Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor

1988 ◽  
Vol 8 (3) ◽  
pp. 1011-1018
Author(s):  
M K Sauer ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Type V

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

scholarly journals Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

1988 ◽  
Vol 8 (3) ◽  
pp. 1011-1018 ◽  
Author(s):  
M K Sauer ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Type V

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

scholarly journals Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis

1992 ◽  
Vol 283 (1) ◽  
pp. 91-98 ◽  
Author(s):  
J A Feild ◽  
R H Reid ◽  
D J Rieman ◽  
T P Kline ◽  
G Sathe ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Receptor Binding ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Function Analysis ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Transforming Growth Factor Α

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

scholarly journals Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity.

1993 ◽  
Vol 121 (3) ◽  
pp. 705-713 ◽  
Author(s):  
P Bellosta ◽  
D Talarico ◽  
D Rogers ◽  
C Basilico
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Growth Factor ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Biologically Active ◽  
Heparin Binding ◽  
Wild Type ◽  
Mammalian Expression

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.

scholarly journals Assignment of intrachain disulfide bonds in platelet-derived growth factor B-chain

1993 ◽  
Vol 268 (18) ◽  
pp. 13372-13377
Author(s):  
A. Ostman ◽  
M. Andersson ◽  
G. Bäckström ◽  
C.H. Heldin
Keyword(s):  
Growth Factor ◽  
Disulfide Bonds ◽  
Platelet Derived Growth Factor ◽  
Factor B

scholarly journals Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

1988 ◽  
Vol 8 (3) ◽  
pp. 1247-1252 ◽  
Author(s):  
E Lazar ◽  
S Watanabe ◽  
S Dalton ◽  
M B Sporn
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Amino Acid ◽  
Growth Factor ◽  
Aspartic Acid ◽  
Transforming Growth Factor ◽  
Biologically Active ◽  
Single Amino Acid ◽  
Transforming Growth Factor Alpha ◽  
Factor Alpha

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 1990-1998 ◽  
Author(s):  
Wolfgang E. Kaminski ◽  
Per Lindahl ◽  
Nancy L. Lin ◽  
Virginia C. Broudy ◽  
Jeffrey R. Crosby ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Liver Growth ◽  
Hematologic Disorders ◽  
Fetal Liver Cells ◽  
Β Receptor

Abstract Platelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents

Parasitology ◽  
1984 ◽  
Vol 88 (1) ◽  
pp. 27-36 ◽  
Author(s):  
R. J. Howard ◽  
J. W. Barnwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Plasmodium Knowlesi ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Ionic Detergents ◽  
Class A ◽  
Anionic Detergents ◽  
Triton X

SUMMARYPlasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200000 and 180000 were detected only after extraction with deoxycholate or SDS.

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex

1990 ◽  
Vol 10 (5) ◽  
pp. 2359-2366
Author(s):  
D K Morrison ◽  
D R Kaplan ◽  
S G Rhee ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Serine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Wild Type ◽  
Receptor Proteins ◽  
Signaling Complex

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone

1991 ◽  
Vol 11 (7) ◽  
pp. 3603-3612
Author(s):  
S Marcus ◽  
G A Caldwell ◽  
D Miller ◽  
C B Xue ◽  
F Naider ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activity ◽  
Methyl Ester ◽  
Total Synthesis ◽  
Biologically Active ◽  
Structural Genes ◽  
Wild Type ◽  
Mating Pheromone ◽  
Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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