The N-terminal regions of eukaryotic acidic phosphoproteins P1 and P2 are crucial for heterodimerization and assembly into the ribosomal GTPase-associated center

Acidic phosphoproteins P1 and P2 form a heterodimer and play a crucial role in assembly of the GTPase-associated center in eukaryotic ribosomes and in ribosomal interaction with translation factors. We investigated the structural elements within P1 and P2 essential for their dimerization and for ribosomal function. Truncation of the N-terminal 10 amino acids in either P1 or P2 and swapping of the N-terminal 10 amino acid sequences between these two proteins disrupted their dimerization, binding to P0 and P0 binding to rRNA. In contrast, truncation of the C-terminal halves of P1 and P2 as well as swapping of these parts between them gave no significant effects. The protein dimers containing the C-terminal truncation mutants or swapped variants were assembled with P0 onto Escherichia coli 50 S subunits deficient in the homologous protein L10 and L7/L12 and gave reduced ribosomal activity in terms of eukaryotic elongation factor dependent GTPase activity and polyphenylalanine synthesis. The results indicate that the N-terminal 10 amino acid sequences of both P1 and P2 are crucial for P1-P2 heterodimerization and for their functional assembly with P0 into the GTPase-associated center, whereas the C-terminal halves of P1 and P2 are not essential for the assembly.     

重组表达的着丝粒蛋白-B N-端抗原在抗着丝粒自身免疫病血清检测中的应用

目的 分析重组表达的CENP-B抗原对抗着丝粒自身免疫病血清的鉴定结果，探讨其作为临床诊断指标的可行性。方法 通过构建原核表达载体，在大肠杆菌中高效表达CENP-B-N端300个氨基酸的1段多肽；纯化包涵体，制备重组表达的CENP-B抗原，并利用ELISA、Western blot的方法对临床诊断为阳性的ACA血清进行鉴定。结果 重组表达的CENP-B N-端抗原对ACA血清的ELISA阳性检出率为99％，Western blot阳性检出准确率为97％。结论 重组表达的CENP-B抗原可以作为抗着丝粒自身免疫病血清临床诊断的1个指标。     

Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro: role of DNA-binding and self-associating activities in kinetochore organization

Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere auto-antigens. In a previous study, we showed that it possesses DNA-binding activity in vitro. Recently, centromere-binding activity was suggested at the C-terminal region in vivo. However, little is known about the role of CENP-C in kinetochore organization. Here, to characterize its biochemical properties, three separate antigenic regions of human CENP-C were expressed in Escherichia coli, affinity purified and used in South-western blotting and chemical cross-linking analyses. We found that the internal DNA-binding domain was composed of two kinds of elements: the core and two flanking stabilizing elements that support the activity. When cross-linked with disuccinimidyl suberate (DSS), the N-terminal region produced the ladder bands of dimerand tetramer: the C-terminal region exclusively produced the dimer band, whereas the internal region was not affected at all. Dimer formation at the C-terminus in the native state was also indicated by gel filtration and the presence of conformation-specific autoantibodies in the patient's sera. These results suggest that human CENP-C consists of three functional units required for kinetochore assembly: a putative N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain.This revised version was published online in November 2005 with corrections to the Cover Date.     

Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3

Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases. CENP-A is one of the major antigens against ACA and a histone H3-like protein. To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera. Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3. Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3-17 (peptide A) and 25-38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA.     

Low prevalence of autoantibodies to CENP-H, -I, -K, -L, -M, -N, -T and -U in a Japanese cohort of anti-centromere positive samples

Objective: The constituents of the centromere region, centromere protein (CENP)-A, -B, and -C, are mainly targeted by anticentromere antibodies (ACA). Many other proteins also assemble around CENP-A nucleosomes in interphase nuclei to form the interphase centromere complex (ICEN). CENP-H, -I, -K, -L, -M, -N, -T, and -U have been reported as the constitutive components of ICEN. In this study, we examined the reactivities of ACA to the 8 CENPs for the purpose of investigating their autoantigenicity.

Methods: Sera from 95 patients with ACA were tested by western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) with the recombinant C-terminal of CENP-B (Ct-CENP-B). Next, the sera were examined for autoantibodies against the 8 CENPs by WB with each recombinant protein. Furthermore, the coiled-coil motifs and granzyme B (GB) cleavage for various CENPs were analyzed with computer tools.

Results: Out of 95 ACA-positive sera, 85 and 93 sera were positive for anti-Ct-CENP-B antibodies in WB and in ELISA, respectively. In WB using the 8 CENPs, no sera reacted to any other 7 CENPs, except 1 serum, which reacted weakly to CENP-T. We were unable to find any obvious relationships between the autoantigenicity of CENPs and coiled-coil-forming probabilities or potential substrates for GB.

Conclusion: This study demonstrates that ACA rarely target the 8 CENPs, in contrast to CENP-B. Protein structures might not contribute in a major way to the autoantigenicity of CENPs.     

血红素加氧酶在大肠杆菌中稳定表达的氨基酸范围的研究

目的　确定在原核细胞中呈可溶性稳定表达但不改变活性的血红素加氧酶氨基酸范围。方法　应用PCR技术构建了鼠血红素加氧酶 1(RHO 1)和人血红素加氧酶 2 (HHO 2 )的N端和C端部分氨基酸缺失的变异型RHO 1和HHO 2 ,将它们在大肠杆菌中表达并纯化后通过光谱扫描法测定变异型酶与野生型酶的催化活性 ,确定RHO 1和HHO 2中的N端和C端缺失不影响其可溶性表达及催化活性的氨基酸范围。结果　△N8RHO 1(N端缺失 8个氨基酸的RHO 1)和△C70RHO 1在大肠杆菌中表达为可溶性蛋白且催化活性与RHO 1野生型相同 ,△N2 0HHO 2和△C77HHO 2在大肠杆菌中也表达为可溶性蛋白且催化活性与HHO 2野生型相同 ,而△N9RHO 1和△C75RHO 1在大肠杆菌中表达为包涵体。结论　RHO 1和HHO 2的N端和C端部分氨基酸缺失的突变型酶对其催化功能没有影响 ,并可在大肠杆菌中稳定可溶性表达。     

Relationship between autoepitope and DNA-binding site on a histone H1 molecule.

Autoepitope and DNA-binding domain on a histone H1 molecule were compared using truncated histone H1 peptides as antigens. At least two epitopes (epitope A, N-terminal side; epitope B, C-terminal side) were found both of which were composed of approximately 20 amino acids. IgM from all 17 anti-histone H1-positive SLE sera reacted with epitope A. IgG from 12 sera reacted with epitope A and IgG from 4 sera reacted with epitope B. In one case, no IgG anti-histone H1 reactivities were found while IgM from the same patient reacted with epitope A. Epitope A had the ability to bind DNA. The reactivities against histone H1 of affinity-purified antiepitope A autoantibodies were inhibited by DNA. These data suggest that some anti-histone H1 antibodies are directed against a histone H1 DNA-binding site, raising the possibility that an idiotype/anti-idiotype network, at least in part, is involved in the generation of anti-histone H1 autoantibodies.     

Anti-centromere antibodies (ACA) in systemic sclerosis patients and their relatives: a serological and HLA study

Autoantibody reactivity to centromere proteins CENP-A, CENP-B and CENP-C was examined in 58 patients with systemic sclerosis (SSc). 218 first degree relatives and 22 spouses, HLA class II typing for HLA-DRB1 and HLA-DQA1 was performed by restriction fragment length polymorphism (RFLP) analysis in 50 families, and HLA-DRB1, HLA-DQA1 and HLA-DQB1 typing was performed by olignucleolitde typing in 44 families. Eleven probands and two relatives had ACA. The two relatives with ACA also had SSc. One relative was an identical twin sister of a pro band with ACA and the other relative was a sister of a proband with ACA. All ACA-positive probands and relatives were female, and all recognized CENP-A, CENP-B and CENP-C. The presence of at least one HLA-DQB1 allele not coding for leueine at position 26 of the first domain appeared necessary, although not sufficient for the generation of ACA, Therefore within SSc families ACA is strongly associated with female gender and disease phenotype, and is at least in part genetically determined.     

Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens.

We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I.     

Antigenic determinants of the 70-kDa subunit of the Ku autoantigen.

Autoantibodies against Ku antigen were found in subsets of sera from patients with rheumatic diseases. The Ku autoantigen was characterized as a DNA-binding protein complex composed of two subunits, 70 and 86 kDa. In this study, we report the amino acid sequences of the 70-kDa subunit that are important for interactions with a monoclonal and autoimmune antibodies. Full-length cDNA and numerous 5' and 3' deletion mutants were expressed in bacteria and the immunoreactivity of the fusion proteins was analyzed by Western blotting. The reactivity of the monoclonal antibody depended on the region between Ile321 and Phe350. Ten autoimmune sera were tested for reactivity with deletion mutants in immunoblots. The reactivity of six sera strongly depended on the C-terminal amino acids and four sera did not show such dependence; however, these C-terminal sequences did not react with the sera when expressed alone. These results strongly suggest the conformational nature of the Ku autoepitopes. Interestingly, the DNA-binding activity of this Ku protein subunit analyzed by Southwestern blot depended on the same C-terminal amino acids that were involved in interactions with autoantibodies, indicating that anti-Ku autoantibodies are directed to conformationally intact Ku antigen. Reactivities of the autoimmune sera with Met1-Arg115, Met116-Val149, and Val149-Arg586 were also observed. These results demonstrate that different amino acid regions can be involved in interactions with autoimmune antibodies.     

Dephosphorylation of phosphotyrosine on STAT1 dimers requires extensive spatial reorientation of the monomers facilitated by the N-terminal domain

We report experiments that infer a radical reorientation of tyrosine-phosphorylated parallel STAT1 dimers to an antiparallel form. Such a change in structure allows easy access to a phosphatase. With differentially epitope-tagged molecules, we show that the two monomers of a dimer remain together during dephosphorylation although they most likely undergo spatial reorientation. Extensive single amino acid mutagenesis within crystallographically established domains, manipulation of amino acids in an unstructured tether that connects the N-terminal domain (ND) to the core of the protein, and the demonstration that overexpressed ND can facilitate dephosphorylation of a core molecule lacking an ND all support this model: When the tyrosine-phosphorylated STAT1 disengages from DNA, the ND dimerizes and somehow assists in freeing the reciprocal pY-SH2 binding between the monomers of the dimer while ND ND dimerization persists. The core of the monomers rotate allowing reciprocal association of the coiled:coil and DNA-binding domains to present pY at the two ends of an antiparallel dimer for ready dephosphorylation.     

Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway inflammation of asthma. iNOS is active only as a homodimer. We previously demonstrated that the region encoded by exons 8 and 9 is critical for dimerization. In this study, alanine-scanning mutagenesis was used to identify critical amino acids in that region by expression of mutant proteins in human embryonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected by Western analysis. Four iNOS mutants with alanine replacing Trp260, Asn261, Tyr267, or Asp280 did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degrees C, followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267, however, migrated only as monomers, suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids in exons 8 and 9 critical for iNOS activity, three of which also influence dimerization. These residues are strictly conserved among all NOS isforms and across species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic activity. These data increase our understanding of the structural elements critical for NO synthesis and lay the groundwork for future studies aimed at downregulation of iNOS activity.     

Immunogenic B-cell epitopes of Babesia bovis rhoptry-associated protein 1 are distinct from sequences conserved between species.

Babesia bovis merozoite apical membrane polypeptide Bv60 was found to be rhoptry associated by immuno-electron microscopy and was redesignated rhoptry-associated protein 1 (RAP-1). The N-terminal 300 amino acids of RAP-1 have a high level of sequence similarity to the same N-terminal region of p58, its homolog from Babesia bigemina. However, the interspecies conserved sequences did not include RAP-1 surface-exposed B-cell epitopes as defined by monoclonal antibodies. Furthermore, neither heterologous B. bigemina immune nor monospecific anti-p58 bovine serum binds to whole RAP-1, indicating that the major B-cell epitopes recognized by these sera are also not encoded by the conserved sequences. Truncated RAP-1, expressed by a subclone encoding the N-terminal 235 amino acids, is weakly bound by antibodies in heterologous sera. A peptide representing the longest conserved amino acid sequence (amino acids 121 to 134) in this region is also weakly bound by antibodies in immune bovine sera, and rabbit antibodies induced by and strongly reactive with the peptide alone fail to bind native or denatured RAP-1. Thus, although the conserved region may contain one or more poorly immunogenic B-cell epitopes, these epitopes are inaccessible to antibody in whole RAP-1. The results indicate that the major immunogenic B-cell epitopes of RAP-1, including surface-accessible epitopes bound by monoclonal antibodies, are distinct from the conserved sequences representing putative functional domains.     

Proteins responsible for anticentromere activity found in the sera of patients with CREST-associated Raynaud''s phenomenon.

Anticentromere antibodies (ACA) present in a high percentage of patients with complete or incomplete CREST scleroderma, and which are presently used in the diagnosis of this disease, also appear in some primary Raynaud's phenomenon patients. Three principal centromeric antigens, CENP-A, CENP-B and CENP-C, have been described as reacting with the sera of these individuals. We attempt to determine whether or not a correlation between the presence of ACA and serum reactivity against one or more of these peptides could be established, and have observed that CENP-A, but not CENP-B or CENP-C, is specifically recognized by all patients sera tested. The fact that this reactivity is clearly detectable at very high serum dilutions, thus eliminating other non-specific interference, suggests that anti-CENP-A activity might be useful in the diagnosis of patients with CREST-associated Raynaud's phenomenon.     

Mutagenesis of the dimer interface residues of tethered and untethered HIV-1 protease result in differential activity and suggest multiple mechanisms of compensation

As is the case for all retroviruses, the protease of HIV-1 is only functional as a homodimer; dimerization of two protease monomers results in the formation of the enzyme active site. This dimer structure is supported primarily by interactions between the first four amino-terminal and the last four carboxy-terminal amino acids. These eight amino acids form a beta-sheet in which hydrophobic residues are oriented towards the core of the molecule and polar residues are directed towards the solvent. Although the structure of the dimer interface has been determined, the forces that support dimerization have not been fully characterized. Here, we describe a tethered construct in which two protease monomers are joined by a 5 amino acid linker. We evaluate the relative role of each dimer interface residue in functional homo- and heterodimers. Our studies indicate that the hydrophobic residues of the dimer interface are particularly important in maintaining enzyme activity and that enzyme activity is more sensitive to substitutions of the C-terminal amino acids. Further, we demonstrate that the presence of the tether is able to compensate for mutations within the dimer interface that inactivate the enzyme.     

Preservation of enzyme activity and antigenicity after mutagenesis of the membrane anchoring domain of GAD65

The smaller isoform of glutamic acid decarboxylase, GAD65, is an important autoantigen implicated in the pathogenesis of type 1 diabetes whereas the larger isoform, GAD67 appears to play no major role. The primary difference between the two isoforms resides in the N-terminal part of the molecule including the GAD65 membrane-anchoring domain. The aim of this study was to generate mutants of the membrane targeting domain spanning amino acids 24 to 31 of GAD65 to determine effects on enzyme activity and antibody recognition. Three GAD65 mutants were generated by substituting two, nine or eleven nucleotides coding for the membrane targeting with the corresponding bases of GAD67. SDS-PAGE and Western blotting wildtype (wt) and mutated GAD65 ascertained that they were of similar size and recognized GAD65-specific antibodies. No difference in enzymatic activity was found between the mutants and wt GAD65. GAD65 antibody positive sera from type 1 diabetes patients immunoprecipitated mutated GAD65 whether two, nine or eleven nucleotides were replaced. Mono-or polyclonal antibodies to the N-terminal region demonstrated that the mutated GAD65 with two or nine nucleotides replaced was immunoprecipitated markedly better than wt whereas no difference was detected using antibodies specific for the PLP-binding site in the middle part of GAD65 or the C-terminal region. Taken together, these data suggest that no major conformational changes have been introduced by mutating the membrane-anchoring domain of GAD65.     

Immunological Characterization of α Antigen of Mycobacterium kansasii: B-Cell Epitope Mapping

Mapping of B-cell epitopes on α antigen of Mycobacterium kansasii (K-α) was carried out by using recombinant truncated K-α fusion peptides. We observed that two immunodominant B-cell epitopes (amino acids 222–268 and 267–306) and one minor epitope (amino acid 249–286) were located in the C-terminal region of K-α. The other three minor B-cell epitopes were mapped in N-terminal (amino acids 80–98 and 99–166) and central (amino acid 174–204) regions of K-α. All defined epitopes were common to Mycobacterium tuberculosis and M. kansasii . Besides these common epitopes, a region in K-α (amino acid 290–319) revealed different reactivities between antibodies against K-α and α antigen of M. tuberculosis . These findings may provide a basis for development of serodiagnosis that can distinguish between M. kansasii and M. tuberculosis infections.     

Immunological characterization of heterochromatin protein p25β autoantibodies and relationship with centromere autoantibodies and pulmonary fibrosis in systemic scleroderma

 Anticentromere antibodies (ACA) are immunological markers for the subset of systemic scleroderma with the symptoms calcinosis cutis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia (CREST). In western blotting, some ACA-positive sera also recognize a doublet of 23 kDa (p23) and 25 kDa (p25) in addition to centromere protein antigens A (17 kDa), B (80 kDa), and C (140 kDa). Two forms of p25 have been shown to be human homologues of Drosophila heterochromatin-associated protein HP1. One form of p25 (p25β) which was recently cloned in this laboratory was used to evaluate anti-p25β antibody response in scleroderma sera. Of 318 scleroderma sera 42 had ACA (13.2%), and 16 of the 42 sera (38%) had anti-p25β antibodies. On the other hand, 5 of 276 ACA-negative sera (1.8%) showed anti-p25β antibody response, demonstrating that anti-p25β antibody is significantly associated with the ACA response (P<10^–8). Clinically the anti-p25β response was significantly associated with the CREST syndrome. Fourteen (36.8%) of 38 CREST patients compared to seven (2.5%) of 280 patients with other forms of scleroderma were anti-p25β antibody positive (P<10^–8). The 14 CREST patients with anti-p25β antibodies had significantly more interstitial lung disease than those without anti-p25β antibodies (P<0.003). There was also a tendency to increased liver involvement. Two dominant autoepitopes in p25β were determined by western blotting using p25β recombinant fragments. In immunofluorescence C-terminal specific antibodies showed staining of heterochromatin, but N-terminal specific antibodies showed no staining. Interestingly, the majority of sera reacted preferentially with one or the other of the two dominant autoepitopes. Received: 6 March 1997 / Accepted: 21 July 1997     

New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein.

Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP). To search for new epitopes in the non-repetitive part of the CS protein, we expressed the non-repetitive regions of the protein in E. coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues. These fusion proteins were obtained at greater than 70% purity by a single Ni-chelate affinity chromatography step. Of the new epitopes defined in the C-terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C-terminal of the protein's central repetitive domain. Pooled sera from inhabitants of a malaria-endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites. Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein. The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates. One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites. Antibodies which react with non-repetitive regions of the CS protein could contribute to maintaining its genetic variability.