scholarly journals An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix.

1995 ◽  
Vol 131 (6) ◽  
pp. 1547-1560 ◽  
Author(s):  
A J Denzer ◽  
M Gesemann ◽  
B Schumacher ◽  
M A Ruegg
Keyword(s):  
Amino Acids ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Molecular Mass ◽  
Motor Neurons ◽  
Acetylcholine Receptors ◽  
Relative Molecular Mass ◽  
Amino Terminal ◽  
Tissue Homogenates ◽  
Receptor Clusters

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

scholarly journals PROTEINS OF PAROTOID GLAND SECRETIONS FROM TOADS OF THE GENUS BUFO

2000 ◽  
pp. 1-7 ◽  
Author(s):  
David Perry
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Mass Range ◽  
Relative Molecular Mass ◽  
Terminal Amino Acid ◽  
Banding Patterns ◽  
Freeze Dried ◽  
Sds Page ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Freeze-dried parotoid gland secretions from toads of the genus Bufo contained large proportions of protein (25-35% by weight). SDS-PAGE suggested that secretions from several species of Bufo contained mixtures of proteins in the relative molecular mass range of approximately 12 - 200 kDa, which exhibited markedly different banding patterns from species to species. These proteins were presumably not discovered before because the previous extraction procedures used with these secretions were designed to examine low molecular mass compounds and would denature the proteins. SDS-PAGE of secretions from B. mauritanicus and B. calamita are shown here. The N-terminal amino acid sequence of one of the bands (approx. 58 kDa) of B. mauritanicus was found to be LPIPAFPGLDHGF and of a B. calamita band (30.5 kDa) was VQVFGLQKEA. No significant similarities to these two sequences and to three separate but partial N-terminal sequences obtained from these species were found in genetic databases.

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

Substrate Preferences for Antiplasmin Cleaving Enzyme Hydrolysis of Peptides Derived from the α2-Antiplasmin Sequence.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1704-1704
Author(s):  
Kenneth W. Jackson ◽  
Victoria J. Christiansen ◽  
Kyung N. Lee ◽  
Christina F. Mason ◽  
Patrick A. McKee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Enzyme Hydrolysis ◽  
Peptide Sequence ◽  
Peptide Hydrolysis ◽  
Cleavage Rate ◽  
Amino Terminal ◽  
Substrate Preferences ◽  
Polymorphic Position ◽  
Hydrolysis Of

Abstract Antiplasmin cleaving enzyme (APCE) is a proteinase that specifically, but slowly cleaves the Pro-Asn bond in long-form α2-antiplasmin (Met-α2AP) in human plasma. This slow cleavage produces a steady-state plasma mixture of Met-α2AP and an N-terminally shortened form of antiplasmin, Asn-α2AP. The Asn-α2AP form crosslinks to fibrin ~13-fold faster than Met-α2AP. A faster crosslink rate causes a greater number of antiplasmin molecules to become bound during fibrin formation, thereby enhancing resistance to fibrinolysis. Inhibition of plasma APCE may decrease the number of antiplasmin molecules crosslinked and result in clots that are more easily removed during fibrinolysis. Therefore, an inhibitor specific for APCE could potentially be used to regulate fibrinolysis. Human Met-α2AP exists in two polymorphic forms at position six in the mature sequence, with arginine predominant, and tryptophan accounting for a lesser percentage. We have determined the relative cleavage rates of synthetic peptides from a peptide library that span the cleavage site. The peptides contained all common amino acids except cysteine in the polymorphic position (P7 position). Arg was the optimal amino acid in this position with a relative cleavage rate ~5–10-fold faster than other amino acids except Lys, which was ~70% of the Arg rate. The P7 position Arg enhancement was also observed when Arg was in the P6 or P5 position, but no enhancement was observed when Arg was moved to positions P8, P4, P3 or P2. It was also determined that APCE is preferentially an endoproteinase rather than an aminodipeptidase, with a 10-fold greater rate of hydrolysis of the internal Pro-Asn bond in the Met-α2AP 1–17 peptide sequence MEPLGRQLTSGP-NQEQV over the Pro-Asn bond penultimate to the amino-terminal bond in the Met-α2AP 11–27 peptide sequence GP-NQEQVSPLTLLKLGN in peptide hydrolysis experiments. We conclude that APCE inhibitors designed with a positive charge placed upstream of the Pro-X scissile bond equivalent to five to seven amino acid residues may prove to be highly potent and specific. In addition, such inhibitors should also prove useful for blocking the activity of the closely related enzyme fibroblast activation protein. This work was supported by NIH grant HL072995.

A Novel Human Actin-Binding Protein Homologue That Binds to Platelet Glycoprotein Ib

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1268-1276 ◽  
Author(s):  
Wen-feng Xu ◽  
Zhi-wei Xie ◽  
Dominic W. Chung ◽  
Earl W. Davie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Protein ◽  
Full Length ◽  
Actin Binding ◽  
Intracellular Domain ◽  
Platelet Surface ◽  
Full Length Cdna ◽  
Actin Binding Protein ◽  
Amino Terminal

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIb. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other. © 1998 by The American Society of Hematology.

scholarly journals Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210.

1993 ◽  
Vol 178 (6) ◽  
pp. 2237-2242 ◽  
Author(s):  
R E Nickowitz ◽  
H J Worman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Primary Biliary Cirrhosis ◽  
Antigenic Determinants ◽  
Nuclear Pore ◽  
Biliary Cirrhosis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies against a 210-kD integral glycoprotein of the nuclear envelope pore membrane. This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies from 25 patients with PBC that recognize gp210 react with the cytoplasmic carboxy-terminal tail while none react with unmodified linear epitopes in the amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25 patients is contained within a stretch of 15 amino acids. The recognized amino acid sequence is homologous to the protein products of the Escherichia coli mutY gene and Salmonella typhimurium mutB gene with an exact identity of six consecutive amino acids, suggesting that anti-gp210 antibodies may arise by molecular mimicry of bacterial antigenic determinants.

Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch

2005 ◽  
Vol 288 (6) ◽  
pp. L1132-L1138 ◽  
Author(s):  
Caitriona A. Buckley ◽  
Farshid N. Rouhani ◽  
Maryann Kaler ◽  
Barbara Adamik ◽  
Feras I. Hawari ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Inhibitory Activity ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Mucin Production ◽  
Inactive State ◽  
Amino Terminal ◽  
Switch Mechanism ◽  
Cysteine Switch

TNF-α-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18–54 (N-TACE18–54) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE18–54 attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE18–54 inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30–48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.

scholarly journals Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I*

Endocrinology ◽  
1997 ◽  
Vol 138 (7) ◽  
pp. 2972-2978 ◽  
Author(s):  
Taek Jeong Nam ◽  
Walker Busby ◽  
David R. Clemmons
Keyword(s):  
Amino Acids ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Growth Factor ◽  
Heparan Sulfate ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Insulin-like growth factor binding protein-5 (IGFBP-5) has been shown to bind to the extracellular matrix (ECM) of both fibroblasts and smooth muscle cells. The ECM-IGFBP-5 interaction is mediated in part by binding to heparan sulfate containing proteoglycans. Because proteoglycans may not be the only components of ECM that bind to IGFBP-5, we have determined its ability to bind to other ECM proteins. When a partially purified mixture of the proteins that were present in fibroblast conditioned medium was purified by IGFBP-5 affinity chromatography, a 55-kDa protein was eluted. Amino acid sequencing of the amino terminal 28 amino acids showed that it was human plasminogen activator inhibitor-1 (PAI-1). To determine if this interaction was specific, purified human PAI-1 was incubated with IGFBP-5 and the IGFBP-5/PAI-1 complex immunoprecipitated with anti-PAI-1 antiserum. When the precipitate was analyzed by immunoblotting using anti-IGFBP-5 antiserum, the intensity of the IGFBP-5 band was substantially increased compared with controls that did not contain human PAI-1. A synthetic IGFBP-5 peptide that contained the amino acid sequence between positions 201 and 218 inhibited IGFBP-5/PAI-1 interaction. Coincubation of IGFBP-5 mutants that contained substitutions for specific basic residues located between positions 201 and 218 with PAI-1 indicated that some of these amino acids were important for binding. Two mutants that contained neutral substitutions for specific basic amino acids within the glycosaminoglycan binding domain had reduced binding to PAI-1. In contrast, three other mutants that also had substitutions for charged residues in the same region had no reduction in binding. Heparin and heparan sulfate inhibited the IGFBP-5/PAI-1 interaction; however, several other glycosaminoglycans had no effect. PAI-1 was determined to be an important ECM component for binding because approximately 27% of total ECM binding could be inhibited with anti-PAI-1 antiserum. Competitive binding studies with unlabeled IGFBP-5 showed that the dissociation constant of PAI-1 for IGFBP-5 was 9.1 × 10−8m. In summary, IGFBP-5 binds specifically to plasminogen activator inhibitor-1. Because this is present in the extracellular matrix of several cell types, it may be one of the important binding components of ECM. PAI-1 binding partially protects IGFBP-5 from proteolysis, suggesting that it is one of the ECM components that is involved in mediating this effect.

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