Identification of CD21-binding peptides with phage display and investigation of binding properties of HPMA copolymer-peptide conjugates

Cancer targeting with peptides has become promising with the emergence of combinatorial peptide techniques such as phage display. Using phage display under stringent screening conditions, we selected five distinct peptides that specifically recognized the CD21 receptor, a cell surface marker of malignant B cell lymphoma. Two highly hydrophobic sequences were excluded (RLAYWCFSGLFLLVC and PVAAVSFVPYLVKTY). The binding affinity toward CD21 of the other three selected peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, and GRVPSMFGGHFFFSR) was analyzed with fluorescence quenching. Their dissociation constants were determined to be within the micromolar range. On the basis of the results of phage ELISA, competitive phage ELISA, and fluorescence quenching, the binding sites of the three selected peptides were found to reside within the first four short consensus repeats of CD21 (SCR1-4). The peptide RMWPSSTVNLSAGRR (P1) was bound to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, a potential drug carrier for chemotherapeutic agents, and the surface binding properties of HPMA copolymer-P1 conjugates were investigated. Specific interactions were observed between HPMA copolymer-P1 conjugates and surface-bound receptor. Binding of HPMA copolymer-P1 conjugates was directly related to the amount of surface (MaxiSorp plate) bound receptor, and the binding of the conjugates could be inhibited by the application of a 3-4 orders-of-magnitude excess of free peptide over the peptide concentration in conjugates. The enhanced binding of polymer-bound peptide was ascribed to multivalent interactions between the HPMA copolymer-P1 conjugate and the surface-bound CD21 receptor.     

Discovery of high-affinity peptide ligands for vancomycin

Vancomycin, an important antibiotic against medically relevant gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, exerts its antibacterial effects by binding with moderate affinity to the C-terminal Lys-D-Ala-D-Ala motif (Kaa) of the bacterial cell wall peptide precursor. Essential for Kaa binding to vancomcyin is the free-carboxyl group on the terminal D-Ala in Kaa. In efforts to identify other Kaa-based peptides which bind vancomycin with higher affinity, we utilized our one-bead-one-compound (OBOC) combinatorial library approach, a method which has been widely used to discover highly specific ligands against various receptors. In standard OBOC peptide libraries, the C-terminal end of the synthesized peptide is tethered to a solid-support/resin, however, this study reports development of a synthetic strategy for generating OBOC peptide libraries with a free D-Ala-D-Ala carboxyl end. We screened these "OBOC inverted" peptide libraries against vancomycin, and discovered a series of peptide ligands with strong consensus, which bind vancomycin. To further optimize these ligands, two highly focused Kaa-containing OBOC combinatorial peptidomimetic libraries were designed, synthesized, and screened against vancomycin under more stringent conditions. Peptidomimetic ligands which bind vancomycin with higher affinity than Kaa were identified. The dissociation constant of one of these ligands, Lys(Ac)-HOCit-Glu-Cha-Lys(3,5-dihydroxybenzoyl)-D-Ala-D-Ala (9), as determined by surface plasmon resonance, was 1.03 microM, roughly a 50-fold improvement in affinity compared to Kaa (K(D) = 50 microM).     

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.     

A Convenient Approach to Prepare Topologically Segregated Bilayer Beads for One-Bead Two-Compound Combinatorial Peptide Libraries

One-bead one-compound (OBOC) combinatorial peptide libraries have been used to identify ligands and modulators for a wide variety of biological targets. While being very efficient with linear peptides, OBOC libraries with N-terminally blocked peptides or with unsequenceable building blocks require encoding. To fully exploit OBOC combinatorial methods with cyclic peptides and peptidomimetics, topologically segregated bilayer beads have been developed. This strategy offers the opportunity to synthesize two compounds per bead, i.e. with one compound exposed on the bead surface for screening, and the other one found within the inner layer as a tag for sequencing and compound identification. Bead segregation often involves the use of unstable derivatives or requires a series of protection–deprotection steps. In order to expedite and optimize bead segregation, the performance of various reagents has been studied. The results obtained herein show that bead segregation can be efficiently performed with commercially available reagents. Finally, in order to control outer/inner layer ratios in segregated beads, the effects of different parameters have been evaluated. We report a straightforward and efficient procedure to prepare topologically segregated bilayer beads in a wide range of controllable, predictable, and reproducible outer versus inner ratios.     

Screening one bead one compound libraries against serum using a flow cytometer: Determination of the minimum antibody concentration required for ligand discovery

One bead one compound (OBOC) libraries can be screened against serum samples to identify ligands to antibodies in this mixture. In this protocol, hit beads are identified by staining with a fluorescent labeled secondary antibody. When screens are conducted against two different sets of serum, antibodies, and ligands to them, can be discovered that distinguish the two populations. The application of DNA-encoding technology to OBOC libraries has allowed the use of 10?µm beads for library preparation and screening, which pass through a standard flow cytometer, allowing the fluorescent hit beads to be separated from beads displaying non-ligands easily. An important issue in using this approach for the discovery of antibody biomarkers is its analytical sensitivity. In other words, how abundant must an IgG be to allow it to be pulled out of serum in an unbiased screen using a flow cytometer? We report here a model study in which monoclonal antibodies with known ligands of varying affinities are doped into serum. We find that for antibody ligands typical of what one isolates from an unbiased combinatorial library, the target antibody must be present at 10–50?nM. True antigens, which bind with significantly higher affinity, can detect much less abundant serum antibodies.     

高通量筛选孤儿受体和膜结合配体的MACS/FACS方法的建立

本文报道了一种用于分离、筛选孤儿受体和膜结合配体的高通量的表达克隆系统(expression cloning system).该系统是以高效表达的逆转录病毒载体为核心,以高通量的磁力分离法（magnetic cell sorter,MACS）和高精确度分选型流式细胞仪（FACS）相结合的独特的筛选方法.具体方法是将配体通过生物反应对固定在磁粒上 （如biotin/streptavidin, Fc/protein A等）.该磁粒的特点是非常微小,相当一个病毒颗粒.当配体蛋白结合到表达受体的BaF3细胞上时,该细胞将停留在MACS磁场中,从而与非受体表达细胞分开.MACS的优点是效率高,可同时操作上亿细胞.这些细胞是无法直接在分选型流式细胞仪上操作的.当这些阳性细胞增殖后,再次用同一配体染色,用更精确的分选型流式细胞仪分离,直至完全纯化.受体基因将用PCR方法,用载体引物克隆.利用该系统可以从1000 000个细胞中筛选出低至2个表达配体/受体的阳性细胞.为验证该系统的可行性,应用该系统在相应的cDNA文库中筛选到2个已知基因的细胞受体.应用诱饵B7-1从人T淋巴细胞cDNA文库中筛选其功能受体CD28.应用B7-H2作为诱饵,从活化的人T淋巴细胞cDNA文库中筛选到ICOS.最终,从纯化的受体表达细胞中分离出受体的cDNA编码序列.这些结果表明,改进的表达克隆系统适合于大规模分离孤儿受体和膜结合配体,以及在后基因组时代用于研究新蛋白的功能.     

Water-soluble polymer–drug conjugates for combination chemotherapy against visceral leishmaniasis

There is a need for new safe, effective and short-course treatments for leishmaniasis; one strategy is to use combination chemotherapy. Polymer–drug conjugates have shown promise for the delivery of anti-leishmanial agents such as amphotericin B. In this paper, we report on the preparation and biological evaluation of polymer–drug conjugates of N-(2-hydroxypropyl)methacrylamide (HPMA), amphotericin B and alendronic acid. The combinatorial polymer–drug conjugates were effective anti-leishmanial agents in vitro and in vivo, but offered no advantage over the single poly(HPMA)–amphotericin B conjugates.     

Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRalphabeta NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Valpha14i-positive and approximately 50% of Valpha14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Valpha14i-negative than Valpha14i-positive NKT cells. In spleen only, subsets of Valpha14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-gamma/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4(-) Valpha14i-negative NKT cells that were L-selectin(high) and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Valpha14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1(+) Valpha14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Valpha14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4(+) or CD4(-)CD8(-) and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Valpha14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.     

CD45RA T-cell activation without proliferation by a partial agonist monoclonal antibody to beta1 integrin

CD45RA T cells are fully co-activated by natural beta1 integrin ligands fibronectin (FN) and VCAM-1, as well as monoclonal antibody (mAb) 19H8, which binds a combinatorial epitope of the alpha4beta1 heterodimer. These integrin ligands stimulate CD3-dependent proliferation and the upregulation of early activation markers CD25 and CD69. However, beta1-specific antibody 33B6, which binds to a similar range of the predominant T-cell integrins as natural ligands FN (alpha4beta1 and alpha5beta1) and VCAM-1 (alpha4beta1), failed to costimulate proliferation in the CD45RA subset, while retaining the ability to costimulate early activation markers CD25 and CD69. After addition of exogenous human interleukin-2 to the culture media, 33B6 costimulation of proliferation is restored. These data provide evidence that a branch of the alpha4beta1 integrin-signaling pathway in CD45RA T cells can be independently regulated and exploited through the use of partial agonist ligands, including mAbs to the integrin heterodimer.     

Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide.

Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes. Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides. Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used. Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently. At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike. Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature. Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.     

Probing the structure and function of human glutaminase-interacting protein: a possible target for drug design

PDZ domains are one of the most ubiquitous protein-protein interaction modules found in living systems. Glutaminase interacting protein (GIP), also known as Tax interacting protein 1 (TIP-1), is a PDZ domain-containing protein, which plays pivotal roles in many aspects of cellular signaling, protein scaffolding and modulation of tumor growth. We report here the overexpression, efficient refolding, single-step purification, and biophysical characterization of recombinant human GIP with three different C-terminal target protein recognition sequence motifs by CD, fluorescence, and high-resolution solution NMR methods. It is clear from our NMR analysis that GIP contains 2 alpha-helices and 6 beta-strands. The three target protein C-terminal recognition motifs employed in our interaction studies are glutaminase, beta-catenin and FAS. This is the first report of GIP recognition of the cell surface protein FAS, which belongs to the tumor necrosis factor (TNF) receptor family and mediates cell apoptosis. The dissociation constant ( K D) values for the binding of GIP with different interacting partners as measured by fluorescence spectroscopy range from 1.66 to 2.64 microM. Significant chemical shift perturbations were observed upon titration of GIP with above three ligands as monitored by 2D {(1)H, (15)N}-HSQC NMR spectroscopy. GIP undergoes a conformational change upon ligand binding.     

Identification of novel beta1 integrin binding sites in the type 1 and type 2 repeats of thrombospondin-1

In addition to the three known beta(1) integrin recognition sites in the N-module of thrombospondin-1 (TSP1), we found that beta(1) integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-beta(1)-specific. Adhesion of cells that express one dominant beta(1) integrin on immobilized type 1 repeats is specifically inhibited by antagonists of that integrin, whereas adhesion of cells that express several beta(1) integrins is partially inhibited by each alpha-subunit-specific antagonist and completely inhibited by combining the antagonists. beta(1) integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-beta(1) specificity and divalent cation dependence for promoting cell adhesion. Adhesion to the type 2 repeats is less sensitive to alpha-subunit antagonists, but a beta(1) blocking antibody and two disintegrins inhibit adhesion to immobilized type 2 repeats. beta(1) integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and beta(1) integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. The widely used TSP1 function blocking antibody A4.1 binds to a site in the third type 2 repeat. A4.1 proximally inhibits beta(1) integrin-dependent adhesion to the type 2 repeats and indirectly inhibits integrin-dependent adhesion mediated by the TSP1 type 1 repeats. Although antibody A4.1 is also an antagonist of CD36 binding to TSP1, these data suggest that some biological activities of A4.1 result from antagonism of these novel beta(1) integrin binding sites.     

Genomic structure, alternative splicing, and physical mapping of the killer cell lectin-like receptor G1 gene (KLRG1), the mouse homologue of MAFA

The mouse killer cell lectin-like receptor G1 (KLRG1), the mouse homologue of the mast cell function-associated antigen (MAFA), is an inhibitory C-type lectin expressed on natural killer (NK) cells and activated CD8 T cells. Here we report the complete nucleotide sequence, alternatively spliced variants, and the physical mapping of the KLRG1 gene in the mouse. The gene spans about 13 kb and consists of five exons. Short interspersed repeats of the B1 and B2 family, a LINE-1-like element, and a (CTT)170 triplet repeat were found in intron sequences. In contrast to human KLRG1 and to the murine KLR family members, mouse KLRG1 locates outside the NK complex on Chromosome 6 between the genes encoding CD9 and CD4.     

Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens

The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.     

Insertion of host-derived costimulatory molecules CD80 (B7.1) and CD86 (B7.2) into human immunodeficiency virus type 1 affects the virus life cycle

Human immunodeficiency virus type 1 (HIV-1) carries virus-encoded and host-derived proteins. Recent advances in the functional characterization of host molecules inserted into mature virus particles have revealed that HIV-1 biology is influenced by the acquisition of host cell membrane components. The CD28/B7 receptor/ligand system is considered one of the fundamental elements of the normal immune response. Two major cell types that harbor HIV-1 in vivo, i.e., monocytes/macrophages and CD4+ T cells, express the costimulatory molecules CD80 (B7.1) and CD86 (B7.2). We investigated whether CD80 and CD86 are efficiently acquired by HIV-1, and if so, whether these host-encoded molecules can contribute to the virus life cycle. Here we provide the first evidence that the insertion of CD80 and CD86 into HIV-1 increases virus infectivity by facilitating the attachment and entry process due to interactions with their two natural ligands, CD28 and CTLA-4. Moreover, we demonstrate that NF-kappaB is induced by CD80- and CD86-bearing virions when they are combined with the engagement of the T-cell receptor/CD3 complex, an event that is inhibited upon surface expression of CTLA-4. Finally, both CD80 and CD86 were found to be efficiently incorporated into R5- and X4-tropic field strains of HIV-1 expanded in cytokine-treated macrophages. Thus, besides direct interactions between the virus envelope glycoproteins and cell surface constituents, such as CD4 and some specific chemokine coreceptors, HIV-1 may attach to target cells via interactions between cell-derived molecules incorporated into virions and their natural ligands. These findings support the theory that HIV-1-associated host proteins alter virus-host dynamics.     

The CD24 antigen discriminates between pre-B and B cells in human bone marrow.

When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).     

Recognition of alpha 2-macroglobulin by the low density lipoprotein receptor-related protein requires the cooperation of two ligand binding cluster regions

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*) and amyloid precursor protein, two ligands genetically linked to Alzheimer's disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as alpha(2)M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha(2)M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of (125)I-labeled alpha(2)M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for alpha(2)M*, and they suggest a strategy for developing specific inhibitors to block alpha(2)M* binding to LRP by identifying molecules capable of binding repeats in cluster I.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha-helix and beta-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)