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Biomedical subjects

C H Schein

Publications and source records attributed to C H Schein.

At least 19 recordsLinked to original sources
PubMed2004

Lymnaea EGF and gigantoxin I, novel invertebrate members of the epidermal growth factor family.

In this review, we compare the sequence and structural relationships of two epidermal growth factor (EGF) family related proteins that have recently been discovered in invertebrate species. The first is L-EGF, a secreted growth factor from the gastropod mollusk Lymnaea stagnalis. The second is a peptide toxin (Gigantoxin I), isolated from the sea anenome Stichodactyla giganteus, which can paralyze crabs. L-EGF and Gigantoxin I share striking sequence similarity with mammalian erbB1 receptor ligands, including most of the essential receptor binding sites. Intriguingly, L-EGF's tertiary structure resembles more the structure of the EGF-like domain of coagulation factors. That is, the secondary and tertiary structure of L-EGF indicates the presence of a double-stranded beta-sheet but also suggests that this protein, in contrast to all other erbB1 ligands, contains a calcium-binding domain. One of the most remarkable features of L-EGF and Gigantoxin I however, is the indication that these protein are synthesized as non-membrane bound secreted peptides. This feature sets L-EGF and Gigantoxin I apart from all other members of the EGF family or EGF-like proteins identified thus far. We discuss sequence similarities and dissimilarities in the light of indications that, despite the more than 600 million years of phylogenetic distance separating both these invertebrates from mammals, Gigantoxin I and L-EGF retain some affinity for the mammalian erbB-family of receptors. Considering that mammalian EGF and its family members are frequently implicated in neoplastic diseases, the increasing number of identified and characterized invertebrate EGF family members may provide valuable leads in the design of erbB receptor antagonists.

Animals↗
PubMed2004

Mollusk-derived growth factor and the new subfamily of adenosine deaminase-related growth factors.

Peptide and protein growth factors play critical roles in the control of proliferation, differentiation and survival of most, if not all, cell types. In this review, we describe a newly isolated growth factor from Aplysia californica, mollusk derived growth factor (MDGF), that is a member of the adenosine deaminase-related growth factor (ADGF) subfamily. Other known subfamily members from a range of invertebrate and vertebrate species include: insect-derived growth factor, Drosophila ADGFs, tsetse salivary growth factors, insect adenosine deaminases (ADAs; Lutzomyia, Culex, Aedes, Anopheles), and cat eye syndrome critical region gene 1 (CECR1) in humans, pigs, and zebrafish. ADGFs from vertebrates and invertebrates contain both an ADA domain and a novel N-terminal region of about 100 amino acids. Catalytic residues involved in ADA activity are conserved in ADGFs, and inhibitors of ADA can block ADGF activity. ADA enzymatic activity has been shown, by inhibitor and site-directed mutagenesis studies, to be related to the ability of ADGFs from many species to stimulate cell proliferation. The available evidence suggests that the conversion of adenosine to inosine (or their analogs) is important for the mitogenic actions of ADGFs. Future investigations of this novel subfamily should lead to the identification of their receptors.

Adenosine Deaminase↗
PubMed2001

Aplysia attractin: biophysical characterization and modeling of a water-borne pheromone.

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.

Amino Acid Sequence↗
PubMed2000

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3.

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

Allergens↗
PubMed2000

MASIA: recognition of common patterns and properties in multiple aligned protein sequences.

SUMMARY: MASIA is a software tool for pattern recognition in multiple aligned protein sequences. MASIA converts a sequence to a properties matrix that can be scanned in both vertical and horizontal steps. Consistent patterns are recognized based on the statistical significance of their occurrence. Preset macros can be altered on-line to seek any combination of amino acid properties or sequence characteristics. MASIA output can be used directly by our programs to predict the 3D structure of proteins. AVAILABILITY: Access MASIA at http://www.scsb.utmb.edu/masia/ma sia.html.

Sequence Alignment↗
PubMed1999

Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.

BACKGROUND: Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1. OBJECTIVE: A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed. METHODS: Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera. RESULTS: Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis. CONCLUSION: The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.

Allergens↗
PubMed1997

From housekeeper to microsurgeon: the diagnostic and therapeutic potential of ribonucleases.

The RNA population in cells is controlled post-transcriptionally by ribonucleases (RNases) of varying specificity. Angiogenin, neurotoxins, and plant allergens are among many proteins with RNase activity or significant homology to known RNases. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases. RNases are regulated by specific activators and inhibitors, including interferons. Many of these regulatory molecules are useful lead compounds for the design of drugs to control tumor angiogenesis, allergic reactions, and viral replication. One RNase (Onconase) and several RNase activators are now in clinical trials for cancer treatment or inhibition of chronic virus infections. Several others, alone or conjugated with specific cell binding molecules, are being developed for their antifungal, antiviral, and antitumor cell activity.

Allergens↗
PubMed1995

Deletions at the C-terminus of interferon gamma reduce RNA binding and activation of double-stranded-RNA cleavage by bovine seminal ribonuclease.

Recombinant interferon gamma (IFN-gamma) from three species activates the cleavage of double stranded (ds-) RNA by the dimeric RNAase isolated from bovine semen (BS-RNAase). Human and bovine IFN-gamma bind RNA tightly enough to inhibit cleavage by RNAase A [Schein, Haugg and Benner (1990) FEBS Lett. 270, 229-232]. Murine IFN-gamma and a proteolytic fragment of human IFN-gamma, both of which lack part of the positively charged C-terminus, bind RNA weakly and do not inhibit RNAase A. Their ability to activate BS-RNAase is proportional to their activity in the anti-viral assay. Two monoclonal antibodies that neutralize the anti-viral activity of human IFN-gamma inhibit the activation of BS-RNAase by both full-length and proteolysed human IFN-gamma. Our results demonstrate that the C-terminus of IFN-gamma contributes to RNA binding and activation of BS-RNAase, as well as to anti-viral activity.

Amino Acid Sequence↗
PubMed1994

Reverse transphosphorylation by ribonuclease A needs an intact p2-binding site. Point mutations at Lys-7 and Arg-10 alter the catalytic properties of the enzyme.

Bovine pancreatic ribonuclease A interacts with RNA along multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. This work gives additional strong support to the existence of the postulated phosphate-binding subsite p2 (Parés, X., Llorens, R., Arús, C., and Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579) and confirms the central role of Lys-7 and Arg-10 in establishing an electrostatic interaction with a phosphate group of the substrate. The effects of charge elimination by Lys-7-->Gln (K7Q) and/or Arg-10-->Gln (R10Q) substitutions in catalytic and ligand-binding properties of ribonuclease A have been studied. The values of Km for cytidine 2',3'-cyclic phosphate and cytidylyl-3',5'-adenosine are not altered but are significantly increased for poly(C). In all cases, kcat values are lower. Synthetic activity, i.e. the reversion of the transphosphorylation reaction, is reduced for K7Q and R10Q mutants and is practically abolished in the double mutant. Finally, the extent of the reaction of the mutants with 6-chloropurine-9-beta-D-ribofuranosyl 5'-monophosphate indicates that the phosphate ionic interaction in p2 is weakened. Thus, p2 modification alters both the catalytic efficiency and the extent of the processes in which an interaction of the phosphate group of the substrate or ligand with the p2-binding subsite is involved.

Amino Acid Sequence↗
PubMed1994

Controlling oligomerization of pharmaceutical proteins.

The degree of oligomerization (or in some cases aggregation) often determines the physiological half-life and uptake rate of a protein preparation. High-resolution crystal structures of insulin and other pharmacologically interesting proteins have aided in the design of mutants with altered quaternary structure and physiological uptake rates. Analysis of the contacts between natural oligomers and protein complexes can indicate sequences that may enhance protein oligomerization. These sequences can be altered to produce monomeric protein.

Amino Acid Sequence↗
PubMed1993

Solubility and secretability.

The solubility and secretability of proteins can often be affected by extremely small changes in their primary structure. Attempts to determine empirical rules for the alteration of protein structure to improve either of these characteristics have met with only partial success. Those (mostly serendipitous) improvements in solubility that have been obtained via mutagenesis cannot be considered to be 'protein engineering'. The most successful examples where directed mutagenesis has been used to alter protein solubility, hemoglobin and insulin, have relied on established crystal structures and a wealth of data about the relationship between sequence and structure of the targeted protein. Currently, optimizing culture growth conditions by trial and error remains the fastest way to improve expression.

Biotechnology↗
PubMed1992

Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease.

A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library. Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E. coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence. Approx. 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation. When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity. Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene. About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells. Thus this eukaryotic signal sequence is both recognized by the E. coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.

Amino Acid Sequence↗
PubMed1991

Optimizing protein folding to the native state in bacteria.

A correctly folded protein is usually both active and soluble. This review focuses on novel ways to improve the folding of recombinant proteins during production in bacteria and includes a few tips for refolding proteins. Major results in correlating protein primary structure with proper folding and stability, and the production of viral antigens and antibodies in bacteria are also discussed.

Amino Acid Sequence↗
PubMed1990

Interferon-gamma activates the cleavage of double-stranded RNA by bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([3H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-gamma (IFN-gamma). Activation is seen with as little as 4-10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-gamma's ability to control cell growth and induce an antiviral state.

Animals↗