Changes in the levels of low-abundance brain proteins induced by kainic acid.

Low-abundance gene products are of interest in proteomic studies, because they are probably involved in disease-related changes and their altered levels or modifications may carry significant biological information. Detection of low-abundance proteins of a proteome is one of the major limitations of proteomics and a scientific challenge. We investigated the changes in the levels of low-abundance rat brain cytosolic proteins after administration of kainic acid, a potent neurotoxin and excitatory amino acid. The cytosolic proteins from controls and animals treated with kainic acid were fractionated on an ion-exchange column. The fractions collected were analyzed by 2D electrophoresis, and the proteins with altered levels were identified by matrix-assisted laser desorption ionization or ion-spray MS. We found a manifold decrease in annexin VII, heat-shock cofactor HOP/p60 and SP-22 and a manifold increase in heparin-binding protein p30. The results suggest, respectively, the involvement of an apoptotic pathway, recruitment of the heat-shock protein machinery, generation of an antioxidant response, and, probably, induction of repair mechanisms. Three of the four proteins with altered levels had not been previously detected in the cytosolic fraction, and detection of the altered levels was possible only after the protein-enriching step.

Expression pattern and localization of beta,beta-carotene 15,15'-dioxygenase in different tissues.

Beta,beta-carotene 15,15'-dioxygenase cleaves beta,beta-carotene into two molecules of retinal, and is the key enzyme in the metabolism of beta,beta-carotene to vitamin A. The enzyme has been known for more than 40 years, yet all attempts to purify the protein to homogeneity have failed. Recently, the successful cloning and sequencing of an enzyme with beta,beta-carotene 15,15'-dioxygenase activity from chicken, as well as from Drosophila, has been reported. Here, we describe in detail our attempt to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent as to allow determination of partial amino acid sequences, which were then used to design degenerate oligonucleotides. Screening of a chicken duodenal expression library yielded a full-length clone containing a coding sequence of 1578 bp. Functional expression in Escherichia coli and in eukaryotic cell lines confirmed that we had cloned the first vertebrate dioxygenase that cleaves beta,beta-carotene at the central 15,15'-double bond. By performing a sequence homology search, the cDNA sequence of the mouse homologue was found as an expressed sequence tag (EST) in the gene bank. At the amino-acid level, the degree of homology between the chicken and mouse sequences is 81%. Thus beta,beta-carotene 15,15'-dioxygenase can be considered as being an enzyme that is evolutionarily rather well conserved. We established the expression pattern of beta,beta-carotene 15,15'-dioxygenase in chicken and mouse tissues with a combination of Northern blots and in situ hybridization. The mRNA for beta,beta-carotene 15,15'-dioxygenase was localized primarily in duodenal villi, as well as in liver and in tubular structures of lung and kidney. These new findings demonstrate that beta,beta-carotene 15,15'-dioxygenase is also expressed in epithelial structures, where it serves to provide the tissue-specific vitamin A supply.

Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors.

Six different protein factors are required in vitro for 3' end formation of mammalian pre-mRNAs by endonucleolytic cleavage and polyadenylation. Five of the factors have been purified and most of their components cloned, but cleavage factor II(m) (CF II(m)) remained uncharacterized. We have purified CF II(m) from HeLa cell nuclear extract by several chromatographic steps. During purification, CF II(m) activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIB(m)) for the cleavage reaction. CF IIA(m) fractions contain the human homologs of two yeast 3' end processing factors, Pcf11p and Clp1p, as well as cleavage factor I(m) (CF I(m)) and several splicing and transcription factors. We report the cloning of hClp1 and show that it is a genuine subunit of CF IIA(m). Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract. hClp1 interacts with CF I(m) and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3' end processing factors within the cleavage complex.

Cloning and expression of beta,beta-carotene 15,15'-dioxygenase.

beta,beta-Carotene 15,15'-dioxygenase cleaves beta-carotene into two molecules of retinal and is therefore the key enzyme in beta-carotene metabolism to vitamin A. In the present study, it was possible to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent that partial amino acid sequence information could be obtained to design degenerate oligonucleotides. With RT-PCR a cDNA fragment could be obtained and used subsequently in a radioactive screening of a chicken duodenal expression library. We cloned the first eukaryotic beta,beta-carotene 15,15'-dioxygenase which symmetrically cleaves beta-carotene at the 15,15'-double bond.

Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells.

Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin. Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop. This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length- and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins. Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate.

Activating interactions in human NK cell recognition: the role of 2B4-CD48.

2B4 is a cell surface glycoprotein of the immunoglobulin superfamily structurally related to CD2-like molecules. It was originally identified in the mouse as a receptor that mediates non-MHC-restricted cytotoxicity by NK cells and CD8+ T cells. Recently, 2B4 was shown to bind CD48 by molecular binding assays and surface plasmon resonance. Here, we have investigated the cell surface expression, biochemical characteristics and function of human 2B4. Our results show that 2B4 is expressed not only on NK cells and CD8+ T cells, but also on monocytes and basophils, indicating a broader role for 2B4 in leukocyte activation. In NK cells, engagement of 2B4 with a specific monoclonal antibody or with CD48 can trigger NK cell-mediated cytotoxicity. The contribution of 2B4-CD48 interaction to target cell lysis by different NK cell clones varies, probably dependent on the relative contribution of other receptor-ligand interactions. In T cells and monocytes, ligation of 2B4 does not lead to T cell or monocyte activation. Thus, it appears that the primary function of 2B4 is to modulate other receptor-ligand interactions to enhance leukocyte activation.

Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance.

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.

A 17-kDa CD4-binding glycoprotein present in human seminal plasma and in breast tumor cells.

We previously isolated gp17, a human seminal plasma glycoprotein, which specifically interacts with the D1-D2 region of CD4, a T cell surface molecule involved in antigen recognition mediated by helper T cells also acting as a receptor for the human immunodeficiency virus. In this study we report that monoclonal antibodies (mAb) reacting with gp17 are able to inhibit the binding of gp17 to immobilized soluble CD4. An immunohistochemical analysis shows that gp17 is also expressed in mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients. A structural characterization of gp17, including amino acid sequencing, indicates that the protein has an extensive structural similarity with a glycoprotein designated as seminal actin-binding protein (SABP), also secreted by male sexual glands. SABP is in turn identical to gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP), a factor known as a highly specific and sensitive marker of primary and metastatic apocrine breast cancer. To establish further the correspondence of gp17 and GCDFP-15/PIP/SABP, the latter was expressed in bacteria from a cloned cDNA and purified by affinity chromatography to either anti-gp17 mAb-Sepharose or CD4-Sepharose. The purified recombinant protein is shown to inhibit the binding of labeled, pure g17 to immobilized soluble CD4. The finding that breast cancer cells express a protein able to interact with the CD4 domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor-infiltrated CD4+ T cells.

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]

Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity.

The extracellular ligand binding domain of the human interferon gamma receptor includes eight cysteine residues forming four disulfide bonds. Only the nonreduced protein binds interferon gamma. We investigated the alignment of the disulfide bonds, using an enzymatically deglycosylated form of a soluble interferon gamma receptor, produced in baculovirus-infected insect cells. The soluble receptor was digested with endoproteinase Glu-C and proteinase K, and the proteolytic fragments were characterized by amino acid sequence analysis and mass spectrometry. It was found that four consecutive disulfide bonds are formed between residues Cys60-Cys68, Cys105-Cys150, Cys178-Cys183, and Cys197-Cys218. We also investigated the role of the disulfide bonds in biological activity of the receptor, using site-directed mutagenesis and by exchanging the cysteine residues for serines. The mutated proteins were expressed in Escherichia coli and analyzed for ligand binding capacity on protein blots. The assays showed that all disulfide bonds are essential for full ligand binding capacity. Double or quadruple mutations at cysteine residues 60 and 68, and residues 178, 183, 197, and 218, respectively, resulted in complete loss of the activity, whereas double mutations at residues 105 and 150, 178 and 183, and 197 and 218, respectively, resulted in a residual activity about 1 order of magnitude lower than that of the wild type. The specific antibodies gamma R38 and gamma R99 detected conformational epitopes stabilized by disulfide bonds involving cysteine residues 60 and 68, and 178 and 183, respectively.

A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity.

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.

The C terminus of the anaphylatoxin C3a generated upon complement activation represents a neoantigenic determinant with diagnostic potential.

Activation of the C component C3 results in generation of the anaphylatoxin C3a. The C3a polypeptide chain consists of 77 amino acids. The active site of this potent mediator, which also has immunoregulatory function resides in its C terminus. This report demonstrates that the C terminus of C3a (C3a-desArg) exposed by proteolytic cleavage from C3 represents a neoantigenic determinant. Two mAb specific for this epitope were obtained after immunization with the synthetic octapeptide (OP) Arg-Ala-Ser-His-Leu-Gly-Leu-Ala [C3a(69-76)] coupled to the carrier keyhole limpet hemocyanin (KLH). These anti-C3a(69-76) antibodies (H453 and H454) reacted in an ELISA system with C3a and KLH-OP but not with C3 or with KLH alone. Free OP efficiently blocked binding of the antibodies to C3a, whereas binding of another anti-C3a mAb (H13) remained unaffected. In immunoblotting analysis, the anti-C3a(69-76) mAb reacted with purified C3a but failed to react with the denatured, noncleaved C3. A novel quantitative C3a-ELISA was established with the anti-C3a(69-76) mAb. It had a sensitivity in the nanogram range (1 to 5 ng/ml). The C3a determination was not impaired by the presence of high concentrations of C3. Therefore, C3 removal was not required in contrast to the previously described C3a assays. This C3a ELISA might facilitate clinical C3a quantitation, e.g., in samples from patients with adult respiratory distress syndrome. In these patients, C3a determination in the early phase of the disease is of diagnostic relevance and has prognostic value.