Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases

Stefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the K(i) values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases.     

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

The various iterations of the HapMap Project and many genome-wide association studies (GWAS) have identified hundreds of potential genes involved in monogenic and multifactorial traits. We constructed an arrayed 115,000-member human genomic library in the PAC shuttle vector pJCPAC-Mam2 that can be propagated in both bacterial and human cells. The library appears to represent a two-fold coverage of the human genome. Transient transfection of a p53-containing PAC clone into p53-null Saos-2 human osteosarcoma cells demonstrated that both p53 mRNA and protein were produced. Additionally, expression of the p53 protein triggered apoptosis in a subset of the Saos-2 cells. This library should serve as a valuable resource to validate potential disease genes identified by GWAS in human cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.     

Human and Mouse Type I Natural Killer T Cell Antigen Receptors Exhibit Different Fine Specificities for CD1d-Antigen Complex

Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4′,4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4′-OH position in comparison with the 3′-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4′-OH > 3′-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.     

Structural Basis for the Interaction of a Hexameric Replicative Helicase with the Regulatory Subunit of Human DNA Polymerase α-Primase

DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.     

Role of metalloproteases in vaccinia virus epitope processing for transporter associated with antigen processing (TAP)-independent human leukocyte antigen (HLA)-B7 class I antigen presentation

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8(+) lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8(+) T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections.     

Structural Requirements for Recognition of Major Histocompatibility Complex Class II by Membrane-associated RING-CH (MARCH) Protein E3 Ligases

MARCH E3 ligases play a key role in controlling MHC class II surface expression by regulated ubiquitination of a lysine residue in the β-chain. Little is known concerning how these enzymes target their specific substrates. Here we show that recognition of HLA-DR by MARCH proteins is complex. Several features associated with the transmembrane domain and bordering regions influence the overall efficiency of receptor internalization. A cluster of residues at the interface of the lipid bilayer and the cytosol plays the most important role in MARCH8 recognition of HLA-DRβ. Variation in this sequence also determines specificity of MARCH9 for HLA-DQ. Residues located in helical face four of HLA-DRβ together with a charged residue at the boundary with the stalk region also contribute significantly to recognition. Truncation analysis suggested that a dileucine-like motif in the DRβ cytoplasmic tail influences the efficiency of co-localization of HLA-DR with MARCH8. The DRβ-encoded acceptor lysine functioned optimally when placed in its natural location relative to the bilayer. In the DRα/DRβ dimer most other amino acids in the cytoplasmic tail could be substituted for alanine with minimal influence on function. Our data support a model whereby multiple features of HLA-DR are involved in substrate recognition by MARCH8. The single most important region is located at the interface between the transmembrane domain and the cytosol. Variation in sequence in this location between different class II isotypes controls efficiency of recognition by different MARCH E3 ligases.     

Target specificity of an autoreactive pathogenic human γδ-T cell receptor in myositis

In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis.     

Immune Related Genes Underpin the Evolution of Adaptive Immunity in Jawless Vertebrates

The study of immune related genes in lampreys and hagfish provides a unique perspective on the evolutionary genetic underpinnings of adaptive immunity and the evolution of vertebrate genomes. Separated from their jawed cousins at the stem of the vertebrate lineage, these jawless vertebrates have many of the gene families and gene regulatory networks associated with the defining morphological and physiological features of vertebrates. These include genes vital for innate immunity, inflammation, wound healing, protein degradation, and the development, signaling and trafficking of lymphocytes. Jawless vertebrates recognize antigen by using leucine-rich repeat (LRR) based variable lymphocyte receptors (VLRs), which are very different from the immunoglobulin (Ig) based T cell receptor (TCR) and B cell receptor (BCR) used for antigen recognition by jawed vertebrates. The somatically constructed VLR genes are expressed in monoallelic fashion by T-like and B-like lymphocytes. Jawless and jawed vertebrates thus share many of the genes that provide the molecular infrastructure and physiological context for adaptive immune responses, yet use entirely different genes and mechanisms of combinatorial assembly to generate diverse repertoires of antigen recognition receptors.     

超声波均质化对仙台病毒ELISA测试中抗原稳定性的影响

目的研究超声波均质化（homogenization）处理对于仙台病毒在ELISA测试中抗原稳定性的影响。方法对BHK-21细胞内扩增培养的仙台病毒通过差速离心,富集后使用超声波做均质化处理,和未处理的病毒分别包被酶标板,使用标准免疫小鼠血清和SPF小鼠血清对包被平板进行测试,比较样品测试孔间测量数据的变异率。结果对免疫血清做梯度稀释,各梯度样品在未经超声处理仙台病毒抗原包被ELISA检测中测量值的变异率在1.97%～6.02%之间;在经超声处理仙台病毒抗原包被ELISA检测中测量值的变异率在0.53%～2.26%之间;SPF血清测量值的变异率均高于免疫血清;所有样品在经超声处理的病毒抗原包被ELISA检测中测量值的变异率均较小。结论超声波处理有效的提高了仙台病毒抗原的均质性,在ELISA测试中提高了抗原的稳定性。