Immunogenicity of enterovirus 70 capsid protein VP1 and its non-overlapping N- and C-terminal fragments.

Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000-10,000 units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20-320units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.

Expression of recombinant protein encoded by LOC387715 in Escherichia coli.

LOC387715 is a hypothetical gene located on human chromosome 10q26.13 that is associated with the development of age-related macular degeneration (AMD). The native open reading frame (ORF) of LOC387715 cDNA - LOC387715(ORF), contains a large number of Escherichia coli (E. coli) rare codons (RC) including 5.6% and 15.0% Group-I and IIa translational problem causative (TPC) RCs, respectively, which forms 3 and 4 simple E. coli rare codon clusters (RCC) where RCs are spaced by 1 and 2 respective non-TPC codons and one complex E. coli RCC where RCs and RCCs are spaced by <5 non-TPC codons. We modified the entire 35 E. coli RCs (6, 16 and 13 Group I, IIa and IIb RCs, respectively) present in LOC387715(ORF) with their optimal or sub-optimal synonymous degenerate codons, and the resulted LOC387715(ORF)m was free from Shine-Dalgarno-like sequence (SDLS) and ribosome binding site complementary sequence (RBSCS). SDS-PAGE and Western blotting analysis demonstrated that LOC387715(ORF)m was capable of highly expressing the recombinant protein rLOC387715 in E. coli. Mass spectrometry analysis indicated that the bacterial expressed rLOC387715 contained the correct and expected amino acid (aa) sequence without aa misincorporation, aa missing or frame-shift. The results suggest that high and authentic expression of LOC387715 recombinant protein in E. coli was achieved by the synonymous modification of its native ORF cDNA sequence for all the 3 groups of bacterial RCs and the simultaneous elimination of SDLS and RBSCS sequences.

Caspase-3 and -7 mediate apoptosis of human Chang's conjunctival cells induced by enterovirus 70.

Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), but not actinomycin D and guanidine.HCl (although guanidine.HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.

Dendritic cell surface calreticulin is a receptor for NY-ESO-1: direct interactions between tumor-associated antigen and the innate immune system.

How the immune system recognizes endogenously arising tumors and elicits adaptive immune responses against nonmutated tumor-associated Ags is poorly understood. In search of intrinsic factors contributing to the immunogenicity of the tumor-associated Ag NY-ESO-1, we found that the NY-ESO-1 protein binds to the surface of immature dendritic cells (DC), macrophages, and monocytes, but not to that of B cells or T cells. Using immunoprecipitation coupled with tandem mass spectrometry, we isolated DC surface calreticulin as the receptor for NY-ESO-1. Calreticulin Abs blocked NY-ESO-1 binding on immature DC and its cross-presentation to CD8+ T cells in vitro. Calreticulin/NY-ESO-1 interactions provide a direct link between NY-ESO-1, the innate immune system, and, potentially, the adaptive immune response against NY-ESO-1.

Surface calreticulin mediates muramyl dipeptide-induced apoptosis in RK13 cells.

Calreticulin (CRT) is a binding protein for apoptotic N-acetylmuramyl-L-alanyl-D-isoglutamine (L,D-MDP) or peptidoglycan in RK(13) cells. CRT on RK(13) cell surface (srCRT) forms complex(es) with tumor necrosis factor receptor 1 (TNFR1) and TNFR-associated death domain (TRADD) protein of the cell membrane. CRT polyclonal or monoclonal antibody binding to RK(13) srCRT dose-dependently inhibited L,D-MDP-induced apoptosis. In RK(13) cells, L,D-MDP up-regulated the TNFR1.TRADD complex of the plasma membrane and subsequently induced cytosolic TRADD-Fas-associated death domain protein complex. Biotinylated srCRT was capable of calcium-dependent binding of Sepharose-immobilized L,D-MDP or peptidoglycan. However, Toll-like receptors TLR-2 and TLR-4, Nod2, and CD14 of RK(13) cells did not specifically bind Sepharose-immobilized L,D-MDP. High concentrations (5-40 mm) of EGTA dose-dependently inhibited free L,D-MDP binding to purified RK(13) cell CRT and promoted free L,D-MDP dissociation from RK(13) cell CRT.MDP complex. Different concentrations of EGTA (0-40 mm) added to Dulbecco's modified essential medium with 1.8 mm calcium or phosphate-buffered saline with 0.18 mm calcium have different effects on medium free calcium concentrations but have identical inhibiting effects on L,D-MDP-induced apoptosis. More inhibition of the L,D-MDP-induced apoptotic DNA ladders and caspase-3 activity in RK(13) cells was obtained with EGTA pretreatment (83%) than just EGTA + L,D-MDP (47%). The knocking down of srCRT by antisense oligonucleotide CRTAS121 (250 nmol/ml) and stealth small interfering RNA CRT_siR479 (150 pm/ml) for 2 days (44 and 66%, respectively), resulted in the inhibition of L,D-MDP-induced caspase-3 activity (47 and 65%, respectively). The results suggest that (a) the binding of L,D-MDP to srCRT is calcium-dependent, i.e. on srCRT-bound calcium, and (b) it is srCRT, not TLR-2, TLR-4, Nod2 or CD14, that mediates L,D-MDP-induced RK(13) cell apoptosis through activating the TNFR1. TRADD-Fas-associated death domain protein apoptotic pathway.

Calreticulin is a binding protein for muramyl dipeptide and peptidoglycan in RK13 cells.

Calreticulin (CRT) was isolated and identified as a protein in rabbit kidney RK(13) cells that binds the apoptogenic bacterial cell wall (BCW) components, muramyl dipeptide (MDP) and peptidoglycan (PG). Mannan-agarose purified RK(13) cell CRT (rCRT) selectively bound sepharose-immobilized L,D-MDP and PG, but not L,L-MDP or D,D-MDP. Purified rCRT and bovine CRT (bCRT) also bound free PG and L,D-MDP demonstrated in bioassays of RK(13) cell apoptosis. The results suggest that, in RK(13) cells, (a) CRT is a specific binding protein for both L,D-MDP and PG and (b) CRT binding L,D-MDP or PG is dependent on the stereoisomeric configuration of the dipeptide (L-alanyl-D-isoglutamine) moiety. In addition, the results also suggest that, in RK(13) cells, the binding of L,D-MDP, L,L-MDP, D,D-MDP, or PG to CRT correlates with their capacities of inducing apoptosis.

Expression of enterovirus 70 capsid protein VP1 in Escherichia coli.

The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.