Amino acid sequence, glycan structure, and proteolytic processing of the lectin of Vatairea macrocarpa seeds

FEBS Letters ◽  
1998 ◽  
Vol 425 (2) ◽  
pp. 286-292 ◽  
Author(s):  
Juan J Calvete ◽  
Cláudia F Santos ◽  
Karlheinz Mann ◽  
Thalles B Grangeiro ◽  
Manfred Nimtz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Processing ◽  
Glycan Structure ◽  
Vatairea Macrocarpa

scholarly journals Amino acid sequence and molecular modelling of glycoprotein IIb-IIIa and fibronectin receptor iso-antagonists from Trimeresurus elegans venom

1996 ◽  
Vol 319 (3) ◽  
pp. 775-782 ◽  
Author(s):  
Andrea SCALONI ◽  
Elisabetta DI MARTINO ◽  
Nadia MIRAGLIA ◽  
Alessandra PELAGALLI ◽  
Rossella DELLA MORTE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Modelling ◽  
Membrane Filtration ◽  
Platelet Rich Plasma ◽  
Biological Activities ◽  
Amino Acid Sequences ◽  
Proteolytic Processing ◽  
Fibronectin Receptor ◽  
Induced Aggregation

Low-molecular-mass Arg-Gly-Asp (RGD)-containing polypeptides were isolated from the venom of Trimeresurus elegans by a simple two-step procedure consisting of membrane filtration and reverse-phase HPLC. A combination of electrospray MS, fast-atom bombardment MS and Edman degradation allowed us to ascertain the presence in the venom of different isoforms and to determine their primary structures. The amino acid sequences resembled the structure of elegantin, the only disintegrin previously reported from the T. elegans venom [Williams, Rucinski, Holt and Niewiarowski (1990) Biochim. Biophys. Acta 1039, 81–89]. MS analyses indicated the occurrence of differential proteolytic processing at both the N-terminus and the C-terminus of the polypeptide chains. The amino acid sequence alignment of the elegantin isoforms with known components of the disintegrin family demonstrated the complete conservation of the 12 cysteine residues involved in disulphide bridges. Molecular modelling of elegantins predicted an overall folding of these molecules quite similar to that reported for the kistrin solution structure. The newly identified polypeptide isoforms strongly inhibited ADP-induced aggregation in both human and canine platelet-rich plasma but showed a different species-dependent specificity. These molecules were also able to inhibit B16-BL6 murine melanoma cell adhesion to immobilized fibronectin. The comparison of the structures and biological activities of elegantin isoforms and kistrin allowed us to highlight some structural features that, in addition to the RGD locus, might be involved in the interaction of these snake-venom polypeptides with the integrin receptors on the platelet and cell surface.

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules

1987 ◽  
Vol 7 (10) ◽  
pp. 3527-3537
Author(s):  
B A Lee ◽  
D W Maher ◽  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Signal Sequence ◽  
Nuclear Transport ◽  
Proteolytic Processing ◽  
Platelet Derived Growth Factor ◽  
Cell Fractionation ◽  
Mutant Proteins

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.

scholarly journals Proteolytic processing of a coleopteran-specific δ-endotoxin produced by Bacillus thuringiensis var. tenebrionis

1989 ◽  
Vol 261 (1) ◽  
pp. 99-105 ◽  
Author(s):  
J Carroll ◽  
J Li ◽  
D J Ellar
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Amino Acid Sequence ◽  
Proteinase Inhibitors ◽  
Cleavage Product ◽  
Proteolytic Processing ◽  
N Terminus ◽  
Delta Endotoxin ◽  
Insect Gut

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.

scholarly journals Improving Proteolytic Cleavage at the 3A/3B Site of the Hepatitis A Virus Polyprotein Impairs Processing and Particle Formation, and the Impairment Can Be Complemented intrans by 3AB and 3ABC

1999 ◽  
Vol 73 (12) ◽  
pp. 9867-9878 ◽  
Author(s):  
Yuri Kusov ◽  
Verena Gauss-Müller
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cleavage Site ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Particle Formation ◽  
Proteolytic Processing ◽  
Cleavage Sites ◽  
Structural Protein ◽  
Hydrophobic Domain

ABSTRACT The orchestrated liberation of viral proteins by 3Cpro-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3Cpro from its precursors and to assess the function of the stable processing intermediates 3AB and 3ABC, we studied the effect of cleavage site mutations on hepatitis A virus (HAV) polyprotein processing, particle formation, and replication. Using the recombinant vaccinia virus system, we showed that the normally retarded cleavage at the 3A/3B junction can be improved by altering the amino acid sequence at the scissile bond such that it matches the preferred HAV 3C cleavage sites. In contrast to the processing products of the wild-type polyprotein, 3ABC was no longer detectable in the mutant. VP0 and VP3 were generated less efficiently, implying that processing of the structural protein precursor P1-2A depends on the presence of stable 3ABC and/or 3AB. In addition, cleavage of 2BC was impaired in 3AB/3ABC-deficient mutants. Formation of HAV particles was not affected in mutants with blocked 3A/3B and/or 3B/3C cleavage sites. However, 3ABC-deficient mutants produced small numbers of HAV particles, which could be augmented by coexpressing 3AB or 3ABC. The hydrophobic domain of 3A that has been proposed to mediate membrane anchorage of the replication complex was crucial for restoration of defective particle formation. In vitro transcripts of the various cleavage site mutants were unable to initiate an infectious cycle, and no progeny viruses were obtained even after blind passages. Taken together, the data suggest that accumulation of uncleaved HAV 3AB and/or 3ABC is pivotal for both viral replication and efficient particle formation.

scholarly journals Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

1987 ◽  
Vol 7 (10) ◽  
pp. 3527-3537 ◽  
Author(s):  
B A Lee ◽  
D W Maher ◽  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Signal Sequence ◽  
Nuclear Transport ◽  
Proteolytic Processing ◽  
Platelet Derived Growth Factor ◽  
Cell Fractionation ◽  
Mutant Proteins

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.

The staining pattern of the tropomyosin sequence is displayed in a new paracrystal

1986 ◽  
Vol 44 ◽  
pp. 150-151
Author(s):  
M.K. Lamvik ◽  
L.L. Klatt
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Staining Pattern ◽  
Banding Patterns ◽  
Mass Thickness ◽  
New Form

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Paris I Dysfibrinogenemia: A Point Mutation in Intron 8 Results in Insertion of a 15 Amino Acid Sequence in the Fibrinogen γ-Chain

1993 ◽  
Vol 69 (03) ◽  
pp. 217-220 ◽  
Author(s):  
Jonathan B Rosenberg ◽  
Peter J Newman ◽  
Michael W Mosesson ◽  
Marie-Claude Guillin ◽  
David L Amrani
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Point Mutation ◽  
Genomic Dna ◽  
Comparative Sequence Analysis ◽  
Chain Gene ◽  
Molecular Defect ◽  
Nucleotide Position ◽  
Normal Individuals ◽  
Polymerase Chain

SummaryParis I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal γ-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the γ-chain region of the Paris I subject’s genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A→G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I γ-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature γparis I chain mRNA, and encodes a 15 amino acid insert after γ350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I γ-chain mRNA also results after translation into a substitution of S for G at position γ351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the γpariS I chain. We conclude that the insertion of this amino acid sequence leads to a conformationallyaltered, and dysfunctional γ-chain in Paris I fibrinogen.

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