Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system.

Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.

Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the in vitro citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6-75 nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination.

[Characterization of anti-mouse prion peptide single chain Fv antibody by phage display].

Antibodies can distinguish not only differences in amino acid sequences (primary structure), but also differences in three-dimensional structure and thus may be useful for detecting the conversion of prion proteins, especially in vivo. For diagnosis, we prepared chicken single chain variable fragment (scFv) antibodies that specifically recognized a prion protein using a phage display approach. As antigen, mouse prion protein (MoPrP) 138-153 containing YYR residues was conjugated with KLH. Total RNA was extracted from the splenocytes of an immunized chicken, and the cDNA of scFv was ligated in a phagemid vector. The phage display scFv library was panned against the peptide antigen four times. Twenty-three scFv phage clones that tested positive using ELISA with the peptide antigen were then reacted with recombinant mouse prion protein (23-231), mouse brain homogenate, mouse neuroblastoma Neuro-2a, recombinant human V129 and M129 prion proteins, and human glyoma T98G using ELISA, immunoblotting analysis, and immunocytochemistry. The results suggested that the scFv phage clones were useful for detecting mouse and human prion proteins.

Characterization and expression analysis of the chicken interleukin-11 receptor alpha chain.

Interleukin-11 (IL-11) is a multifunctional cytokine involved in various pathways in blood cells, their precursors and many other cell types in vitro and in vivo. The effects of IL-11 are largely mediated by the IL-11 receptor alpha-chain (IL-11Ralpha). In this study, a putative cDNA sequence encoding the 414 amino acid propeptide of chicken IL-11R (chIL-11R) was identified. The predicted 414 amino acid sequence showed 42-43% sequence identity with mammalian homologues. In a domain search of the molecule, two fibronectin (FN) type-III domains were identified in the C- terminal portion. On comparison with mammalian IL-11R, 4 conserved cysteine residues and a WSXWS motif were observed within the FN type-III domains. Expression analysis revealed that chIL-11Ralpha is strongly expressed in brain, heart, lung, liver, glandular stomach, kidney, the immature testis, ovary and chicken blastodermal cells (CBCs) after 1-day-cultivation. These findings strongly indicate that the identified chicken cDNA sequence encodes chIL-11R alpha-chain homologue.

Biological activity of recombinant chicken interleukin-6 in chicken hybridoma cells.

Interleukin-6 (IL-6), a multipotential cytokine that plays roles in regulating immune responses, acute phase reactions and hematopoiesis, induces proliferation and antibody production in hybridoma cells. The biological activities of the recombinant chicken IL-6 (rchIL-6) were determined using murine and chicken hybridoma cells. Cell proliferation and tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3) were induced by rchIL-6 in the IL-6-dependent murine hybridoma cell line MH60, whereas the recombinant protein exhibited no significant cell proliferation activity in chicken hybridoma cells but induced antibody production and tyrosine phosphorylation of STAT3. The lack of cell proliferation induced by rchIL-6 in HUC2-13 cells may have been because the cell line was not IL-6-dependent in contrast to MH60 cells. These results suggest that rchIL-6 may be useful for promoting antibody production of chicken hybridoma cells as well as for creating chicken hybridomas by cell fusion.

Establishment of a chicken monoclonal antibody panel against mammalian prion protein.

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.

Development of recombinant chicken IgY from single chain fragment of variable region for diagnosis of BSE.

We generated two recombinant chicken IgYs, designated Ab3-15 and Ab4-19, against mammalian prion protein (PrP) from the single chain fragment of variable region (scFv) antibodies. These two antibodies recognized PrP(Sc) from bovine spongiform encephalopathy (BSE) in cattle and were more sensitive than the corresponding scFv antibodies. These antibodies also recognized PrP(Sc) from other scrapie-infected mammals. These results indicate that Ab3-15 and Ab4-19 are useful for diagnosis of BSE as well as other prion diseases.

Stable production of recombinant chicken antibody in CHO-K1 cell line.

When compared with mammalian IgG, chicken IgY is advantageous in terms of cross-reactivity. In this study, two plasmids were constructed for expression of recombinant chicken IgY derived from a chicken hybridoma. The first was for expression of the light (L) chain, and the other was for the heavy (H) chain with a histidine (His) tag at the carboxy-terminal. After transfection of recombinant chicken IgY gene into Chinese hamster ovary cells, a transfectant designated HF33 that secreted the specific antibody was selected. HF33 cells produced recombinant IgY with His tag at 10-15 microg/10(6) cells/24 h. On Western blotting analysis, the recombinant IgY was detected as one band for the H chain and two bands for the L chain. The recombinant IgY was successfully purified in a one-step procedure using a nickel-affinity resin. These results indicate that the present recombinant chicken IgY is useful for further applications.

Excision of foreign gene product with cathepsin D in chicken hepatoma cell line.

To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens.

Generation of a mouse monoclonal antibody against chicken interleukin-6.

A monoclonal antibody (MAb) specific for chicken interleukin-6 (chIL-6) was generated by using Balb/c mice immunized with recombinant chIL-6 (rchIL-6). On Western blot analysis, the MAb, designated E3, reacted with rchIL-6 but not with recombinant murine IL-6 (rmIL-6). The MAb E3 also reacted with supernatant of the chicken macrophage-like cell line HD11 stimulated with lipopolysaccaride. The rchIL-6-induced phosphorylation of STAT3 in a chicken hybridoma cell line was inhibited by addition of MAb E3 in a dose-dependent manner. These results indicate that the MAb E3 specific for chIL-6 is useful for detection and functional analysis of chIL-6.

Inhibition of prion propagation in scrapie-infected mouse neuroblastoma cell lines using mouse monoclonal antibodies against prion protein.

We screened six mouse monoclonal antibodies (mAbs) against prion protein (PrP), which were previously established in our laboratory, for inhibitory activity against PrP(Sc)-accumulation in scrapie-infected cell lines and identified two mAbs, 3S9 and 2H9, as possessing this inhibitory activity. mAb 3S9 recognized an epitope including 154th tyrosine in the helix 1 region of PrP, while mAb 2H9 recognized a discontinuous region that included helix 1. In three scrapie-infected cell lines infected with different mouse-adapted scrapie strains, mAb 3S9 strongly inhibited accumulation of PrP(Sc), while mAb 2H9 moderately inhibited accumulation of PrP(Sc), indicating that inhibition of prion propagation by mAbs may be dependent on PrP(Sc) characteristics. Furthermore, mAb 3S9 completely excluded PrP(Sc) from these cell lines. These results suggest that mAbs 3S9 and 2H9 might be useful for clarifying the mechanisms of prion propagation and prevention by PrP-specific antibodies, and for tracing the conversion of PrP(C) to other PrP(Sc) isoforms.

Application of tyramide signal amplification for detection of N-glycolylneuraminic acid in human hepatocellular carcinoma.

BACKGROUND: N-Acetylneuraminic acid and N-glycolylneuraminic acid (NeuGc) are the most common sialic acids in mammals, and NeuGc has attracted attention as a tumor-associated antigen. METHODS: In frozen liver sections from patients with hepatocellular carcinoma, glycolipid-type NeuGc was detected on the surface of liver cancer cells in 9 of 17 samples (52.9%) by immunostaining, using two chicken monoclonal antibodies against NeuGc and the tyramide signal amplification method. When conventional immunostaining without amplification was used, all 17 specimens tested were negative. RESULTS: Increased serum levels of anti-NeuGc IgG and/or IgM were observed in 13 of the 17 patients with hepatocellular carcinoma (76.5%). The presence of these antibodies was mostly attributed to the expression of NeuGc on hepatocellular carcinoma cells. Of the subjects with small HCCs (diameter 3 cm or less), 6 of 10 were positive for serum anti-NeuGc antibodies; however, 1 of these was negative for both serum Alpha-fetoprotein (AFP) and for prothrombin-induced vitamin K antagonist II (PIVKA-II). There was no correlation between serum AFP- or PIVKA-II, levels and the presence of NeuGc or anti-NeuGc IgG and/or IgM. CONCLUSION: The tyramide signal amplification method is useful for the immunohistochemical detection of low-level NeuGc expression by hepatocellular carcinoma cells. We therefore consider that measurement of serum levels of anti-NeuGc antibodies is clinically meaningful and that anti-NeuGc antibody may be a useful screening test, in combination with AFP and PIVKA-II, for the early diagnosis of hepatocellular carcinoma.

Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state.

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.