Isolation and characterization of rat olfactory marker protein

The olfactory marker protein was isolated and characterized from rat olfactory bulbs. Its properties and those of the olfactory marker protein isolated from the mouse are described. The rat protein was less acidic (pI = 5.0) than the mouse protein (pI = 4.7). However, the amino acid compositions were very similar: in both proteins arginine plus lysine accounted for 13 mol% and glutamate plus aspartate for 30 mol% of the total residues. Molecular weights of both proteins estimated by sodium dodecyl sulfate gel electrophoresis were indistinguishable and estimated to be 16,500. The molecular weight of the native rat olfactory marker protein estimated by gel filtration techniques was 30,000, which is identical to the molecular weight of the native mouse and garfish olfactory marker proteins. This suggested a dimeric structure. The purified rat and mouse proteins behaved like species of 35,000 molecular weight on gel filtration.     

The cloning and characterization of a new stress response protein. A mammalian member of a family of theta class glutathione s-transferase-like proteins

Using differential display, a cDNA fragment was identified as being overexpressed in a mouse lymphoma cell line that had gained resistance to cell death after exposure to a variety of agents used in cancer therapy. The full-length cDNA of 1.1 kb that was cloned contained an open reading frame coding for a previously unidentified 28-kDa mammalian protein, p28. p28 showed significant homologies to a large family of stress response proteins that contain a glutathione S-transferase (GST) domain. In correspondence with the sequence homology, p28 was found to bind glutathione; however, GST or glutathione peroxidase activity could not be demonstrated. Northern analysis of the mRNA of this protein showed abundant expression in mouse heart and liver tissues, whereas anti-p28 antibody binding identified p28 expression in mouse 3T3 cells and early passage mouse embryo fibroblasts. Subcellular protein fractionation revealed p28 localization in the cytoplasm, but with thermal stress p28 relocated to the nuclear fraction of cellular proteins. Based on sequence homology and protein activity we conclude that p28 acts as a small stress response protein, likely involved in cellular redox homeostasis, and belongs to a family of GST-like proteins related to class theta GSTs.     

Steroid binding by mouse salivary proteins

The goals of this study were to determine the steroid-binding specificity of the mouse salivary androgen-binding protein (ABP) family and to ascertain whether there might be other proteins in mouse saliva capable of binding steroids. The optimal conditions for testosterone binding by mouse salivary proteins were determined using a small-scale chromatography system to separate bound from unbound steroid. Testosterone binding appeared to be biphasic but was directly proportional to saliva concentration, with an optimum temperature of 37 degrees C in the second phase. These results were used to develop a steroid-binding protocol to study the steroid specificity of salivary proteins separated by electrophoresis. The ABP family bound testosterone and progesterone well and HO-progesterone and DHT to a lesser extent but did not bind either cholesterol or estradiol. Steroid structural comparisons suggest that binding by ABP is governed by the A ring of the steroid. Another protein that is not a member of the ABP family bound cholesterol specifically but no protein that specifically bound estradiol was observed.     

Physical mapping of RFLP markers on four chromosome arms in maize using terminal deficiencies

Lactational function in the mammary epithelial cell is subject to complex regulation, most probably involving multiple extracellular and intracellular proteins that act at any of a number of levels. Although some of these proteins have been identified it is likely that additional controllers of lactation exist, but have yet to be discovered. In an effort to identify such proteins, a search was made for non-milk lactation-associated or prolactin-responsive proteins in primary mouse mammary epithelial cells and the mouse mammary epithelial cell line, COMMA-D using two-dimensional electrophoresis on large-format gels. These analyses revealed 12 proteins whose rate of synthesis was dependent on lactation state or on response to prolactin. Two of these (p77 and p63) were lactation-associated in primary cells and prolactin-responsive in COMMA-D cells. These two proteins were identified by amino acid sequencing as glucose-regulated protein 78 (GRP78) and protein disulphide isomerase (PDI). The localization of these proteins in the endoplasmic reticulum and their presence in other secretory cell types and tissues suggests that they have a function in the processing or secretion of milk proteins.     

Molecular identity of a sperm acrosome antigen recognized by HS-63 monoclonal antibody

The molecular identity of mouse sperm acrosome antigen recognized by HS-63 monoclonal antibody was analyzed by various biochemical, immunological and molecular biological methods. When its cognate antigen, MSA-63 was isolated from mouse testis by immunoaffinity chromatography, a group of protein spots with wide range of molecular sizes and isoelectric points were identified. Through previous studies, it was established that most of these protein spots were actin-like molecules co-purified with MSA-63 protein from mouse testis. To analyze the molecular size heterogeneity of the isolated MSA-63 proteins, rabbit antisera against a computer-predicted antigenic synthetic peptide (amino acid residue No. 160-171) and a recombinant glutathione S-transferase (GST) fusion protein (GST-63) were raised. These two antisera and those raised against the isolated MSA-63 protein were used as the probes in comparative Western blot assay, indirect immunofluorescent assay and enzyme-linked immunosorbent assay (ELISA). Using ELISA, antisera against GST-63 and computer-predicted antigenic synthetic peptides were shown to cross-react with affinity-isolated MSA-63 protein coated on microwells. However, little immunological cross-reactivity was observed between GST-63 fusion protein and the synthetic peptide. By using a Western blot assay, two major protein bands of 22 and 32 kDa, respectively were commonly detected on mouse testis homogenate strips by both anti-MSA-63 and anti-GST-63. In addition, anti-MSA-63 also recognized several protein bands with molecular masses greater than 35 kDa. The results of this study suggested that the molecular heterogeneity of MSA-63 protein isolated from mouse testis and sperm, is due to a series of post-translational modifications on a single gene product. These modifications may include glycosylations, proteolytic digestions and tight non-covalent associations with other testicular cytoskeletal proteins, such as actins.     

Evidence of quinone metabolites of naphthalene covalently bound to sulfur nucleophiles of proteins of murine Clara cells after exposure to naphthalene

Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.     

Immunocytochemical characterisation of rabbit and mouse embryonic fibroblasts

By using indirect immunofluorescence we demonstrated the localisation of extracellular matrix (ECM) proteins (laminin--LAM, collagen IV--COL IV, fibronectin--FN) and the basic fibroblast growth factor (bFGF) in rabbit and mouse primary embryonic fibroblasts (PEF). Proliferating mitotically arrested PEF (by mitomycin C) were compared in both species. The stability of protein expression was ascertained during the first five successive passages. In addition, STO cells (i.e. permanent line of irradiated mouse fibroblasts) were similarly analysed. Rabbit PEF showed very high extracellular staining for FN and a negligible cytoplasmic positivity for LAM and COL IV. A totally reversed staining pattern for ECM proteins was found in mouse PEF. A dense cytoplasmic granulation (concentrated around the nucleus) was revealed for LAM and COL IV and almost no reaction for FN. The staining patterns were very stable at the culture conditions we applied. They were maintained during the first five successive passages in proliferating as well as non-proliferating mouse and rabbit PEF and were independent of cell concentration (individually dispersed cells versus cells in a confluent layer). STO cells showed the same staining for ECM proteins as the mouse PEF, thus confirming their origin from the same animal species. Light granular staining for bFGF was found in the cytoplasm of proliferating and mitotically arrested rabbit and mouse PEF and STO cells. The differences in expression of ECM proteins between the rabbit and mouse PEF, as well as the synthesis of bFGF, should be taken into consideration when these cells are used in vitro as a feeder layer for various cells (e.g. embryonic stem cells).     

Identification of protein A-binding components in Spisula oocytes

Components involved in sustaining meiosis arrest of oocytes were determined. Proteins that bind to protein A from meiosis-arrested and 5-HT-matured Spisula oocytes were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Meiosis-arrested oocytes contained three doublets of proteins with estimated Mrs of 43 and 45, 38 and 40, and 21 and 23 kDa. In 5 HT-matured oocytes the 21 and 23 and 38 and 40 kDa proteins were retained; whereas the 43 and 45 kDa proteins were absent. The protein A-bound proteins did not interact with antibodies against the various subclasses of human, mouse, rat and rabbit IgG or human Fc fragment. The amino acid sequence of the N-terminus of the 43 kDa protein was determined to be NH2-VLRIGSGMXDT. Comparison of this sequence with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) showed no homology with any reported protein. The protein A-bound components from meiosis-arrested oocytes were incubated in vitro with [gamma-32P]ATP. Only the 68 kDa protein was radiophosphorylated. This protein was not detected in 5-HT-matured oocytes. The disappearance of the 43, 45, and 68 kDa proteins in 5-HT-matured oocytes suggests that these components may be involved in sustaining meiosis meiosis. A unique property of these proteins is that they interact with protein A and are distinctly different from immunoglobulin.     

Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines

BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.     

Activation of a murine autoreactive B cell by immunization with human recombinant autoantigen La/SS-B: characterization of the autoepitope

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.     

Definition of family of coronin-related proteins conserved between humans and mice: close genetic linkage between coronin-2 and CD45-associated protein

Cell adhesion and signal transduction are coordinated processes that may be linked through regulatory elements such as actin-binding proteins. One such protein that may fulfill this role is coronin. In Dictyostelium discoideum, coronin is involved in cellular processes such as mitosis, cell motility, and phagocytosis. In addition, a human coronin, p57, has been described which interacts with the p47 component of phox proteins and may be involved in the formation of phagocytic vacuoles. Here, we describe a family of four mouse proteins which share 38% identity with Dictyostelium coronin and thus are designated coronin-1, -2, -3, and -4. The gene for coronin-2 is localized to mouse chromosome 19, 5' of the gene for CD45-associated protein. All the coronin proteins contain five highly conserved WD domains. However, their carboxyl regions are quite distinct. Three of the four proteins are ubiquitously expressed, whereas coronin-1, the mouse ortholog of p57, demonstrates expression restricted to hematopoietic cells. Comparison of expressed sequence tag cDNAs indicates that coronin-1, -2, -3, and -4 are highly conserved between mice and humans.     

Primary structure of matrilin-3, a new member of a family of extracellular matrix proteins related to cartilage matrix protein (matrilin-1) and von Willebrand factor

A mouse cDNA encoding for matrilin-3, the third member of the novel matrilin family of extracellular matrix proteins, was cloned. The protein precursor of 481 amino acids consists of a putative signal peptide, a short positively charged sequence, a single vWFA-like domain followed by four epidermal growth factor-like modules and a potential coiled-coil alpha-helical oligomerization domain at the C-terminus. It is the smallest member of the matrilin family with a predicted Mr of the mature protein of 48 902. The primary structure of a C-terminal portion of 310 amino acids of the human matrilin-3 was determined and showed a sequence identity to the mouse matrilin-3 of 84.8%. Northern blot hybridization of mouse matrilin-3 mRNA showed a 2.9 kb mRNA expressed in sternum, femur and trachea and indicates a cartilage-specific expression.     

A sec1-related vesicle-transport protein that is expressed predominantly in epithelial cells

Sec1-related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Sec1-related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc-18-2/Munc-18b/muSec1 protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associates in vitro with syntaxins 1A, 2, and 3. In situ hybridization analysis of embryonic mouse tissues revealed prominent expression of the munc-18-2 mRNA in the epithelia of several tissues. Cell-fractionation studies demonstrated that the majority of Munc-18-2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc-18-2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS-1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin 1A, it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc-18-2 as a predominantly epithelial vesicle-transport protein with a polarized distribution and provided novel in vivo evidence for the association of Sec1-related proteins with members of the syntaxin family.     

Structure of mouse Mx1 protein. Molecular assembly and GTP-dependent conformational change

Mouse Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection. The Mx1 protein purified from interferon-induced A2G mouse liver exhibited GTPase activity as did the Mx1 protein purified from the Mx1 cDNA-expressing Escherichia coli (Nakayama, M., Nagata, K., Kato, A., and Ishihama, A. (1991) J. Biol. Chem. 266, 21404-21408; Nakayama, M., Nagata, K., and Ishihama, A. (1992) Virus Res. 22, 227-234). The Mx1 protein purified from both mouse liver and Mx1-cDNA expressing E. coli was found to exist as assembled polymeric states judged from gel filtration pattern. By making a set of deletion derivatives of the Mx1 cDNA, the main motif for self-assembly of the Mx1 protein was mapped between amino acid residues 51-99. This motif is highly conserved not only in the Mx family of proteins but also in Mx-related proteins. The polymeric form of Mx1 from E. coli was observed as "horseshoe"-like structure by negative staining microscopy. When the Mx1 protein was incubated with GTP, this horseshoe structure was transformed to larger and tightly stacked helical forms. Electron microscopic analysis of immunostained liver of the interferon-induced mice indicated that the Mx1 protein exists in nuclei, forming giant complexes of about half the size of nucleoli.     

Nuclear proteins from liver and kidney bind a 37 bp sequence in the 5' upstream region of the mGAT1 gene

The mouse GABA transporter (mGAT1) gene has been shown to be exclusively expressed in brain by Northern and Western blot analyses. The interactions between the 5' flanking region of the mGAT1 gene and nuclear proteins from different mouse tissues were studied by means of gel-shift assay. Our results show that nuclear protein factors from non-nervous tissues can specifically recognize a 37 bp sequence that is conserved in the 5' flanking region between the human and mouse GAT1 genes. Similar nuclear protein factors were also found to exist in rat, rabbit and pig.