Structural basis for the blockage of IL-2 signaling by therapeutic antibody basiliximab.

IL-2 signaling plays a central role in the initiation and activation of immune responses. Correspondingly, blockage of this pathway leads to inhibition of the immune system and would provide some therapeutic benefits. Basiliximab (Simulect), a therapeutic mAb drug with specificity against IL-2R alpha of T cells, was approved by U.S. Food and Drug Administration in 1998. It has been proven to be effective in the suppression of the IL-2 pathway and hence has been widely used to prevent allograft rejection in organ transplantation, especially in kidney transplants. In this study, we report the crystal structure of the basiliximab Fab in complex with the ectodomain of IL-2R alpha at 2.9 A resolution. In the complex structure, the Fab interacts with IL-2R alpha with extensive hydrophobic and hydrophilic interactions, accounting for a high binding affinity of 0.14 nM. The Ag binding site of basiliximab consists of all six CDR loops that form a large binding interface with a central shallow hydrophobic groove surrounded by four hydrophilic patches. The discontinuous epitope is composed of several segments from the D1 domain and a minor segment from the D2 domain that overlap with most of the regions responsible for the interactions with IL-2. Thus, basiliximab binding can completely block the interactions of IL-2 with IL-2R alpha and hence inhibit the activation of the IL-2 signal pathway. The structural results also provide important implications for the development of improved and new IL-2R alpha-targeted mAb drugs.

Efalizumab binding to the LFA-1 alphaL I domain blocks ICAM-1 binding via steric hindrance.

Lymphocyte function-associated antigen 1 (LFA-1) plays important roles in immune cell adhesion, trafficking, and activation and is a therapeutic target for the treatment of multiple autoimmune diseases. Efalizumab is one of the most efficacious antibody drugs for treating psoriasis, a very common skin disease, through inhibition of the binding of LFA-1 to the ligand intercellular adhesion molecule 1 (ICAM-1). We report here the crystal structures of the Efalizumab Fab alone and in complex with the LFA-1 alpha(L) I domain, which reveal the molecular mechanism of inhibition of LFA-1 by Efalizumab. The Fab binds with an epitope on the inserted (I) domain that is distinct from the ligand-binding site. Efalizumab binding blocks the binding of LFA-1 to ICAM-1 via steric hindrance between its light chain and ICAM-1 domain 2 and thus inhibits the activities of LFA-1. These results have important implications for the development of improved antibodies and new therapeutic strategies for the treatment of autoimmune diseases.

Structural modeling and biochemical studies reveal insights into the molecular basis of the recognition of beta-2-microglobulin by antibody BBM.1.

Human beta-2-microglobulin (beta2m) is the light chain of human leucocyte antigen-I (HLA-I). It can disassociate from HLA-I and accumulate to cause serious dialysis-related amyloidosis (DRA) in long-term hemodialysis patients. Monoclonal antibody (mAb) BBM.1 can recognize both free-form and HLA-I associated beta2m. It can be used for specific elimination of beta2m from serum and can induce apoptosis of several types of tumor cells, and thus has great therapeutic potential. In this study, we constructed structural models of the BBM.1 Fv (fragment of the variable domain) and the BBM.1 Fv-beta2m complex, followed by biochemical evaluation. Analysis of the optimal complex model reveals that the previously identified immunodominant residues Glu(44) and Arg(45) of beta2m have direct interactions with BBM.1, while Asp(38) exerts its function mainly via stabilization of Arg(45). In addition, Arg(81) of beta2m is a newly identified immunodominant residue to have direct interaction with BBM.1. Further modeling study shows no steric conflict between the antibody and the HLA-I heavy chain. These results provide insights into the molecular basis of the recognition of beta2m by BBM.1 and explain why BBM.1 can bind both free-form and HLA-1 associated beta2m. This information could be exploited in the engineering and improvement of BBM.1 and the development of other beta2m-targeting mAbs for therapeutic purposes.

Crystal structure of chimeric antibody C2H7 Fab in complex with a CD20 peptide.

Anti-CD20 monoclonal antibodies have been proven to be efficient in the treatment of certain B-cell lymphomas and autoimmune diseases. Intriguingly, these antibodies seem to exert diverse functions with narrow epitope specificity. This study is to investigate the molecular basis of the fine specificity of 2H7 derived antibodies which are of great therapeutic potential. We show that chimeric 2H7 (C2H7) can mediate complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity effects on CD20 positive human Burkitt lymphoma cells and the Fab fragment can well recognize and bind to an epitope peptide of the extracellular loop of CD20. The crystal structure of C2H7 in complex with the CD20 epitope peptide was determined at 2.6A resolution. The bound peptide displays a circular conformation and the binding specificity is mainly contributed by the (170)ANPS(173) motif and the disulfide bond of the peptide which maintains the unique conformation of the peptide. Compared with the complex structure of another anti-CD20 monoclonal antibody Rituximab with the same epitope peptide which was previously determined, the major differences lie in the CDR loop H3 of C2H7 which stretches outward against the interface. Correspondingly, the pocket which accommodates the peptide becomes wider and the peptide moves toward loop H3 and thus is more distant from loops H1 and H2. The hydrogen-bonding interactions are also quite different from those observed in the Rituximab-epitope peptide complex, and both the hydrophilic and hydrophobic interactions are less intense. Our data not only reveal the molecular basis for the fine specificity of C2H7 to CD20, but also provide valuable information for further improvement of antibodies derived from 2H7.

The vaccines-associated Arthus reaction.

The Arthus reaction is a rare adverse reaction that usually occurs after vaccination with large and more severe local reactions, belonging to type Ⅲ hypersensitivity reaction. This reaction is characterized by pain, swelling, induration (Tissue that becomes firm) and edema, even accompanied by severe necrosis or ulceration at the injection sites. However, most of mild cases generally can be cured without treatment, and only severe cases need to be treated with anti-allergy. Therefore, this adverse reaction is often ignored by people.We searched PubMed, Web of Science and Chinese database (CNKI database and Wan Fang database) for published studies using the terms "Arthus reaction" or "Arthus phenomenon", combined with "vaccine", with no date or language restrictions for all publications before January 28, 2019. Only 30 cases of Arthus reaction were found, of which only one case died.4 cases of Arthus reaction post-dose-1 were reported in the review. The proportion of Arthus reaction occurred after the first, second and third injections in those case reports was 13.3%, 50.0%, and 23.3%, respectively. Arthus reaction was determined according to the clinical symptoms (The symptoms which were observed by the researchers, such as red, swelling and painful with itching at or around the injection sites). The specific causes of Arthus reaction after one dose of vaccination are not described in detail in literatures. Therefore, it could be hypothesized that the case has a pre-existing specific IgG (Such as pre-existing antibody, etc.) to cause the Arthus reaction.And 17 reported cases were observed in children younger than 6 y. In addition, we collected only 18 cases of bacterial vaccine-induced Arthus reaction and 12 cases of viral vaccines. However, there are no other data (Such as the total number and incidence rate of vaccination) in literatures, so we cannot compare statistically significant differences. At presents, no previous reviews of vaccine-induced Arthus reaction have been found. Thus, a systematic review about vaccine-associated Arthus reaction is urgently needed to deepen people's understanding and concern of this phenomenon. In this manuscript, we retrospectively reviewed the description of the discovery process and mechanisms of Arthus reaction, a description of the characteristics of Arthus reaction cases, reporting the Arthus reaction cases in China during 2010-2015, diagnostic criteria and general treatment, preventive measures of Arthus reaction, and challenges remaining to be investigated in the future.

Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site.

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

Risk factors of fatigue status among Chinese adolescents.

In recent years, fatigue is common among adolescents. The aim of this study is to evaluate fatigue status and find related factors of fatigue among students ranged from 13-26 years from Wuhu, China. This is a case-control, cross-sectional observational study. The students from six middle schools (high school? 26 years old?) in Wuhu city were recruited, Self-Rating Fatigue Scale (SFS) was used to measure the fatigue status among students ranged from 13-26 years, and some demographic characteristics of students also was determined. A total of 726 students are included in our study. A significant difference was observed between fatigue status and grade, a balanced diet, the partial eclipse, picky for food, lack of sleep, excessive fatigue, drinking (P < 0.05). The risk factors of fatigue status include myopia, partial eclipse, picky for food, lacking of sleep, drinking; grade while a balanced diet is the protective factor of fatigue. Therefore, the school should pay more attention to the fatigue among students in middle school in China, and take some properly measures to reduce the fatigue.

The Sequence Analysis and Expression of cDNA of Human Cardiac Myosin Light Chain 1 and the Preparation of Monoclonal Antibody to the Expressed Product.

The nucleotide sequence of cDNA of ventricular myosin light chain 1 of Chinese patients was analyzed. Two remarkable differences in deduced amino acid sequence were found by comparison with amino acid sequence reported previously by Jackowski. The cDNA was expressed in E.coli and the expressed product was used for production of specific polyclonal and monoclonal antibodies. Using both of the poly- and monoclonal antibodies, as well as the expressed product, diagnosis kits for Acute Myocardial infarction was constructed (China patent application number 98 122066.5). Detailed studies on physiological function of cardiac myosin light chain 1 is under way in our lab.

Binding of Human Cardiac Myosin Light Chain 1 to Heavy Chain and Actin.

The cloning and sequence analysis of Chinese human cardiac myosin light chain 1 (CCMLC1) was previously reported. In this paper the cDNA of CCMLC1 was used as template and both of cDNAs of N and C terminal fragments of CCMLC1, each containing 98 amino acid residues, were obtained by PCR. Using the expressed products of both fragments, the binding experiments of two fragments to cardiacmyosin heavy chain of rat, human cardiac actin and to monoclonal antibody raised against CCMLC1, have been performed, respectively, by means of precipitation with GST-Sepharose beads. The results showed that all the heavy chain, actin and monoclonal antibody bound the N terminal fragment of CCMLC1 at different sites. Under experimental conditions, the binding of CCMLC1 with actin could affect the subsequent binding of CCMLC1 to heavy chain in topologically.

Localization of PS9 in Rice Ragged Stunt Oryzavirus and Its Role in Virus Transmission by Brown Planthopper.

A cDNA of rice ragged stunt oryzavirus(RRSV) coding for its protein PS9 was prepared and expressed in E.coli as a fusion protein then purified and cleaved by factor-Xa to a 38kD polypeptide. Using the IgG of the antiserum raised against the expressed fusion protein the gold immuno-labelling experiments provided a direct evidence that the 38kD polypeptide is one of the major proteins forming the virus spikes. Virus transmission experiments in brown planthopper fed with PS9 showed the inhibition of transmission of virus by the PS9 protein suggesting that the spike proteins of the virus may be essential for the virus infection.

Prevalence of obesity among secondary school students from 2009 to 2014 in China: a meta-analysis.

BACKGROUND: In recent years, obesity was a major public health problem in many countries. It was estimated that 8% of their children are obese. However, little is known about the overall prevalence of obesity among secondary students in China, the aim of this study was to evaluate the overall obesity prevalence of student from Chinese secondary school. METHODS: Publications from 2009 to 2014 on the obesity prevalence among secondary school students in China were retrieved from PubMed, online Chinese periodical full-text databases of VIP, CNKI and Wan fang. Meta Analyst was used analyze the total rates of obesity for Chinese secondary school. RESULTS: After evaluation of the quality of the articles, 32 papers were finally included in our study, and the total sample sizes on the obesity investigation were 218317 (107631 male and 110686 female), in which 27455(14865 male and 12590 female) were obesity. Meta-analyst findings showed that the pooled prevalence of obesity in secondary school students are 8.4 %( 95% CI: 6.2%- 11.3%) and 4.8 %( 95% CI: 3.2%-7.2%) for boy and girl respectively. CONCLUSION: Our results suggest that school and government related department should pay more attention to the obesity among secondary school students in China, and take some properly measures should to curve the trend growth of obesity.

Antecedentes: En los últimos años, la obesidad es un importante problema de salud pública en muchos países. Se calcula que el 8% de los niños son obesos.Sin embargo, poco se sabe acerca de la prevalencia de la obesidad entre los alumnos de secundaria en China, el objetivo de este estudio fue evaluar la prevalencia de la obesidad en general chino, estudiante de la escuela secundaria. Métodos: Publicaciones de 2009 a 2014 en la prevalencia de obesidad entre los estudiantes de la escuela secundaria en China fueron recuperados de PubMed, Online Chinese periódico de bases de datos de texto completo de VIP, CNKI y Wan Fang.Analista del meta fue utilizado analizar el total de las tasas de obesidad para chinos de la escuela secundaria. Resultados: Después de la evaluación de la calidad de los artículos, 33 papeles fueron finalmente incluidos en nuestro estudio, y el total de los tamaños de muestra sobre la obesidad investigación fueron 218317 (107631 macho y 110686 hembra), en la que 27455 (14863 macho y 12590 mujeres) fueron la obesidad.Los resultados mostraron que el analista del meta la prevalencia de la obesidad en estudiantes de secundaria son 8,4% (IC del 95%: 6,2% - 11,3%) y 4,8% (IC del 95%: 3,2% - 7,2%) para chico y chica, respectivamente. Conclusión: Nuestros resultados sugieren que la escuela y Gobierno related Departamento debería prestar más atención a la obesidad entre los estudiantes de la escuela secundaria en China, y tomar algunas medidas adecuadamente a la curva de la tendencia de crecimiento de la obesidad.

Structural basis for recognition of CD20 by therapeutic antibody Rituximab.

Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-A resolution. The combining site of the Fab consists of four complementarity determining regions that form a large, deep pocket to accommodate the epitope peptide. The bound peptide assumes a unique cyclic conformation that is constrained by a disulfide bond and a rigid proline residue (Pro(172)). The (170)ANPS(173) motif of CD20 is deeply embedded into the pocket on the antibody surface and plays an essential role in the recognition and binding of Rituximab. The antigen-antibody interactions involve both hydrogen bonds and van der Waals contacts and display a high degree of structural and chemical complementarity. These results provide a molecular basis for the specific recognition of CD20 by Rituximab as well as valuable information for development of improved antibody drugs with better specificity and higher affinity.

Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.