Immunological detection of a cysteine protease in the skin and other tissues

Summary Monospecific antibody directed to cysteine protease of 2-day-old rat epidermis recently characterized as being different from the proteases previously reported was produced in rabbits. By immunofluorescence microscopy and immunoperoxidase staining with an avidin-biotin-peroxidase method the protease was found to be present in the epidermis of rodents of different ages as well as that of humans, but not in the dermis. The staining in germinative cells was more intense than in cells in the superficial layers. It appeared as irregular patches in the nuclei and stained more diffusely in the cytoplasm where small granular components, strongly stained, were identified. The staining patterns in granular cells showed accumulation of the antigen in a granular form. The morphology and distribution of granules resembled those of keratohyalin-like granules in the nucleus and dense homogenous deposits in the cytoplasm. In cornified cells the reaction product was localized by the plasma membrane where concentration of the dense homogenous deposits occurred, suggesting that the cysteine protease is one component of the unique and characteristic structure of differentiated keratinocytes. In addition, the cysteine protease antigen having the same molecular weight as the epidermal enzyme was detected in liver, kidney and lung indicating a wider tissue distribution of the protease. The significance of the protease in regulation of cellular functions remains to be investigated.     

Synthesis and evaluation of immunostimulant plasmalogen lysophosphatidylethanolamine and analogues for natural killer T cells

Plasmalogen lysophosphatidylethanolamine (pLPE) had been identified as a self antigen for natural killer T cells (NKT cells). It is very important in the development, maturation and activation of NKT cells in thymus. Besides, pLPE is a novel type of antigen for NKT cells. To evaluate the structure–activity relationship (SAR) of this new antigen, pLPE and its analogues referred to different aliphatic chains and linkages at the sn-1 position of the glycerol backbone were synthesized, and the biological activities of these analogues was characterized. It is discovered that the linkages between phosphate and lipid moiety are not important for the antigens’ activities. The pLPE analogues 1, 3, 4, 7 and 9, which have additional double bonds on lipid parts, were identified as new NKT agonists. Moreover, the analogues 4, 7 and 9 were discovered as potent Th2 activators for NKT cells.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Sheep antibody response to cell wall antigens expressed in vivo by Pasteurella haemolytica serotype A2

Abstract Cells of Pasteurella haemolytica A2 were harvested directly from the pleural fluid of sheep with pneumonic pasteurellosis and the protein composition of their envelopes was examined by SDS-PAGE. Several high molecular-weight proteins expressed in pleural fluid were absent when the same isolate was grown in nutrient broth, but were produced during culture in iron restricted media. When the cell wall envelopes were examined by immunoblotting with sera and lung washes of lambs recovering from pneumonic pasteurellosis, antibodies to the major outer membrane proteins, the iron-regulated proteins and one other 'in vivo'-expressed antigen were present. These results have implications for the formulation of effective vaccines against pasteurellosis in sheep.     

不同狂犬病毒株抗原反应性的比较研究

应用ELISA法对285份免疫前及健康人血清和742份以不同毒株制备的狂犬病疫苗(aG株、CaG株、CTN株,PM株)全程免疫后人群的血清标本进行抗狂犬病毒抗体的检测。结果显示CTN株病毒抗原对不同毒株狂犬病疫苗免疫后血清的抗体阳性检出率均能达到90％以上,且与SNT效价的相关性较好;而aG株抗原对除aG株以外其它毒株狂犬病疫苗免疫后血清的阳性检出率仅为50％左右。因此不同毒株狂犬病毒抗原的反应性存在明显差异,选用纯度高、活性好的CTN株病毒或两株病毒以不同比例混合作为ELISA检测用抗原,测定疫苗免疫后人群的抗体水平,可获得满意的结果。     

The Dok-3/Grb2 Protein Signal Module Attenuates Lyn Kinase-dependent Activation of Syk Kinase in B Cell Antigen Receptor Microclusters

Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5′ phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.     

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.