophores that block acidification of endosomes, inhibit FMDV infection (5, 11-13), indicating that genome release is dependent on endosomal acidification. In addition, internalized FMDV particles colocalize with markers from early and recycling endosomes (8, 51, 52) and FMDV infection is reduced by expression of a dominant negative mutant of Rab5 (33), suggesting that FMDV may release its genome from these compartments.The FMDV capsid comprises 60 copies of each of the four structural proteins (VP1 to VP4) arranged in an icosahedral lattice of 12 pentameric subunits. FMDV particles are highly acid labile and disassemble at pH values slightly below neutrality (13). Acid lability is not a feature of the capsids of other picornaviruses, such as Enterovirus. Pentameric subunits are intermediates of FMDV assembly and disassembly (64). A high density of His residues is found close to the interpentameric interface. Protonation of these residues at the acidic pH in the endosomes has been proposed to trigger acid-induced capsid disassembly by electrostatic repulsion between the protonated His side chains (1). His 142 (H142) in VP3 of type A FMDV is involved in a His-α-helix dipole interaction, which is likely to influence the acid lability of FMDV (13). In silico predictions suggested that H142 and H145 in VP3 may have the greatest effect on this process (63). Experimental evidence of the involvement of H142 of VP3 in acid-induced disassembly of FMDV has also been reported (20). Concomitantly with capsid disassembly into pentameric intermediates, internal protein VP4 and viral RNA are released. VP4 is a highly hydrophobic and myristoylated protein (7) whose release has been suggested to mediate membrane permeabilization and ion channel formation, thus facilitating the endosomal exit of viral RNA (15, 16, 34).Besides providing information about the endosomal pH requirements for the release of virus genomes, drugs modifying endosomal acidification can reveal the molecular changes associated with viral resistance to their action. These analyses may also address whether the balance between acid lability and capsid stability required for completion of virus replication allows FMDV, which disassembles at a pH close to neutrality, to escape inhibition by drugs raising the endosomal pH. In this work, we have isolated and characterized FMDV mutants that are able to escape from the inhibition of endosomal acidification exerted by NH₄Cl, a lysosomotropic weak base that raises endolysosomal pH and impairs uncoating and infection of viruses that require transit through acidic endosomal compartments for penetration (5, 26, 53). These mutants showed an increased acid lability, which is likely to allow them to uncoat at more-alkaline pH values. A single amino acid substitution close to the interpentameric interfaces in the capsid of one of these mutants was responsible for a total resistance to the elevation in endosomal pH caused by NH₄Cl treatment and for the acid-labile phenotype.     

Insights into binding modes of tumstatin peptide T7 with the active site of αvβ3 integrin

Through interaction with the active site of α_vβ₃ integrin, tumstatin T7 peptide inhibits both the angiogenesis and the proliferation of tumour cells. In this work, docking in conjunction with molecular dynamics simulation was used to explore the binding mode of T7 peptide and α_vβ₃ integrin. The binding mode analysis revealed that the residues Ser⁹⁰, Arg⁹¹, Asp⁹³ and Tyr⁹⁴ in T7 peptide, and (α)-Asp¹⁵⁰, (β)-Arg²¹⁴, (α)-Asp¹⁴⁸ (α)-Gln²¹⁴ and (α)-Glu¹²³ in the active site of α_vβ₃ integrin were most likely the key interaction sites. The hydroxyl of Tyr⁹⁴ coordinates α_vβ₃ via a Mn²⁺ ion, revealing that Mn²⁺ is also an important factor for the interaction. The insight into these key interaction sites not only suggests that the active site of α_vβ₃ integrin can bind to molecules through multiple binding mechanisms, but also provides some useful information for structure-based drug design.     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

Mutations on the Switch III region and the alpha3 helix of Galpha16 differentially affect receptor coupling and regulation of downstream effectors

Background

Gα₁₆ can activate phospholipase Cβ (PLCβ) directly like Gα_q. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα₁₆. Previous studies on Gα_q have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα₁₆ might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.

Results

Alanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα₁₆. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα₁₆ was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G₁₆ and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.

Conclusion

The integrity of the switch III region and α3 helix of Gα₁₆ is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα₁₆ to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G₁₆. The studied region was shown to bear multiple functionally important roles of G₁₆.     

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin

Osteopontin (OPN) is a ubiquitously expressed, multifunctional, and highly phosphorylated protein. OPN contains two neighboring integrin-binding motifs, RGD and SVVYGLR, which mediate interaction with cells. Phosphorylation and proteolytic processing affect the integrin-binding activities of OPN. Here we report that the kinase, FAM20C, phosphorylates Ser¹⁴⁶ in the ¹⁴³RGDSVVYGLR¹⁵² motif of OPN and that Ser¹⁴⁶ is phosphorylated in vivo in human and bovine milk. Ser¹⁴⁶ is located right next to the RGD motif and close by the regulatory thrombin and plasmin cleavage sites in the OPN sequence. Phosphorylation of Ser¹⁴⁶ could potentially affect the proteolytic processing and the integrin-binding activities of OPN. We show that phosphorylation of Ser¹⁴⁶ does not affect the susceptibility of OPN for thrombin or plasmin cleavage. However, phosphorylation of Ser¹⁴⁶ significantly reduces the RGD-mediated interaction with the α_vβ₃ integrin in MDA-MB-435 and Moαv cells. This suggests a new mechanism by which specific phosphorylation of OPN can regulate interaction with the α_vβ₃ integrin and thereby affect OPN-cell interaction.     

Evidence for positive selection in the capsid protein-coding region of the foot-and-mouth disease virus (FMDV) subjected to experimental passage regimens

We present sequence data from two genomic regions of foot-and-mouth disease virus (FMDV) subjected to several experimental passage regimens. Maximum-likelihood estimates of the nonsynonymous-to-synonymous rate ratio parameter (d(N)/d(S)) suggested the action of positive selection on some antigenic sites of the FMDV capsid during some experimental passages. These antigenic sites showed an accumulation of convergent amino acid replacements during massive serial cytolytic passages and also in persistent infections of FMDV in cell culture. This accumulation was most significant at the antigenic site A (the G-H loop of capsid VP1), which includes an Arg-Gly-Asp (RGD) cellular recognition motif. Our analyses also identified a subregion of VP3, part of the fivefold axis of FMDV particles, that also appeared to be subjected to positive selection of amino acid replacements. From these results, we can conclude that under the restrictive conditions imposed either by the presence of the monoclonal antibodies, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in some capsid-coding regions can be positively selected toward an increase of those mutants with a higher capability to infect the cell.     

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin α_IIbβ₃, and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (α_vβ₃, α_vβ₅, and α_IIbβ₃). By comparing K562 cells expressing a common α subunit (Kα_vβ₃, Kα_vβ₅) with cells expressing a common β subunit (Kα_vβ₃, Kα_IIbβ₃), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5–7.5 μg/ml consistently enhanced the adhesion of β₃expressing cells (Kα_vβ₃,Kα_IIbβ₃). In contrast, UH at 0.5–7.5 μg/ml inhibited Kα_vβ₅adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kα_vβ₃cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the β subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.     

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.