Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.     

Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

Of the inhibitory checkpoints in the immune system, programmed death 1 (PD-1) is one of the most promising targets for cancer immunotherapy. The anti-PD-1 antibodies currently approved for clinical use or under development bind to human PD-1 (hPD-1), but not murine PD-1. To facilitate studies in murine models, we developed a functional antibody against both human and murine PD-1, and compared the epitopes of such antibody to a counterpart that only bound to hPD-1. To quickly identify the epitopes of the 2 antibodies, we used alanine scanning and mammalian cell expression cassette. The epitope identification was based on PD-1-binding ELISA and supported by affinity ranking of surface plasmon resonance results. The hPD-1 epitopes of the 2 functional antibodies were also compared with the binding region on hPD-1 that is responsible for PD-L1 interaction. In silico modeling were conducted to explain the different binding modes of the 2 antibodies, suggesting a potential mechanism of the antibody cross-species binding.     

肝癌特异性鼠源及人源化单链抗体基因的克隆与表达

为探讨肝癌特异性鼠源及其人源化单链抗体基因的表达策略并比较二者的结合能力,在3种载体中分别以融合、分泌及胞内表达的方式进行了研究;对复性后的单链抗体以抗原捕获ELISA法进行检测。结果表明,在3种载体中表达的鼠源及人源化单链抗体都是包含体,诱导物浓度及培养温度不影响表达形式;抗原捕获细胞ELISA表明人源化的单链抗体和鼠源单链抗体有相近的抗原结合能力。结论是:在大肠杆菌中表达的基因工程单链抗体的可溶性可能主要由自身氨基酸一级序列决定;先前的设计所采取的人源化方案没有影响到鼠源抗体的CDRs的天然构象,表达的人源化单链抗体提供了免疫原性评价及临床应用的基础。     

Generation and characterization of a murine monoclonal antibody specific for the human T1-CD5 molecule

Murine monoclonal antibodies (MoAbs) have found widespread applications in the characterization of the molecular and functional features of lymphocyte differentiation antigens. The present paper summarizes the results of our work dealing with the production and selection of a murine MoAb recognizing a molecule expressed during the whole differentiative life of T lymphocytes. The MoAb CB01 resulted to be specific for an apparently unique epitope of the T-cell specific membrane glycoprotein T1-CD5.     

Relationship between the structural conformation of monoclonal antibody layers and antigen binding capacity

Neutron reflection has been used to determine the pH-dependent structural conformation of monoclonal antibody layers adsorbed at the hydrophilic silicon oxide/solution interface, within the pH range 4-8, over which the silicon oxide surface carried weak negative charges and the net charge on the antibody reversed. The depth resolution achieved, by use of D2O as solvent to enhance the neutron contrast of the adsorbed antibody layer, was around 2-3 A. The results have been correlated with the ellipsometric measurement of antigen binding capacity (AgBC). The antibody was a mouse monoclonal anti-hCG (human chorionic gonadotropin) directed against the beta subunit of hCG, with molecular weight of 150 000 and isoelectric point around pH 6.0. At pH 4, the adsorbed antibody could be described as a single layer 40 A thick, consistent with an almost perfect flat-on orientation with all three fragments (Fc, Fab) lying flat on the surface. With increasing pH, the antibody layer swelled (65 A at pH 6, 75 A at pH 8) and could be described as three sublayers of different protein density, consistent with some twisting of molecules so that some fragments became more loosely attached to the surface. At pH 8, the repulsive interaction between protein and surface was reflected in a significantly decreased total adsorbed amount. The dominant effect acting to increase AgBC was decreased surface packing density. The effect of the conformational changes revealed at different pH was less important. The results have shown that within the flat-on orientation adopted by the adsorbed antibody, steric hindrance is the main constraint on binding, restricting the access of the antigen to active sites within the antibody layer.     

Guided selection of human antibody light chains against TAG-72 using a phage display chain shuffling approach

To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.     

Affinity purification and structural characterization of a specific binding protein for human growth hormone in human serum

A highly specific binding protein for human growth hormone (hGH) has been isolated from human serum by hGH-affinity chromatography. A purification of approximately 1500-fold with a 30-40% recovery was obtained with essentially no alteration in binding characteristics. Covalent cross-linking of 125I-hGH to the binding protein, followed by analysis by SDS-polyacrylamide gel electrophoresis and autoradiography, revealed two specifically labeled complexes. Allowing for a 1:1 binding stoichiometry the binding proteins themselves had mean mol wts of 57,000 and 69,300. These increased slightly to mol wt 60,300 and 72,000 respectively in the presence of 100 mM dithiothreitol, suggesting the presence of intramolecular but not inter-subunit disulfide linkages. These data confirm the presence of the hGH binding protein(s) in human serum and define their gross structural nature.     

Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class.The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.     

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin

We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.     

Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv–pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC–CLEIA (chemiluminescent enzyme immunoassay). The IC₁₅ was 0.012 ± 0.02 μg/L, and the IC₅₀ was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv–antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.     

Identification of novel members reveals the structural and functional divergence of lepidopteran-specific Lipoprotein_11 family

30K proteins (30KPs) are classified into the lepidopteran-specific Lipoprotein_11 family. They are involved in various physiological processes such as energy storage, embryonic development, and immune response in the silkworm. To date, 30KPs were only found in Bombyx mori and Manduca sexta. Moreover, the C-termini of ENF peptide binding proteins (ENF-BPs) show similarity to 30KPs. ENF peptides are multifunctional insect cytokines and involved in growth regulation and defense reaction, whereas ENF-BPs act as active regulators of ENF peptides. In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified 73 30KP homologous genes in 12 lepidopteran species, of which 56 are novel members. The structural and phylogenetic analyses revealed that these genes could be classified into three groups: ENF-BP genes, typical 30KP genes, and serine/threonine-rich 30KP (S/T-rich 30KP) genes. The C-terminal regions are common to all the three subfamilies, but the N-termini are highly variable. We found a novel subfamily of Lipoprotein_11 and named it S/T-rich 30KP according to its exclusive S/T-rich domain in the N terminus. ENF-BP was also found to contain a special domain in the N terminus, which is homologous to Pp-0912 of Pseudomonas putida. Microarray data and semi-quantitative RT-PCR showed that the three groups have their respective temporal–spatial expression patterns. S/T-rich 30KP genes have enriched expression in the mature testis and might be involved in spermiogenesis or fertilization. Typical 30KP genes are expressed mainly in the fat body and integument at the larvae and pupae stages. ENF-BP genes are expressed predominantly in the hemocyte. The differential spatial–temporal expression profiles revealed the functional divergence of three Lipoprotein_11 subfamilies.     

Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1

Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.     

Preparation of polyclonal antibody specific for NOR1 and detection of its expression pattern in human tissues and nasopharyngeal carcinoma

Oxidored-nitro domain containing protein 1 （NORI） gene is a novel nitroreductase gene first isolated from nasopharyngeal carcinoma （NPC）. It plays an important role in the formation of chemical carcinogen and the carcinogenesis of NPC for its nitrosation function. Overexpression of the wild-type NOR1 gene in nasopharyngeal carcinoma cells is effective to inhibit cell growth and proliferation. In this study, for the first time, we generated a highly specific NOR1 antibody and analyzed NOR1 distribution in the human tissues and NPC biopsies. The results showed that NOR1 protein is predominantly expressed in human nasopharynx and tracheal tissues. Human heart, liver, spleen, stomach, colon, kidney, skeletal muscle, thymus, and pancreas are all deficient of NOR1 protein. More importantly, we performed immunohistochemistry assay of NOR1 protein expression in the NPC tissues, and the result showed that NOR1 protein is frequently down-expressed in NPC. These data shed light on the selectivity of potential physiological functions of NOR1 and provides an indispensable reference to the carcinogenesis process of NPC and to identify or validate tissue-specific drug targets.     

Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

Background  

Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA.     

A selection index for gene expression evolution and its application to the divergence between humans and chimpanzees

The importance of gene regulation in animal evolution is a matter of long-standing interest, but measuring the impact of selection on gene expression has proven a challenge. Here, we propose a selection index of gene expression as a straightforward method for assessing the mode and strength of selection operating on gene expression levels. The index is based on the widely used McDonald-Kreitman test and requires the estimation of four quantities: the within-species and between-species expression variances as well as the sequence heterozygosity and divergence of neutrally evolving sequences. We apply the method to data from human and chimpanzee lymphoblastoid cell lines and show that gene expression is in general under strong stabilizing selection. We also demonstrate how the same framework can be used to estimate the proportion of adaptive gene expression evolution.     

Mapping the interaction between murine IgA and murine secretory component carrying epitope substitutions reveals a role of domains II and III in covalent binding to IgA.

We have identified sites for epitope insertion in the murine secretory component (SC) by replacing individual surface-exposed loops in domains I, II, and III with the FLAG sequence (Crottet, P., Peitsch, M. C., Servis, C., and Corthésy, B. (1999) J. Biol. Chem. 274, 31445-31455). We had previously shown that epitope-carrying SC reassociated with dimeric IgA (IgA(d)) can serve as a mucosal delivery vehicle. When analyzing the capacity of SC mutants to associate with IgA(d), we found that all domain II and III mutants bound specifically with immobilized IgA(d), and their affinity for IgA(d) was comparable to that of the wild type protein (IC(50) approximately 1 nM). We conclude that domains II and III in SC are permissive to local mutation and represent convenient sites to antigenize the SC molecule. No mutant bound to monomeric IgA. SC mutants exposing the FLAG at their surface maintained this property once bound to IgA(d), thereby defining regions not required for high affinity binding to IgA(d). Association of IgA(d) with SC mutants carrying a buried FLAG did not expose de novo the epitope, consistent with limited, local changes in the SC structure upon binding. Only wild type and two mutant SCs bound covalently to IgA(d), thus implicating domains II and III in the correct positioning of the reactive cysteine in SC. This establishes that the integrity of murine SC domains II and III is not essential to preserve specific IgA(d) binding but is necessary for covalency to take place. Finally, SC mutants existing in the monomeric and dimeric forms exhibited the same IgA(d) binding capacity as monomeric wild type SC known to bind with a 1:1 stoichiometry.     

Establishment of the human hepatocellular carcinoma cell line and its characteristics

c-Hc-4 has been established and maintained for more than seven years. The hepatocellular carcinoma originated in 45-year old man with liver cirrhosis. The cell grew in vitro forming a sheet of monolayered cells and firmly attaching to the inner surface of cultured flasks. Morphologically they showed epithelial-like pattern. The doubling time was about 20 hours. Their modal chromosome number was 58. Serial heterologous transplantation in nude mice was successful. The histological finding was almost the same patterns as those in the primary tumor. The cultured cells produced alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA).