The human Ku autoantigen shares amino acid sequence homology with fungal,but not bacterial and viral,proteins

Context: Molecular mimicry between autoantigens and microbial antigens is a possible triggering mechanism of autoimmunity. Human Ku is a DNA-associated autoantigen targeted by autoantibodies in patients with systemic lupus erythematosus and related disorders; available data are consistent with a role of molecular mimicry in the pathogenesis of Anti-Ku autoimmunity, but no research exist on this topic.

Objective: We aimed to define the most probable microbial triggers of anti-Ku autoimmunity via molecular mimicry. Materials and methods. We performed a computer-assisted search for amino acid sequence homologies between the two subunits of human Ku and proteins of known human pathogens.

Results: Some fungal, but no bacterial or viral, proteins have statistically significant amino acid sequence homology with the p70 or p80 subunit of Ku. Twenty-six fungal proteins contain long segments highly homologous to p70 (14 proteins) or p80 (12 proteins) and belong to human pathogens (Aspergillus clavatus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Chaetomium globosum, Cryptococcus neoformans, Coccidioides immitis, Malassezia globosa, Neosartorya fischeri, Penicillium chrysogenum Wisconsin, Penicillium marneffei, and Yarrowia lipolytica). Twelve p70-homologous and eleven p80-homologous segments span at least one T cell epitope-containing part of the respective human Ku subunit (in the other cases, overlap is almost complete).

Discussion and Conclusion: We postulate that, in genetically predisposed persons, infection by the above fungi can be a trigger in the onset of anti-Ku autoimmunity via molecular mimicry between fungal proteins and the Ku autoantigen. Due to the low frequency of anti-Ku autoimmunity, multicentric collaboration is necessary to verify our hypothesis.     

The melanoma progression-associated antigen P3.58 is identical to the intercellular adhesion molecule, ICAM-1

The 89kd cell surface glycoprotein, P3.58, is expressed on human malignant melanomas in situ where it is associated with an increased risk of metastatic disease. Monoclonal antibodies detecting denatured P3.58 were produced and used to isolate a P3.58 encoding cDNA clone from a human melanoma lambda expression library. Sequencing of the cDNA revealed that the P3.58 antigen is identical to the leukocyte intercellular adhesion molecule 1 (ICAM-1).     

Interaction of the chromatin compaction-inducing domain (LR domain) of Ki-67 antigen with HP1 proteins

BACKGROUND: The LR domain of marsupial chmadrin is defined by its C-terminal amino acid sequence, which contains several pairs of leucine (L) and arginine (R) residues. The LR domain of chmadrin causes a significant compaction of chromatin over the entire length of chromosomes when it is overproduced. The possible human homologue of chmadrin, Ki-67 antigen (pKi-67), also has a stretch of LR pairs, but with no obvious overall similarity, at its C-terminus. RESULTS: The LR domain of human pKi-67 also induced chromatin compaction, both in human and marsupial cells. A yeast two-hybrid assay and an in vitro binding assay demonstrated that the human LR domain binds to heterochromatin protein 1 (HP1), a well-characterized molecule as a mediator of heterochromatin formation. In fixed cells stained with specific antibodies, the pKi-67 was found to be co-localized partially with HP1 at foci on chromosomes in an early G1 phase. Time-lapse observation in living cells co-expressing the fluorescently tagged proteins showed that the LR domain formed foci on chromosomes over a limited period of the cell cycle from the telophase to early G1 phase and that HP1 subsequently accumulated at these foci of the LR domain. CONCLUSIONS: Marsupial chmadrin and human pKi-67 induce chromatin compaction across species, possibly via the interaction of its LR domain with HP1.     

Synthetic peptide homologous to the envelope proteins of retroviruses shares a cross-reacting epitope with the CD4 receptor.

A synthetic peptide (CKS-17) homologous to a highly conserved region of the retroviral transmembrane protein p15E was tested for its effect on receptor expression on monocytes. The CKS-17 amino acid sequence is present in several retroviruses including human T-cell lymphotropic virus types I and II and human immunodeficiency virus. The CKS-17 peptide has been previously shown to inhibit monocyte superoxide production, natural killer cell activity, polyclonal B-cell activation, and monocyte-mediated killing by inactivation of interleukin-1. In this study, we demonstrated that the anti-CD4 monoclonal antibody OKT4 binds strongly in vitro to CKS-17-treated human blood monocytes, whereas other antibodies tested were not reactive. This observed binding was the result of direct interaction of OKT4 antibody with the CKS-17 peptide. Moreover, a partial homology was found in amino acid sequence analysis of the CD4 epitope and the CKS-17 peptide.     

The oct-1 homeo domain contacts only part of the octamer sequence and full oct-1 DNA-binding activity requires the POU-specific domain

The ubiquitous octamer-binding protein oct-1 contains a POU domain required for DNA binding, which can be subdivided into a POU-specific domain and a POU homeo domain. We have overproduced the POU domain and the POU homeo domain in a vaccinia expression system, purified both polypeptides to near homogeneity, and compared their DNA-binding properties. In contrast to the POU domain, the homeo domain protects only part of the octamer sequence in the Ad2 origin against breakdown by DNase I or hydroxyl radicals. Analysis of purine contacts by DMS and DEPC interference assays shows that the Ad2 octamer can be divided into two regions: one that is recognized both by the POU domain and the homeo domain in an identical fashion, and one that is only recognized by the POU domain. This suggests that the POU-specific domain is responsible for the additional contacts located at one side of the octamer. In agreement with this, mutating the first 3 nucleotides (ATG) of the octamer affected binding by the POU domain but not by the homeo domain. The apparent binding affinities to different octamer sites were compared. The homeo domain binds 600-fold less efficiently to the canonical octamer sequence (ATGCAAAT) than the POU domain. The difference is only sevenfold for the Ad2 octamer, whereas both Kd values are almost identical for the HSV ICP4 TAATGARAT motif. Both the POU and homeo domains recognize target sequences for mammalian homeo box proteins. We conclude that the octamer can act as a bipartite recognition sequence for oct-1 and that the POU-specific domain contributes to the binding affinity, as well as to the specificity, by providing additional contacts.     

Myeloma class switch variant X63.2aRI-16 secretes IgG2a and IgG1 with identical VDJ sequence

The myeloma line X63.2aRI-16 isolated in vitro as spontaneous tertiary class switch variant (γ₁ → γ_2b → γ_2a → γ_2a,γ₁) of X63 (IgG₁,x) by fluorescence-activated cell sorting using class-specific antisera expresses two heavy chains. X63.2aRI-16 secretes IgG_2a,x as does the parental cell line X63.2a-25, a hybrid molecule containing γ_2a and γ₁ heavy chains and IgG₁,x. The chain of the latter protein is the product of a reverse class switch in regard to the embryonic order of the C_H genes. We purified the immunoglobulins of X63.2aRI-16, isolated the CNBr fragments of both heavy chains and determined the complete amino acid sequence of the V_H domains and of the C_H domains up to the first subclass-specific residues. The remaining CNBr fragments of the C_H domains were characterized by amino acid analyses. It was found that both heavy chains of the double producer possess identical V_H domains and C_H domains characteristic for the subclasses γ_2a and γ₁, respectively. The identities of the two VDJ sequences expressed in X63.2aRI-16 cells suggest that the reverse class switch event to γ₁ cannot simply be explained on the basis of a deletion model involving only a single C_H locus.     

HLA‐A*11:53 is shown to be identical to the corrected A*11:02:01 allele sequence

According to the IMGT/HLA Database, the DNA sequence of A*11:53 is identical to A*11:02:01 in exons 2, 3, 4 and 5 except at codon 276. A*11:53 was reported as a rare variant of A*11, while A*11:02:01 was understood to be the second most frequently observed variant of A*11 after A*11:01:01 in Taiwanese. We sequenced HLA‐A locus exons 2, 3, 4 and 5 of Taiwanese blood donors (n = 50) previously typed to carry A*11:02:01. We found out all of their sequences are identical to A*11:53 in exons 2, 3, 4 and 5′ part of exon 5 including codon 276.     

Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins

Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have named tlpA (for TIR-like protein A), was identified in the Salmonella enterica serovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-kappaB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterized Salmonella virulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomal tlpA gene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1beta secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disrupted tlpA also displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-kappaB activity and IL-1beta secretion that contributes to serovar Enteritidis virulence.     

Recombinant TGF-beta 1 is synthesized as a two-component latent complex that shares some structural features with the native platelet latent TGF-beta 1 complex

The entire coding region of the human transforming growth factor beta 1 (TGF-beta 1) precursor cDNA has been stably expressed in a human renal carcinoma cell line. Like platelet TGF-beta 1, the recombinant TGF-beta 1 is secreted in a biologically latent form. Immunoblot analysis and gel-filtration indicate that the recombinant latent TGF-beta 1 is a 100-kDa complex in which active 25-kDa TGF-beta 1 is noncovalently associated with the remaining 75 kDa of the processed precursor. Unlike the platelet latent complex, the recombinant latent complex contains no 135-kDa component. Thus, the processed precursor peptide alone is sufficient to confer latency on active TGF-beta 1, and the 135-kDa platelet component has a different role. The processed precursor is similarly glycosylated in recombinant and platelet complexes, and in both has an exposed heparin binding site that may be involved in targeting of the latent complex. Finally, acid activation of recombinant and platelet complexes is reversible, suggesting that the activation process does not cause major structural modifications in the components of the latent complex.     

Purification and some properties of a non-o1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin.

Cholera-like enterotoxin was isolated and purified from the culture supernatant of a non-O1 strain of Vibrio cholerae, E8498, isolated from the environment. Enterotoxin was purified by aluminum hydroxide absorption and elution and successive gel filtrations on Sephadex G-100, Bio-Gel A-5m, and Sephadex G-75. Purified enterotoxin gave a single stained band on polyacrylamide gel disc electrophoresis, and the mobility was the same as that of cholera enterotoxin. The specific biological activity of the purified enterotoxin was almost the same as that of cholera enterotoxin in the Chinese hamster ovary cell assay, fluid accumulation in mouse ligated intestine, increase in vascular permeability in rabbit skin, and passive immune hemolysis. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed that the purified enterotoxin consisted of subunits A and B, identical to those of cholera enterotoxin, and Ouchterlony double gel diffusion tests indicated that the two toxins were immunologically identical. Enterotoxins prepared from several non-O1 strains isolated from human patients were also immunologically identical to cholera enterotoxin.     

Variation in the presence of neuraminidase genes among Streptococcus pneumoniae isolates with identical sequence types

Streptococcus pneumoniae frequently colonizes the upper respiratory tract of young children and is an important cause of otitis media and invasive disease. Carriage is more common than disease, yet the genetic factors that predispose a given clone for disease are not known. The relationship between capsule type, genetic background, and virulence is complex, and important questions remain regarding how pneumococcal clones differ in their ability to cause disease. Pneumococcal neuraminidase cleaves sialic acid-containing substrates and is thought to be important for pneumococcal virulence. We describe the distribution of multilocus sequence types (ST), capsule type, and neuraminidase genes among 342 carriage, middle ear, blood, and cerebrospinal fluid (CSF) pneumococcal strains from young children. We found 149 STs among our S. pneumoniae isolates. nanA was present in all strains, while nanB and nanC were present in 96% and 51% of isolates, respectively. The distribution of nanC varied among the strain collections from different tissue sources (P = 0.03). The prevalence of nanC was 1.41 (95% confidence interval, 1.11, 1.79) times higher among CSF isolates than among carriage isolates. We identified isolates of the same ST that differed in the presence of nanB and nanC. These studies demonstrate that virulence determinants, other than capsule loci, vary among strains of identical ST. Our studies suggest that the presence of nanC may be important for tissue-specific virulence. Studies that both incorporate MLST and take into account additional virulence determinants will provide a greater understanding of the pneumococcal virulence potential.     

Computer simulations to predict the availability of peptides with known HLA class I motifs possibly generated by proteolysis of HIV-1 proteins in infected cells

Cytotoxic T cells that recognize HIV-1 peptides generated from all viral proteins were reported. To predict the cleavage pattern of HIV-1 proteins by cytoplasmic proteasomes into peptides with motifs fitting known HLA class I molecules, the computer program Findpatterns was used. In this paper the combined amino acids patterns for proteolytic cleavages and the HLA class I haplotype-restricted peptides motifs are studied. It was noted that peptides with motifs of HLA class I A2 and A68 were abundant compared with HLA class 5B2, B8, B53, and B35.     

T and B cells target identical regions of the non-collagenous domain 1 of type VII collagen in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a severe immunobullous disease and is caused by IgG against type VII collagen (Col VII) of anchoring fibrils. In this study, utilizing ELISA and immunoblot, 13/15 EBA sera but 0/20 bullous pemphigoid sera and 0/30 healthy control sera showed IgG reactivity with distinct recombinant subregions of the non-collagenous domain 1 (NC1) of Col VII. In two EBA patients, IgG titers against Col VII-NC1 were grossly correlated to clinical disease activity. Moreover, Col VII-reactive T cells were identified in a representative EBA patient which recognized identical subdomains of Col VII-NC1. These findings strongly suggest that (1) the Col VII-NC1 ELISA is a powerful tool for making the diagnosis of EBA, (2) Col VII-specific IgG grossly relates to disease activity and (3) IgG reactivity is associated with T cell recognition of identical subdomains of Col VII-NC1.     

Expression of the early lymphocyte activation antigen CD69, a C-type lectin,is regulated by mRNA degradation associated with AU-rich sequence motifs

CD69 is the earliest inducible cell surface glycoprotein acquired during lymphoid activation both in vitro and in vivo under physiological conditions and inflammation. This molecule is involved in lymphocyte proliferation, and functions as a signal-transmitting receptor in T and B lymphocytes, NK cells and platelets. Molecular cloning of CD69 cDNA revealed that this antigen is a type-II integral membrane protein with a C-type lectin domain in the extracellular region. The expression time course of CD69 mRNA has previously been reported to be transient, peaking around 3 h after induction in T lymphocytes, and declining to nearly resting levels by 8 h. We describe herein studies on the stability of the CD69 mRNA in phorbol ester-activated T lymphocytes. The level of CD69 mRNA in these cells declined rapidly with a half-life of less than 60 min. This finding is consistent with the presence of several AU-rich sequence motifs in the 3′ untranslated region (3′UTR), which have been implicated in the selective destabilization of short-lived mRNA of mammalian cytokines, and proto-oncogenes. We have therefore introduced a fragment of the 3′UTR of the human CD69 cDNA, which contains the AU-rich sequence motifs, into the 3′UTR of the rabbit β-globin gene. This inserted sequence causes the otherwise stable β-globin mRNA to become unstable in vivo. A similar destabilizing effect is observed when the 51-nucleotide AU sequence from the mRNA of the human cytokine granulocyte/macrophage colony-stimulating factor is used as a positive control. Furthermore, the introduction of 194-bp fragment from the CD69 3′UTR containing most of the AU-rich motifs was sufficient to induce the destabilizing effect. We propose that the selective degradation pathway involved in the regulation of the expression of cytokines and proto-oncogenes is implicated in the rapid degradation of CD69 mRNA in activated T lymphocytes. This pathway may constitute a general mechanism to regulate the expression of inducible molecules involved in inflammatory processes.