A Drosophila melanogaster gene encodes a protein homologous to the mouse t complex polypeptide 1

We have isolated and sequenced a cDNA from Drosophila melanogaster that is homologous to the mouse Tcp-1 gene encoding the t complex polypeptide 1, TCP-1. The Drosophila gene maps by in situ hybridization to bands 94B1-2 of the polytene chromosomes. It shares 66% nucleotide sequence identity with the mouse gene. The predicted Drosophila protein consists of 557 amino acids and shares 72% identity with the mouse polypeptide. The TCP-1 polypeptide appears to be highly conserved in evolution from mammals to simple eukaryotes because the Drosophila gene probe also detects related sequences in DNA from the yeast, Saccharomyces cerevisiae. The presence of TCP-1-related polypeptides in organisms such as Drosophila and yeast should facilitate biochemical and genetic analysis of its function.     

Sequence and structural homology between a mouse T-complex protein TCP-1 and the 'chaperonin' family of bacterial (GroEL, 60-65 kDa heat shock antigen) and eukaryotic proteins

A mammalian cytoplasmic protein TCP-1, encoded by a gene within the mouse t-complex, has been found to exhibit highly significant (p much less than 0.00001) sequence homology to the 'chaperonin' family of bacterial and eukaryotic proteins (viz. groEL protein of E. coli, rubisco subunit binding protein of plant chloroplasts, yeast hsp58 and mammalian P1 proteins and 60-65 kDa mycobacterial antigen). With the introduction of few gaps, the amino acid sequence of TCP-1 shows between 60-63% similarity (17-20% identical residues and 42-45% conserved substitutions) throughout its length to various chaperonin proteins, indicating a common evolutionary origin. The sequence data also suggest that in contrast to the endosymbiotic origin of mitochondrial and chloroplast chaperonins, the cytoplasmic TCP-1 may have directly descended from the common universal ancestor via eukaryotic lineage. The observed similarity between TCP-1 and the 60-65 kDa bacterial 'common antigen' is also of importance from the viewpoint of immune/autoimmune response.     

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells.

The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus.     

Chromosomal assignment of the murine gene encoding the transformation-related protein p53.

p53 is a transformation-related protein that is encoded by the cellular genome and is synthesized at elevated levels in a wide range of different cell line types and in primary tumors of various species. By using several independently established anti-p53 monoclonal antibodies, it was possible to distinguish between p53 of mouse origin and p53 of Chinese hamster origin. By analysis of a series of mouse X Chinese hamster hybrid cell lines containing various mouse chromosomes, we mapped the p53 gene product to mouse chromosome 11.     

The t complex polypeptide 1 (TCP-1) is associated with the cytoplasmic aspect of Golgi membranes

The t complex polypeptide 1 (TCP-1) is a protein of unknown function expressed in large amounts during spermatogenesis. Rat monoclonal antibodies recognizing TCP-1 have been prepared and used to immunoprecipitate and Western blot a 57 kd protein from germ cell and tissue culture cell extracts. In tissue culture cells, indirect immunofluorescent localization of antigen indicated a perinuclear distribution similar to that of the Golgi apparatus. Analysis of the TCP-1 distribution in tissue culture cells showed that the polypeptide was associated with the cytoplasmic aspect of membranes of the trans-Golgi network (TGN). The distribution in spermatids suggested that TCP-1 was localized to structures often associated with the developing acrosome. The TCP-1 antigenic epitopes are highly conserved, allowing the protein to be identified in cells across a wide variety of vertebrate species and tissues. These experiments suggest that TCP-1 may be essential for transport of proteins through the exocytic pathway in all cells and required in large amounts for acrosome formation in developing spermatids.     

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.     

Cell-specific modulation of papovavirus replication by tumor suppressor protein p53

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.     

Localization of calcium-binding protein parvalbumin-immunoreactive neurons in mouse and hamster visual cortex

Calcium-binding proteins are thought to play important roles in regulating intracellular calcium in the central nervous system. In the present study, we investigated the distribution and morphology of neurons containing parvalbumin in the visual cortex of mouse and hamster. The calcium-binding proteins were localized using immunocytochemistry. Parvalbumin-immunoreactive neurons were located in all layers except layer I. The highest density of parvalbumin immunoreactivity was found in layer V of both mouse and hamster. The labeled neurons varied in morphology. The majority of the parvalbumin-immunoreactive neurons both in mouse and hamster visual cortex was stellate and round, or oval with multipolar dendrites. These results indicate that the calcium-binding protein parvalbumin is contained in specific layers and in selective cell types of the mouse and hamster visual cortex. The distribution of parvalbumin in the mouse visual cortex is very similar to that of hamster.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.     

T-complex polypeptide-1 interacts with the erythrocyte cytoskeleton in response to elevated temperatures

Chaperonins are double ring complexes composed of highly conserved 60-kDa protein subunits that are divided into two subgroups. Group II chaperonins are found in archaea and the cytoplasm of eukarya and are believed to function like other chaperonins as part of a protein folding system. We report here that human erythrocytes contain the group II chaperonin T-complex polypeptide 1 (TCP-1) and that this complex translocates from the cytoplasm to the cytoskeleton in response to heat treatment in the absence of overt cell damage. Identification as TCP-1 was determined by immunodetection for TCP-1alpha and corroborated by mass spectroscopy peptide sequencing. Direct visualization by immunofluorescence confirmed peripherally localized TCP-1 in response to heat treatment. Temperatures ranging from 37-50 degrees C were demonstrated to have distinct kinetic profiles of induced translocation. Heat-induced binding was shown by Triton shell analysis to be specifically associated with the cytoskeletal proteins. Furthermore, the binding was reversible following removal of the stimulatory condition. A stabilizing process is hypothesized based on the known interactions of chaperonins.     

A scaffold for the Chinese hamster genome

Chinese hamster ovary (CHO) cells are a prevalent tool in biological research and are among the most widely used host cell lines for production of recombinant therapeutic proteins. While research in other organisms has been revolutionized through the development of DNA sequence-based tools, the lack of comparable genomic resources for the Chinese hamster has impeded similar work in CHO cell lines. A comparative genomics approach, based upon the completely sequenced mouse genome, can facilitate genomic work in this important organism. Using chromosome synteny to define regions of conserved linkage between Chinese hamster and mouse chromosomes, a working scaffold for the Chinese hamster genome has been developed. Mapping CHO and Chinese hamster sequences to the mouse genome creates direct access to relevant information in public databases. Additionally, mapping gene expression data onto a chromosome scaffold affords the ability to interpret information in a genomic context, potentially revealing important structural and regulatory features in the Chinese hamster genome. Further development of this genomic scaffold will provide opportunities to use biomolecular tools for research in CHO cell lines today and will be an asset to future efforts to sequence the Chinese hamster genome.     

Molecular cloning and sequence analysis of a haploid expressed gene encoding t complex polypeptide 1

The mouse t haplotypes show defects in spermatogenesis attributed to multiple loci on chromosome 17. We have cloned the gene for an abundant testicular germ cell protein, t complex polypeptide 1, which has a variant form in t haplotypes, TCP-1A. A cDNA clone, pB1.4, which hybridizes to a 19S mRNA that is abundant in haploid cells during mouse spermatogenesis, derives from the 3' end of the mRNA encoding TCP-1B. The Tcp-1 gene appears to be a member of a novel gene family and shows multiple changes between the predicted amino acid sequences of TCP-1B and TCP-1A. An additional Taq1 site is created by a T to C transition in the predicted open reading frame of the Tcp-1a gene. The resultant RFLP has allowed typing of the Tcp-1 gene cluster in 54 complete and partial t haplotype chromosomes. DNA sequence comparison of the Tcp-1 genes suggests that the t haplotype chromosome arose within the genus Mus more than one million years ago.     

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.     

Genetic mapping of the gene for androgen-binding protein/sex hormone-binding globulin to mouse chromosome 11

Southern blot analysis of DNAs from Chinese hamster x mouse and rat x mouse somatic cell hybrids showed that the mouse gene encoding androgen-binding protein/sex hormone-binding globulin (ABP-SHBG) is on Chromosome 11. Progeny from an intersubspecies backcross were analyzed to position this locus, termed Shbg, between Il-3 and Int-4 in the middle of this chromosome. Shbg is thus closely linked to several neurological mutations, one of which, Tr, is also associated with male sterility. The recent finding that ABP-SHBG is found throughout the rat brain raises the possibility that one of these mutations may be due to a defect in Shbg.     

A transmembrane tight junction protein selectively expressed on endothelial cells and platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.     

Mitochondrial binding of a protein affected in mutants resistant to the microtubule inhibitor podophyllotoxin

Specific antibodies to a protein P1 Mr approximately equal to 63,000) from Chinese hamster ovary cells, which is affected in mutants resistant to the microtubule inhibitor, podophyllotoxin, and behaves like a microtubule-related protein by certain criteria [14], have been raised. The antibody reacts specifically with the P1 protein in one- and two-dimensional immunoblots, and a cross-reacting protein of similar molecular mass and electrophoretic mobility is also found in cells from various vertebrate and invertebrate species. The observed similarity in the peptide maps of the cross-reacting protein from human, mouse, Chinese hamster and chicken cells indicates that the structure of this protein should be highly conserved. However, no P1-antibody cross-reacting protein was observed in plants (corn, mung), fungus (Neurospora crassa), yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli and Salmonella typhimurium). Immunofluorescence studies with the P1-antibody show that, in interphase cells of various cross-reacting species, it bound specifically to mitochondria which were associated and distributed on and along the length of microtubules. Similar association and codistribution of mitochondria and microtubules were not observed in mitotic cells. Some implications of the mitochondrial localization of the protein P1 and the observed association between microtubules and mitochondria are discussed.     

Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities

In the biopharmaceutical industry, recombinant protein drugs are commonly produced in Chinese hamster ovary (CHO) cells. During the development process, removal of CHO cell-derived proteins from the biopharmaceutical product is monitored using multi-product immunoassays. Such immunoassays are developed by raising antibodies to a single CHO cell protein preparation. However, these assays are utilized to monitor CHO cell protein impurities during the recovery of products from different CHO cell lines. To address whether underlying differences between CHO cell lines result in sufficient protein expression changes to exclude the suitability of multi-product immunoassays, a comparative proteomics study of three independently generated CHO cell lines was performed. Statistical analysis of over 1000 proteins resolved by 2-D PAGE demonstrated that the protein expression profiles of three different CHO cell lines exhibit very few differences in protein expression. Only 11 qualitative changes in protein expression and 26 quantitative changes greater than two-fold were observed. Identification of protein spots by mass spectrometry revealed that many of the observed changes were due to post-translational modifications rather than expression of novel proteins in each cell line. These results suggest that multi-product immunoassays are suitable for monitoring host cell proteins in biopharmaceuticals produced in different CHO cell lines.     

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.