Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.     

乳抗氧化肽的分离纯化与结构鉴定

本文检测了不同分子量范围的WPI(乳清分离蛋白)酶解物的抗氧化活性,结果表明各个组分显示出不同的抗氧化活性,其中分子量小于5 kDa的组分最强。采用凝胶过滤色谱对分子量小于5 kDa的WPI酶解物进行分离,抗氧化活性强的组分继续采用RP-HPLC进行纯化。通过MALDI-TOF-MS与氨基酸组成分析鉴定该活性肽为His-Ile-Arg。     

Novel Immunodominant Peptide Presentation Strategy: a Featured HLA-A*2402-Restricted Cytotoxic T-Lymphocyte Epitope Stabilized by Intrachain Hydrogen Bonds from Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β₂-microglobulin (β₂m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.In 2003, severe and acute respiratory syndrome (SARS), emerging from China, caused a global outbreak, affecting 29 countries, with over 8,000 human cases and greater than 800 deaths (5, 9, 24, 33, 37). Thanks to the unprecedented global collaboration coordinated by the WHO, SARS coronavirus (SARS-CoV), a novel member of Coronaviridae family, was rapidly confirmed to be the etiological agent for the SARS epidemic (36). Soon after the identification of the causative agent, SARS was controlled and then quickly announced to be conquered through international cooperation on epidemiological processes (9). However, the role that human immunity played in the clearance of SARS-CoV and whether the memory immunity will persist for the potential reemergence of SARS are not yet well understood.In viral infections, CD8⁺ cytotoxic T lymphocytes (CTLs) are essential to the control of infectious disease. Virus-specific CD8⁺ T cells recognize peptides which have 8 to 11 amino acids, in most cases presented by major histocompatibility complex (MHC) class I molecules. However, identification of virus-specific CD8⁺ T-cell epitopes remains a complicated and time-consuming process. Various strategies have been developed to define CTL epitopes so far. One of the most common practices to determine immunodominant CTL epitopes on a large scale is based on screening and functional analysis of overlapping 15- to 20-mer peptides covering an entire viral proteome or a given set of immunogenic proteins (19, 23, 32). However, peptides identified through this method are too long to be naturally processed CTL epitopes, and the definition of MHC class I restriction of these peptides still requires further analysis. Rapid and efficient strategies should be developed for the determination of naturally presented CTL epitopes in the context of any given HLA allele of interest. Furthermore, no other HLA alleles except HLA-A2-restricted CTL epitopes have been reported for SARS-CoV-derived proteins (16, 22, 31, 43, 46, 47, 49). This is primarily because of the limitation of the experimental methods for the other HLA alleles. HLA-A24 is one of the most common HLA-A alleles throughout the world, especially in East Asia, where SARS-CoV emerged, second only to HLA-A2 (30). The development of a fast and valid method to screen and identify HLA-A24-restricted epitopes would greatly contribute to the understanding of the specific CTL epitope-stimulated response and widen the application of the epitope-based vaccine among a more universal population (17). A genomewide scanning of HLA binding peptides from SARS-CoV has been performed by Sylvester-Hvid and colleagues, through which dozens of peptides with major HLA supertypes, including HLA-A24 binding capability, have been identified (41).There are strong indications that different peptide ligands, such as peptides with distinct immunodominance, can elicit a diverse specific T-cell repertoire, and even subtle changes in the same peptide can have a profound effect on the response (25, 44). Furthermore, a broader T-cell receptor (TCR) repertoire to a virus-specific peptide-MHC complex can keep the host resistant to the virus and limit the emergence of virus immune-escape mutants (29, 34, 38). Recent studies have demonstrated that the diversity of the selected TCR repertoire (designated as T-cell receptor bias) is clearly influenced by the conformational characteristics of the bound peptide in the MHC groove. Peptides with a flat, featureless surface when presented by MHC generate only limited TCR diversity in a mature repertoire, while featured peptides with exposed residues (without extreme bulges) protruding outside the pMHC landscapes are rather associated with the more diverse T-cell repertoire (15, 28, 39, 44, 45). Therefore, being able to determine the binding features of a peptide to MHC and describe the peptide-MHC topology will help us understand the immunodominance of a given peptide and demonstrate the peptide presentation strategy of the host.Structural proteins of SARS-CoV, such as spike, membrane, and nucleocapsid (N), have been demonstrated as factors of the antigenicity of the virus, as compared with the nonstructural proteins (12, 20). Coronavirus nucleocapsid (N) protein is a highly phosphorylated protein which not only is responsible for construction of the ribonucleoprotein complex by interacting with the viral genome and regulating the synthesis of viral RNA and protein, but also serves as a potent immunogen that induces humoral and cellular immunity (13, 14, 26, 48). The CD8⁺ T-cell epitopes derived from SARS-CoV N protein defined so far mainly cluster in two major immunogenic regions (4, 21, 23, 31, 32, 43). One of them, residues 219 to 235, comprises most of the N protein-derived minimal CTL epitopes identified so far—N220-228, N223-231, N227-235, etc.—all of which are HLA-A*0201 restricted (4, 43). The other region, residues 331 to 365, also includes high-immunogenicity peptides that can induce memory T-lymphocyte responses against SARS-CoV (21, 23, 32). However, until now, no minimal CTL epitope with a given HLA allele restriction has been investigated in this region.Here, based on previously defined immunogenic regions derived from SARS-CoV N protein (21), we identified an HLA-A*2402-restricted epitope, N1 (residues 346 to 354), in the region through a distinct strategy using structural and functional approaches. The binding affinity with HLA-A*2402 molecules and the cellular immunogenicity of the peptide were demonstrated in a series of assays. The X-ray crystal structure of HLA-A*2402 complexed with peptide N1 has shown a novel host strategy to present an immunodominant CTL epitope by intrachain hydrogen bond as a featured epitope.     

Epitope Identification from Fixed-complexity Random-sequence Peptide Microarrays

Antibodies play an important role in modern science and medicine. They are essential in many biological assays and have emerged as an important class of therapeutics. Unfortunately, current methods for mapping antibody epitopes require costly synthesis or enrichment steps, and no low-cost universal platform exists. In order to address this, we tested a random-sequence peptide microarray consisting of over 330,000 unique peptide sequences sampling 83% of all possible tetramers and 27% of pentamers. It is a single, unbiased platform that can be used in many different types of tests, it does not rely on informatic selection of peptides for a particular proteome, and it does not require iterative rounds of selection.In order to optimize the platform, we developed an algorithm that considers the significance of k-length peptide subsequences (k-mers) within selected peptides that come from the microarray. We tested eight monoclonal antibodies and seven infectious disease cohorts. The method correctly identified five of the eight monoclonal epitopes and identified both reported and unreported epitope candidates in the infectious disease cohorts. This algorithm could greatly enhance the utility of random-sequence peptide microarrays by enabling rapid epitope mapping and antigen identification.Antibodies play a central role in the immune system and in modern health care and medical research. They are commonly used as affinity reagents in research and diagnostic applications and have emerged as an important class of therapeutics (1). When new affinity reagents are being generated, it is useful to know the target sequence (epitope) bound by the antibody in question. Many methods have been developed to accomplish this, including peptide tiling and phage, bacteria, and mRNA display (2–4). Especially for newly discovered diseases, such as Middle East respiratory syndrome (5), knowing the epitope(s) that elicits a humoral response enables the production of diagnostics and vaccines. Large-scale mapping of cohorts infected with the same disease may guide the development of universal vaccines for flu and other infections. Crystal structure and B-cell sequencing provide the most detailed information about antibody targeting, but in practice these are cost prohibitive and rarely done. Library-panning-type approaches use bacteria or phages to display peptide sequences, avoiding costly crystallization or synthesis steps, and are common approaches for linear epitope mapping (3, 6). Recently, bacterial display methods have been used to discover antigens in celiac disease (2). Tools for probing the “memory” of the immune system could reveal a wealth of information about an individual''s health status and antibody repertoire. Although display techniques are effective and result in highly accurate and specific linear epitope determination (7, 8), they have hidden and poorly understood biases regarding sequence populations (9–11) and rely on selection steps that eliminate certain sequences in favor of others. This creates issues with cost and reliability at scale, and information is discarded as the selection process becomes increasingly stringent. As a rapid identification method, panning is not optimal.Peptide array technologies provide an alternative approach. They are simple and reproducible, they provide information about binders and non-binders, and they can be low cost if mass produced, but they represent a smaller sequence library than phage display and contain only linear sequences. This might seem like a disadvantage, but in practice, linear epitopes are actually quite common in nature, and even mimotopes can provide useful, if indirect, information about non-linear epitopes. Microarrays containing hundreds of thousands of peptides are becoming more accessible, reducing the impact of smaller libraries. Additionally, microarrays are capable of displaying interactions between antibodies and peptides with short, gapped sequences containing four to six anchor residues, which seem to cover a sizable class of antibodies (12, 13).To date the most common approach to designing peptide microarrays has been to tile sequences from a known protein or proteome of interest and find sequences that bind the target (4, 14–17). Recently this technique has been scaled to whole proteomes using arrays containing millions of sequences (14, 16). This approach is effective on a single-protein scale, but problems arise when one is looking for specific epitope sequences in the presence of millions of other peptides. Cross-reactivity of antibodies to non-target peptides often obscures the eliciting antigen (14). This might be due in part to the fact that tiled peptides are fundamentally different from folded proteins, and inaccessible parts of a protein are likely to be exposed when linear pieces of it are tiled. Additionally, there are many common n-mers across apparently unrelated pathogens. It might be possible to address this problem using motif-based discovery rather than peptide-based discovery. Short motifs (4- to 5-mers) will likely appear multiple times in a given peptide library. Longer sequences (6- to 12-mers) should appear more rarely. We propose that a platform for epitope discovery should focus on representing as many unique short motifs as possible, rather than providing longer, overlapping sequences from a particular set of proteins.Previously our group used random-sequence peptide microarrays to diagnose disease using immunosignatures (18, 19). The immunosignaturing effect relies on the interaction of serum antibodies with random-sequence peptides bound to a microarray. When properly trained on well-validated cohorts, this indirect information provides very discerning and predictive information about disease states in blinded individuals (18, 20–23). Although immunosignatures are sensitive and specific as a diagnostic tool, a link has not been established between immunosignature profiles and actual sequences of signature peptides. This was attempted in a previous study by our group in which we evaluated an array of 10,000 17-mer peptides as a platform for epitope mapping. Although useful for predicting linear sequences for some monoclonal antibodies, it offered virtually no predictive power in serum samples from mice immunized to a known antigen (24). Since then, advances in in situ synthesis techniques have enabled our group to produce microarrays containing several million peptides per slide (25). These arrays contain >27% of possible pentamers and 83% of possible tetramers. Although it lacks the majority of pentamers, this is a fairly dense sampling of short peptide sequences that might be useful for epitope mapping.Here we report on a general approach that uses random sequence peptide arrays to map epitopes. We demonstrated this by identifying epitope sequences from a set of monoclonal antibodies. We then used the same technique with different disease cohorts containing antibodies of unknown specificity, revealing both previously discovered and new epitopes. The study described here is the first attempt at deciphering a microarray with fixed but random peptide sequences for epitopes that does not a priori assume a set of eliciting proteins.     

A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP^32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP^32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP^32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP^32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP^32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP^32–41 internalization routes. ECP^32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.     

Identification and Enhancement of HLA-A2.1-Restricted CTL Epitopes in a New Human Cancer Antigen-POTE

Identification of CD8⁺ T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272–280 and POTE 323–331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2⁺ human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.     

Designing Potent Derivatives of Ovokinin(2-7), an Anti-hypertensive Peptide Derived from Ovalbumin

We obtained a potent anti-hypertensive peptide, RPFHPF, by replacing the amino acid residues of ovokinin(2-7) (RADHPF), an orally active anti-hypertensive peptide derived from ovalbumin. After intravenous administration in anesthetized Wistar rats, the designed peptide [Pro², Phe³]-ovokinin(2-7) had a long-lasting hypotensive activity at a dose of 10 mg/kg, while that of ovokinin(2-7) was only transient even at a dose of 100 mg/kg. After oral administration in conscious spontaneously hypertensive rats (SHRs), [Pro², Phe³]-ovokinin(2-7) significantly lowered the systolic blood pressure in a dose-dependent manner. It is noteworthy that the minimum effective dose of [Pro², Phe³]-ovokinin(2-7) was 0.3 mg/kg, about one-thirtieth of that of ovokinin(2-7). On the other hand, orally administered [Pro², Phe³]-ovokinin(2-7) did not show any significant hypotensive effect in normotensive Wistar-Kyoto rats (WKYs) even at a dose of 3 mg/kg. Taken together, [Pro², Phe³]-ovokinin(2-7) proved to be an ideal, potent anti-hypertensive peptide with little effect on normal blood pressure when given orally.     

The Peptide Derived from erbB2 Auto-Inhibitor Herstatin Shared in the Same Epitope and Function with Functional Antibody 2C4

Previous studies have shown that different epitopes of HER2 exhibit distinct functions and that the epitope bound by the antibody 2C4 plays a role in formation of hetereodimers between HER2 and other receptors of the HER family. In this study, we used computer modeling to determine that the epitope of HER2 which the C-terminal 79 amino acids of herstatin (named HSTC79) binds is similar to that bound by 2C4. Based on these theoretical results, recombinant HSTC79 fused with GST was expressed in Escherichia coli and purified by affinity chromatography. Experimental analysis showed that HSTC79 did specifically bind to HER2 and that the epitope of HER2 identified by HSTC79 was near that identified by 2C4. Furthermore, HSTC79 inhibited the growth of HER2-overexpressing cells. These results highlight the fact that the binding site architecture and certain key residues of HER2 may be very helpful for understanding the protein’s biological role and providing insights for designing novel inhibitors of HER2.     

EGFR T790M Mutation as a Possible Target for Immunotherapy; Identification of HLA-A*0201-Restricted T Cell Epitopes Derived from the EGFR T790M Mutation

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non–small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.     

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG₁/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.     

高致病性禽流感病毒血凝素蛋白广谱中和表位模拟肽的筛选与鉴定

以H5N1型禽流感病毒HA蛋白广谱中和单抗8H5为基础,利用噬菌体展示肽库技术及类病毒颗粒融合表达技术研究HA模拟表位。ELISA检测结果显示:筛选获得模拟HA表位的模拟肽123,进行类病毒颗粒融合蛋白表达后,仍具有与8H5单抗特异结合的能力。免疫荧光检测结果说明,类病毒颗粒免疫小鼠后产生了能与HA交叉反应的抗体。禽流感病毒HA模拟表位的研究与性质的分析及类病毒颗粒融合蛋白的表达与活性分析、免疫原性分析,都为研制禽流感通用表位疫苗奠定了基础。     

食品蛋白质中血管紧张素转化酶抑制肽的研究

血管紧张素转化酶Ⅰ (angiotensinIconvertingenzyme ,简称ACE)在人体血压调节过程中起重要的生理作用。源于食品蛋白质中的血管紧张素转化酶抑制肽 (angiotensinIconvertingenzymeinhibitorypeptides ,简称ACEIP)有明显的降血压作用 ,这些肽是通过抑制ACE的活性起降血压作用。文章中综述了来源于各种食品蛋白质的ACEIP的最新研究进展以及两种主要制备方法和评价方法 ,并对食品蛋白质中ACEIP的应用前景进行了展望     

Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.     

Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library

The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC_1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif ⁴⁸DTTQGRFD⁵⁵ shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of ⁴⁸DTTQGRFD⁵⁵. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection.     

高效缺氧诱导表达载体的构建与鉴定

目的：构建缺氧诱导表达载体,以介导报告基因在缺氧环境下的特异、高效表达。方法：通过分子生物学方法,将鼠磷酸甘油酸激酶基因的缺氧应答元件（HRE）和最小CMV（mCMV）启动子重组,构建增强型绿色荧光蛋白（EGFP）或萤光素酶报告基因的可诱导载体;通过酶切鉴定和测序分析,证实载体获得正确的构建;将重组载体转染HeLa细胞,观察EGFP荧光强度并检测萤光素酶活性。结果：HRE／mCMV启动子调控的报告基因载体具有特异和高效的诱导活性。结论：构建了可以进行特异和高效缺氧诱导的报告基因载体,为其进一步的开发和应用奠定了实验基础。     

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

Background

The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.

Methods and Results

This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched ²¹³SVQYHPL²¹⁹ of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that ²¹³SVQYHPL²¹⁹ was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.

Conclusions and Significance

We identified ²¹³SVQYHPL²¹⁹ as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.     

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvβ3 signaling, this collagen epitope promoted αvβ3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvβ3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself.