Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.     

Insight into the free-radical-scavenging mechanism of hydroxyl-substituent Schiff bases in the free-radical-induced hemolysis of erythrocytes

This work aimed to explore the mechanism by which hydroxyl-substituent Schiff bases scavenge free-radicals. Thus, four Schiff bases, that is benzylidene aniline (BAN), 2-(phenyliminomethyl)phenol (BAH), 4-benzimidoylphenol (PBH) and 2-benzimidoylphenol (OBH), were applied to protect human erythrocytes against 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced hemolysis. The results revealed that the --OH attached to the ortho-position of methylene in Schiff base scavenges 1.46 radicals per molecule, the --OH attached to the para-position of the N atom scavenges 2.94 radicals and the --OH attached to the ortho-position of the N atom scavenges 3.63 radicals. In addition, four Schiff bases were used together with some familiar antioxidants, such as 6-hydroxyl-2,5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox), L-ascorbic acid (VC), alpha-tocopherol (TOH) and L-ascorbyl-6-laurate (VC-12) in AAPH-induced hemolysis of erythrocytes. It was found that, except for BAN+VC-12, BAH + VC-12, OBH + VC-12 and PBH+TOH, all the other combinations protected erythrocytes more perfectly than when used individually. This result demonstrated that a promotive protection existed between Schiff base and other antioxidants and this improved their ability to scavenge free-radicals. Finally, IC(50) values of the aforementioned Schiff bases together with 2-((o-hydroxylphenylimino) methyl)phenol (OSAP) and 2-((p-hydroxylphenylimino)methyl)phenol (PSAP) were determined by reaction with two radical species, that is, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS(+.)) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH). The results implied that the molecular framework of a Schiff base and an --OH attached to the ortho-position of methylene were apt to reduce radicals, but the --OH attached to the aniline ring in a Schiff base was prone to scavenge radicals directly.     

Actin cross-linking domain of Aeromonas hydrophila repeat in toxin A (RtxA) induces host cell rounding and apoptosis

The repeat in toxin (Rtx) of an environmental isolate ATCC 7966 of Aeromonas hydrophila consists of six genes (rtxACHBDE) organized in an operon similar to the gene organization found for the Rtx of the Vibrio species. The first gene in this operon (rtxA) encodes an exotoxin in vibrios, while other genes code for proteins needed for proper activation of RtxA and in secretion of this toxin from Vibrio cholerae. However, the RtxA of ATCC 7966, as well as from the clinical isolate SSU of A. hydrophila, was exclusively expressed and produced during co-infection of this pathogen with the host, e.g., HeLa cells, indicating that rtxA gene expression required host cell contact. Within the RtxA, an actin cross-linking domain (ACD) exists and to investigate the functionality of this domain, several truncated versions of ACD were generated to discern its minimal biological active region. Such genetically modified genes encoding ACD, which were truncated on either the NH(2) or the COOH terminal, as well as on both ends, were expressed from a bidirectional promoter of the pBI-enhanced green fluorescent protein (EGFP) vector in a HeLa-Tet-Off cell system. We demonstrated that only the full-length ACD of RtxA from A. hydrophila catalyzed the covalent cross-linking of the host cellular actin, whereas the ACD truncated on the NH(2), COOH or both ends did not exhibit such actin cross-linking characteristics. Further, we showed that the full-length ACD of A. hydrophila RtxA disrupted the actin cytoskeleton of HeLa cells, resulting in their rounding phenotype. Finally, our data provided evidence that the full-length ACD of RtxA induced host cell apoptosis. Our study is the first to report that A. hydrophila possesses a functional RtxA having an ACD that contributes to the host cell apoptosis, and hence could represent a potential virulence factor of this emerging human pathogen.     

Crystal structure of lactoperoxidase at 2.4 A resolution

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to R_cryst and R_free factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by anti-phosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined α-helices of varying lengths including a helix, H_2a, unique to LPO, and two short antiparallel β-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 Å from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 Å. Two histidine residues and six buried water molecules are connected through a hydrogen-bonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry.     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

真菌漆酶的酶活测定方法评价

目前真菌漆酶酶活的测定方法多样,没有统一的标准,致使不同研究之间的漆酶酶活无法进行比较分析,也造成漆酶产品在酶活质量意义上的混乱。因此,对测定真菌漆酶酶活的各种不同的分光光度法进行了综述和比较分析,认为采用ABTS法作为测定漆酶酶活的方法较具合理性和科学性,建议作为漆酶酶活测定的统一方法。     

红背叶提取物的体外抗氧化活性

采用DPPH法、FRAP法、ABTS法和清除羟基自由基法四种抗氧化活性方法,测定了红背叶不同溶剂萃取60%EtOH提取物得到的部位清除自由基的能力.结果表明,乙酸乙酯提取物的抗氧化能力最强,强于阳性对照VC和Trolox;其次是60%乙醇提取物,其抗氧化能力基本与阳性对照BHA相当.     

硬枝树花粗提物的抗氧化作用

采用DPPH和ABTS法对硬枝树花石油醚(30~60)、二氯甲烷、乙酸乙酯、甲醇等4个溶剂依次提取得到的浸膏物,进行抗氧化活性测定试验。结果表明:甲醇提取物,乙酸乙酯和二氯甲烷提取物在0.4~1.0 mg/mL检测浓度范围内对.DPPH的清除效果和质量浓度呈现一定的量效关系。甲醇提取物在1 mg/mL时,清除率高达93.99%,二氯甲烷提取物在1 mg/mL时清除率为92.32%,均高于同质量浓度的BHT。乙酸乙酯和二氯甲烷提取物在0.4~1.0mg/mL检测浓度范围内对ABTS.+自由基清除率与质量浓度呈现一定的量效关系,在1 mg/mL时,二氯甲烷提取物对ABTS.+自由基清除率为65.6%,乙酸乙酯提取物对ABTS.+自由基清除率为48%。     

硬枝树花的化学成分及抗氧化研究

以梯度提取的方法对硬枝树花进行石油醚(30–60)、二氯甲烷、乙酸乙酯提取,浸膏经过色谱柱层析分离纯化得到6个单体化合物,分别为松萝酸(1)、黑茶溃素(2)、巴尔巴地衣酸(3)、去甲环萝酸(4)、地弗地衣酸(5)、藻纹苔酸(6),并且都是首次从硬枝树花中分离得到。通过对清除DPPH?和ABTS?+能力的测定,对这6个化合物体外抗氧化活性进行评价。结果表明:藻纹苔酸在检测浓度范围内的?DPPH的清除率和终浓度呈现一定的量效关系,在55μmol/L时,藻纹苔酸的清除率为57.05%(P0.01)。黑茶溃素、巴尔巴地衣酸、去甲环萝酸在13.75–55μmol/L检测浓度范围内的DPPH自由基清除率与终浓度呈量效关系。松萝酸对DPPH?的清除能力较强,在3.4375μmol/L及27.5μmol/L时,DPPH自由基清除率达到74.78%(P0.01)及74.82%(P0.01),接近对照组BHT浓度为55μmol/L的清除率。地弗地衣酸、藻纹苔酸、巴尔巴地衣酸的ABTS?+自由基清除率与终浓度呈现一定的量效关系,在浓度为55μmol/L时,地弗地衣酸ABTS?+自由基清除率为50.79%(P0.01),藻纹苔酸为44.53%(P0.01),巴尔巴地衣酸为44.34%(P0.01),在13.75–55μmol/L浓度范围内,黑茶溃素ABTS?+自由基的清除率随着浓度的增大而逐渐增大。硬枝树花中含有多样的苯骈呋喃类和缩酚酸类化合物及其衍生物,是具有开发利用潜力的抗氧化剂资源。     

Preparation and properties of monoclonal antibodies to myelin basic protein and its peptides

Techniques are described which have enabled the production and characterisation of monoclonal antibodies to myelin basic protein. These are shown by enzyme immunoassay to react with six different epitopes. Two of these are to peptide 82–91, a region claimed to be present in the spinal fluid of patients with demyelinating disease. One of these, an IgG_2a, is shown to react only with peptides in which the 91–92 phe-phe bond has broken. The other, an IgM, also reacts with whole myelin basic protein. The IgG_2a antibody is shown to have an affinity suitable for use in immunoassay of peptide 82–91. The enzyme immunoassay procedures described help to minimise the work load involved in the preparation and characterisation of monoclonal antibodies to this protein.