A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies

Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.     

Prediction of the coding sequences of mouse homologues of KIAA gene: I. The complete nucleotide sequences of 100 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a human cDNA project to predict protein-coding sequences in long cDNAs (> 4 kb) since 1994. The number of these newly identified human genes exceeds 2000 and these genes are known as KIAA genes. As an extension of this project, we herein report characterization of cDNAs derived from mouse KIAA-homologous genes. A primary aim of this study was to prepare a set of mouse. KIAA-homologous cDNAs that could be used to analyze the physiological roles of KIAA genes in mice. In addition, comparison of the structures of mouse and human KIAA cDNAs might enable us to evaluate the integrity of KIAA cDNAs more convincingly. In this study, we selected mouse KIAA-homologous cDNA clones to be sequenced by screening a library of terminal sequences of mouse cDNAs in size-fractionated libraries. We present the entire sequences of 100 cDNA clones thus selected and predict their protein-coding sequences. The average size of the 100 cDNA sequences reached 5.1 kb and that of mouse KIAA-homologous proteins predicted from these cDNAs was 989 amino acid residues.     

Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.     

InCeP: Intracellular Pathway Based on mKIAA Protein-Protein Interactions

Since December 2001, we have been conducting a project to isolateand determine entire sequences of mouse KIAA cDNA clones, whichencode polypeptides corresponding to human KIAA proteins. Theultimate goal of this project has been elucidation of the functionsof KIAA proteins. To address this issue, we have been generating‘libraries’ of antibodies against mKIAA proteins.We have, to date, already generated >800 antibodies. Usingour ‘libraries’ of antibodies, we are now identifyingendogenous mKIAA protein–protein interactions. In thepresent study, novel interactions were identified by MS/MS analysisfollowing immunoprecipitation with anti-mKIAA antibodies. Theinteractions with biologically known molecules should enableus to predict the function of mKIAA/KIAA proteins, includinghypothetical proteins identified in our cDNA project. Theseinteractions are subsequently used for construction of an intracellularpathway related to the mKIAA protein, and the pathway is distributedthrough the InCeP (IntraCellular Pathway based on mKIAA protein–proteininteractions) database. Users can freely access the InCeP throughthe internet and download the graphical display as well as thecurated information.     

Influences of amino acid features of glutathione S-transferase fusion proteins on their solubility

We have previously described our strategy for high-throughput (HT) production of recombinant antigens for anti-mKIAA antibody generation, which involves using shotgun fragments generated during entire sequencing of mKIAA cDNAs. We applied this strategy to 1628 mouse KIAA (mKIAA) cDNA fragments, and 84.2% of the GST-mKIAA fusion proteins were successfully purified. The solubility of the proteins was predicted by a small-scale bacterial culture, and a large-scale culture was then performed according to the expected results. Among them, 43.8% of the proteins were purified as a soluble form and 56.2% as an insoluble form. The average yield of the soluble proteins was 0.15 nmol/mL of bacterial culture, and that of the insoluble proteins was 0.55 nmol/mL Statistical analysis of the data revealed a significant correlation between amino acid features of the recombinant proteins and their solubility. To achieve the most effective and feasible protein expression, we constructed a decision tree in which the analyzed data were reflected. The information described here may provide practical guidelines for HT production of recombinant proteins.     

Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes. We herein present the entire sequences and the chromosome loci of 500 mKIAA cDNA clones and 13 novel cDNA clones that were incidentally identified during this project. The average size of the 513 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 816 amino acid residues. By comparison of the predicted CDSs between mouse and human KIAAs, 12 mKIAA cDNA clones were assumed to be differently spliced isoforms of the human cDNA clones. The comparison of mouse and human sequences also revealed that four pairs of human KIAA cDNAs are derived from single genes. Notably, a homology search against the public database indicated that 4 out of 13 novel cDNA clones were homologous to the disease-related genes.     

Utilization of mammalian cells for efficient and reliable evaluation of specificity of antibodies to unravel the cellular function of mKIAA proteins

Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (>4 kb) with unknown functions. To facilitate the functional analysis of these human clones, we have isolated and determined the structures of their respective mouse homologues (mKIAA genes). Furthermore, we have comprehensively raised antibodies against the translated mKIAA proteins in order to establish a platform for their functional analysis. Since the specificity of these antibodies is critical for subsequent analyses of protein function, here we introduce two assays utilizing mammalian cells to improve their evaluation. First, we have established a semi-high-throughput production of C-terminally FLAG epitope-tagged proteins for Western blotting using specially designed mammalian expression vectors. Secondly, we have utilized immunofluorescence staining of mouse cells to analyze the subcellular localization of endogenous mKIAA proteins. Importantly, these methods allow us to detect potential posttranslational modification of the mKIAA/KIAA proteins and to predict their biological function based on their subcellular localization.     

High throughput monoclonal antibody generation by immunizing multiple antigens

Recognizing proteins via the production of highly specific monoclonal antibodies (mAbs) is crucial to identifying proteins for proteomic research. However, traditional mAb generation is time-consuming with low efficiency. In this study, we assessed the high throughput method of producing mAbs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell fusion. We selected eight proteins that play important roles in human physiological or pathological processes. These proteins were mixed and simultaneously administered to one mouse. We observed the immunizing period for 10 d, and determined the effect of liquid medium and semi-solid medium in hybridoma generation. As a result, all eight immunogens induced antibodies in the immunized mouse in one cell fusion, we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay (ELISA) screening, hybridomas against five out of eight showed specific positive in Western-blotting assays. This indicates that we generated mAbs specific to eight proteins in one cell fusion, greatly increasing the efficiency of mAb generation. Furthermore, we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to antigens. Our study established high-throughput and time-saving methods for production of mAbs. These results provide alternative approaches for increasing the efficacy of mAb generation.     

A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence analysis because it was polymorphic in Southern blot analyses of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa-2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with β2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for either a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolated genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.     

Epitope-selected monospecific antibodies to recombinant antigens from Toxoplasma gondii reacted with dense granules of tachyzoites.

Epitope-selected monospecific antibodies were applied to investigate the localization of antigenic molecules in Toxoplasma gondii by immunoelectron microscopy. Eighty cDNA clones encoding antigenic polypeptides were immunoscreened from lambda gt11 expression library with T. gondii infected mouse sera. Twenty different clones with no crossreactivity were selected from eighty clones. Monospecific antibodies to antigens derived from respective cDNA clones extracted from infected mouse sera by the epitope selection method were used in Western blot analysis and immunoelectron microscopy. Eleven antigens were detected with epitope-selected antibodies in lysates of T. gondii tachyzoites. Five of the antigens with molecular weights of 60, 40, 35, 28, and 27 KD were localized in the dense granules. Monospecific antibodies purified by the epitope selection method were useful for investigating the localization of antigens without preparation of a monoclonal antibody from a hybridoma.     

Mouse monoclonal antibodies to bovine blood group antigens: additional results

Seven fusions of mouse myeloma cells with spleen cells from mice immunized with bovine red cells yielded 61 clones producing discriminant antibodies out of total of 651 secreting clones. Although antigenic factors of all known bovine blood group systems were present on the donors' cells, the antibodies identified reacted with antigenic factors from only five systems, A, B, F, S and Z. The antibody specificities produced by more than two clones were anti-A1 or -A2 (21 clones), -S (9),- Z(6),-G' (3) and -V1 (3). The absence of clones secreting antibodies to antigens of the other systems, especially the complex C system, remains unexplained. The properties of the antibodies reacting with antigens of the S system (anti-SU", anti-SUU') and of the B system (O-like antibodies) are in accordance with previous interpretations of polyclonal sera and with present knowledge of the genetic map of the B system.     

Brugia malayi: recombinant antigens expressed by genomic DNA clones

A Brugia malayi genomic DNA expression library was screened with rabbit antiserum generated against live infective larvae and 33 clones were identified. Five randomly selected clones were characterized in detail by Western blot, DNA and RNA analyses. The fusion proteins produced by each of these recombinant DNA clones are expressed by different genomic sequences. A profile of antigenic cross-reactivities of all 33 recombinant clones was compiled using a battery of antisera, including sera from humans infected with B. malayi. A high percentage of clones were cross-reactive with antisera against the filarial parasites B. pahangi, Dirofilaria immitis, and Onchocerca volvulus. We have made a preliminary identification of three categories of recombinant clones encoding (1) antigens that were cross-reactive with some or all antisera tested, (2) antigens that were specific to the Brugia genus, and (3) antigens that appeared to be specific to B. malayi. These recombinant antigens are candidates for further studies in filarial immunoprophylaxis and diagnosis.