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F R Masiarz

Publications and source records attributed to F R Masiarz.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of recombinant human insulin-like growth factor binding proteins 4, 5, and 6 produced in yeast.

The insulin-like growth factor binding protein (IGFBP) family comprises six structurally distinct, but highly homologous proteins. They have been identified in serum and other biological fluids, tissue extracts, and cell culture media. We have recently cloned cDNAs encoding human IGFBP-4, -5, and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating processing of the fusion proteins. High-performance liquid chromatography-purified rhIGFBPs had virually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs. Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1, i.e. 25-100 times higher than the IGF I and II affinities of the type I IGF receptor. When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, but rhIGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant. The results of this study show that the primary effect of the three rhIGFBPs is the attenuation of IGF activity and suggest that IGFBPs contribute to the control of IGF-mediated cell growth and metabolism.

Amino Acid Sequence↗
PubMed1992

Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.

Amino Acid Sequence↗
PubMed1991

A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.

The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P'1 or P'2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.

3C Viral Proteases↗
PubMed1991

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments.

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.

Amino Acid Sequence↗
PubMed1991

Identification and molecular cloning of two new 30-kDa insulin-like growth factor binding proteins isolated from adult human serum.

Three insulin-like growth factor binding proteins (IGFBP) with apparent molecular masses of 24, 28-30, and 30 kDa, nonreduced, have been isolated from human serum. The 15 NH2-terminal amino acids of the 24-kDa binding protein are identical with those of the 30-kDa BP. The apparent molecular mass of the latter is reduced to 24 kDa by N-glycanase, suggesting that the 30-kDa BP is the glycosylated form of the isolated 24-kDa BP. The complete amino acid sequences derived from the cloned cDNAs represent two new IGFBPs. They are tentatively termed IGFBP-4 and -5. The prepeptide sequences of BP-4 and -5 contain 27 and 21, the mature proteins 213 and 237 amino acids, respectively (Mr = 22,610 and 25,980). The NH2- and COOH-terminal thirds of BP-4 and -5 display pronounced homology to the other three human BPs. 16 of the 16-20 cysteines and 37 of the 213-289 amino acids (12.8-17.1%) are conserved in all five mature BPs. 10 amino acid positions located in the NH2-terminal region and shared by BP-1, -2, -3, and -5 are different in BP-4. These differences may account for the preferential affinity of BP-4 for IGF II. A most intriguing homology exists between the COOH-terminal quarter of the five IGFBPs, 10 repetitive domains of human thyroglobulin, a gastrointestinal tumor-associated antigen, and the invariant chain of the class II histocompatibility antigen. The cDNAs of five human IGFBPs are now available. They will allow their expression and production in sufficient quantities for in vivo studies to unravel their role in growth and metabolism.

Amino Acid Sequence↗
PubMed1991

The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts.

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.

Amino Acid Sequence↗
PubMed1990

Structure determination of O-linked glycopeptides by tandem mass spectrometry.

A new method for characterizing O-linked glycopeptides without chemical degradation is presented. Collision-induced dissociation (CID) analysis of intact O-linked glycopeptides containing mono- and disaccharides was performed. For glycopeptides containing one hexose unit, both the peptide sequence and the site of attachment of the sugar moiety were obtained from a single high-energy CID spectrum. However, in a glycopeptide bearing multiple sugar residues per site, the CID spectrum was dominated by fragments resulting from cleavages of the carbohydrate substituents and the gas-phase deglycosylated peptide, thus obviating the concomitant observation of peptide sequence ions. Hence, information on the structures of the carbohydrate substituents was obtained, but not on the sites of attachment of these residues to the peptide. Subsequent CID analysis of the gas-phase deglycosylated peptide ion can be used to obtain the sequence of the peptide backbone from the same sample. This method holds promise for simultaneously determining the carbohydrate structure and the peptide sequence of intact O-linked glycopeptides without chemical degradation.

Amino Acid Sequence↗
PubMed1990

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme.

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.

Amino Acid Sequence↗
PubMed1990

Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis.

High-performance tandem mass spectrometry has been employed to structurally characterize intact O-linked glycopeptides and establish the complexity and extent of glycosylation for recombinant human platelet-derived growth factor B chain (rhPDGF-B) expressed in yeast. In addition, liquid secondary ion mass spectrometry (LSIMS) and Edman degradation have been employed to verify the protein sequence. LSIMS of high-performance liquid chromatographically fractionated proteolytic digests confirmed the complete amino acid sequence predicted by the human PDGF-B gene structure. Potential glycopeptides (as indicated by a mass shift of 162 or 324 Da from the mass of a predicted cleavage product) were sequenced using tandem mass spectrometry and Edman degradation. Ultraviolet matrix laser desorption mass spectrometry of rhPDGF-B dimer was used to determine the molecular weight distribution for the intact recombinant glycoprotein. In addition to the presence of unmodified peptides, corresponding peptides bearing monomannosyl moieties were found on serine 26 and threonines 20, 63, 88, 90 and 101. Further, dimannosyl moieties were found on threonines 6 and 63. These data reveal the presence of O-linked glycosylation at sites which do not fortify the concept of a consensus sequence involving proline residues, but which strengthen the concept of secondary and tertiary structure requirements. The advantages of high-energy collisionally induced dissociation analysis of O-linked glycopeptides over conventional base elimination and borohydride reduction and other mass spectrometric techniques are presented for the first time.

Amino Acid Sequence↗
PubMed1990

Yeast clathrin has a distinctive light chain that is important for cell growth.

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.

Amino Acid Sequence↗
PubMed1989

Soluble lactose-binding vertebrate lectins: a growing family.

Extracts of rat intestine contain nine soluble lactose-binding lectins with subunit molecular weights ranging from 14,500 to 19,000 that were purified by affinity chromatography and ion-exchange chromatography. Two of them are either identical with or closely related to other known rat lectins. A third appears to be the isolated carbohydrate-binding C-terminal domain of a known lectin but lacks the N-terminal domain presumed to mediate a different function. The others have not been described previously. Among them, the major rat intestinal lectin, RI-H, and a related protein, RI-G, have N-terminal amino acid sequences with similarities to sequences found in other known rat lectins. Therefore, these results introduce new members of a growing family of these structurally homologous soluble lactose-binding proteins.

Amino Acid Sequence↗
PubMed1989

The human cytomegalovirus strain Towne glycoprotein H gene encodes glycoprotein p86.

The gene encoding the glycoprotein H (gH) homologue of CMV strain Towne was cloned, sequenced, and expressed. The predicted 742 amino acid gH protein had characteristics typical of a membrane glycoprotein including hydrophobic signal and transmembrane domains and six possible N-linked glycosylation sites. The CMV (Towne) gH gene had a 95% nucleotide identity and a 96.6% amino acid identity with the CMV (AD169) gH gene, as described by M. P. Cranage, G. L. Smith, S. E. Bell, H. Hart, C. Brown, A. T. Bankier, P. Tomlinson, B. G. Barrell, and T. C. Minson (1988, J. Virol. 62, 1416-1422). Transcriptional analysis of the gH gene revealed that the 2.9-kilobase (kb) gH transcript was not detected until late after CMV infection, indicating that the kinetics of gH expression were typical of the late class of CMV genes. The gH gene was expressed in COS cells using a vector in which transcription was driven by the SV40 early promoter. The expression of gH was detected by immunofluorescence using the virus neutralizing murine monoclonal antibody 1G6, which is specific for an 86-kilodalton (kDa) CMV virion membrane protein (p86). Amino acid sequence analysis of p86 tryptic peptides revealed sequence identity with peptides from the deduced gH amino acid sequence, confirming that the gH gene encodes p86. These results indicate that CMV gH can induce virus neutralizing antibodies and establishes gH as a candidate antigen for a subunit vaccine against CMV.

Amino Acid Sequence↗
PubMed1988

Expression and processing of biologically active fibroblast growth factors in the yeast Saccharomyces cerevisiae.

Chemically synthesized genes for bovine and human fibroblast growth factors (FGFs) were expressed in heterologous microorganisms. Although the intracellular expression or secretion of acidic and basic FGFs in Escherichia coli or Saccharomyces cerevisiae yielded recombinant growth factors with high biological activity, the resulting proteins had structural microheterogeneity due to modified amino termini. Expression of amino-terminal extended forms of human acidic and basic FGFs in S. cerevisiae gave rise to soluble, but cell-associated polypeptides, with potent biological activity. These yeast-derived proteins were processed in vivo by removal of initiation codon-derived methionine residues and by amino-terminal acetylation. Both of these processes have been observed in mammalian tissues. The yeast systems described here, therefore, provide a good model system for the expression of FGFs as intracellular proteins, but more importantly they give high levels of authentically processed human FGFs with many potential medical applications. Since the recombinant proteins have all the biological activities of their native counterparts, their possible applications in wound healing, tissue grafting, nerve regeneration, and treatment of ischemia are discussed.

Amino Acid Sequence↗
PubMed1988

Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.

The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5' direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.

Amino Acid Sequence↗
PubMed1988

Characterization of recombinant human epidermal growth factor produced in yeast.

Four different forms of human epidermal growth factor (h-EGF) are found in the culture medium of a recombinant strain of Saccharomyces cerevisiae. These forms were characterized after purification using reverse-phase high-performance liquid chromatography. The most abundant form of secreted recombinant h-EGF has leucine at the carboxyl terminus and is identical with gamma-urogastrone. A second species is identical with the most abundant form except that it lacks the carboxyl-terminal leucine. This form appears to be the product of a carboxypeptidase found in the growth medium. The other two forms of recombinant h-EGF are the respective oxidation products of the above where the single methionine residue has been converted to methionine sulfoxide. These four forms of recombinant h-EGF are fully active; they bind to the EGF receptor of A431 cells as well as stimulate mitotic activity of human foreskin fibroblasts with equal specific activity. The location of the disulfide bonds in the predominant form of recombinant h-EGF was determined following digestion with thermolysin. The amino acid compositions of the resulting peptides showed that the placement of disulfide bonds in recombinant h-EGF is identical with that in murine EGF.

Amino Acid Sequence↗
PubMed1988

Characterization of ribosomal frameshifting in HIV-1 gag-pol expression.

Based on precedents from other retroviruses, the precursor of the human immunodeficiency virus (HIV-1) reverse transcriptase is predicted to be a polyprotein with a relative molecular mass (Mr) of 160,000 (160K) encoded by both the viral pol gene and the upstream gag gene. These two genes lie in different translational reading frames, with the 3' end of gag overlapping the 5' end of pol by 205 or 241 nucleotides. Thus, production of the gag-pol fusion protein would require either messenger RNA processing or translational frameshifting. The latter mechanism has been shown in the synthesis of the gag-pol proteins of two other retroviruses, Rous sarcoma virus (RSV) and mouse mammary tumour virus (MMTV). Here we report that translation of HIV-1 RNA synthesized in vitro by SP6 RNA polymerase yields significant amounts of a gag-pol fusion protein, indicating that efficient ribosomal frameshifting also occurs within the HIV-1 gag-pol overlap region. Site-directed mutagenesis and amino-acid sequencing localized the site of frameshifting to a UUA leucine codon near the 5' end of the overlap.

Amino Acid Sequence↗
PubMed1988

Human cytomegalovirus strain Towne glycoprotein B is processed by proteolytic cleavage.

The gene encoding glycoprotein B of human cytomegalovirus (CMV) strain Towne was cloned, sequenced, and expressed in order to study potential targets for viral neutralization. Secondary structure analysis of the 907 amino acid protein predicted a 24 amino acid N-terminal signal sequence and a potential transmembrane region composed of two domains, 34 and 21 amino acids. The CMV (Towne) gB gene had a 94% nucleotide similarity and a 95% amino acid similarity to the CMV (AD169) gB gene [as described by M.P. Cranage et al. (1986, EMBO J. 5, 3057-3063)]. Transcriptional analysis of the CMV (Towne) gB coding strand revealed that the gB message (3.9 kb), was transcribed from this region as early as 4 hr postinfection, and well in advance of gB protein synthesis. Full-length and truncated versions of the gB gene were expressed in COS cells using expression vectors where transcription was driven by the SV40 early promoter or the CMV major immediate early promoter. Expression was detected by immunofluorescence and ELISA using the virus neutralizing murine monoclonal antibody 15D8 (L. Rasmussen, J. Mullenax, R. Nelson, and T.C. Merigan, 1985, J. Virol. 55, 274-280). This antibody had been shown previously to recognize a 55-kDa CMV virion protein and a related 130-kDa intracellular precursor. Amino acid sequence analysis of the N-terminus of the 55-kDa viral glycoprotein (gp55) showed that gp55 is derived from gB (gp130) by proteolytic cleavage and represents the C-terminal region of gp130. The truncated version of gB expressed in COS and CHO cells was also processed by proteolytic cleavage as demonstrated by Western blotting. Our study localizes the epitope recognized by 15D8 to within a 186 amino acid fragment of the gp55 protein. These results indicate that CMV gB is a target for neutralization and establishes gp55 as a candidate component for use in a subunit vaccine.

Amino Acid Sequence↗