Role of polycationic C-terminal portion in the structure and activity of recombinant human interferon-gamma.

Arakawa, Tsutomu; Hsu, Yeuh-Rong; Parker, Carol; Lai, Por-Hsiung

doi:10.1016/s0021-9258(19)83943-1

Cited by 64 publications

(8 citation statements)

References 12 publications

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“…It is notable that previous data on the role of the carboxyl terminus in the biological activity of rHuIFN 7 has been somewhat controversial. Thus, for example, although a 1000-fold loss in antiviral activity was reported after removal of the carboxyl terminus of rHuIFN 7 by tryptic cleavage (Arakawa et al, 1986), it was not established in these studies that a second, lower molecular weight component (Mr 14400) reported to be present in the tryptic digest did not interfere with the determination of antiviral activity, as, e.g., by competition with native rHuIFN 7 for binding to the receptor. These results were in disagreement with the report of Rose et al (1983), who identified by gas chromatographic/mass spectrometric analysis a truncated derivative of rHuIFN 7 that terminated at position 131 but yet retained full antiviral activity.…”

Section: Discussionmentioning

confidence: 84%

“…Possibilities include direct interaction with the rHuIFN 7 receptor, involvement in the triggering of the biological response, or secondary effects on the conformational integrity of these regions. In the studies reported by Arakawa et al (1986) the lack of effect of removal of the carboxyl terminus of rHuIFN 7 by tryptic cleavage on either the circular dichroism spectrum of rHuIFN 7 or its elution profile from gel filtration indicated that gross conformational changes or alterations in the state of association did not occur. Thus, these data provide additional support for a more direct involvement of the carboxyl terminus in receptor interaction.…”

Section: Discussionmentioning

confidence: 98%

“…In distinction, the genetic engineering of a form of rHuIFN y lacking the 19 carboxyl-terminal amino acid residues resulted in a significant depression in both antiviral and antiproliferative activities (Burton et al, 1985;Czarniecki et al, 1985). Similarly, Arakawa et al (1986) have recently observed that tryptic digestion of rHuIFN y produced a variant lacking ca.…”

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confidence: 82%

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Evidence for a polypeptide segment at the carboxyl terminus of recombinant human .gamma. interferon involved in expression of biological activity

Seelig¹,

Wijdenes²,

Nagabhushan³

et al. 1988

Biochemistry

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A panel of 18 murine monoclonal antibodies was raised in BALB/c mice to the full-length, 146 amino acid residue recombinant human gamma interferon (rHuIFN gamma-A). Two monoclonal antibodies, designated 47N3-6 and 30N47-1, were purified from ascites tumors and further characterized. Antibody 47N3-6 neutralized both the antiviral and antiproliferative activities of rHuIFN gamma-A. Both Western blotting and enzyme-linked immunosorbent assays indicated that antibody 47N3-6 could bind to rHuIFN gamma-A as well as to a genetically engineered truncated form lacking the first three amino-terminal residues (rHuIFN gamma-D) but did not recognize a genetically engineered variant terminating at residue 131 (rHuIFN gamma-B). This antibody also demonstrated binding to a 15 amino acid residue oligopeptide, designated F-1, corresponding to residues 132-146 at the carboxyl terminus of rHuIFN gamma-A. Chemical cleavage of peptide F-1 with cyanogen bromide produced two fragments that were separated by reversed-phase high-pressure liquid chromatography. Dot-blot analysis indicated that antibody 47N3-6 could bind to a fragment, KRKRSQHse, derived from residues 132-137 of rHuIFN gamma-A, but could bind only weakly to the cyanogen bromide fragment corresponding to residues 138-146. It was consistent with these results that antibody 47N3-6 demonstrated binding to a form lacking the five carboxyl-terminal amino acids (rHuIFN gamma-D') but did not bind to a synthetic polypeptide corresponding to residues 138-146. Peptide F-1 exhibited neither antiviral nor antiproliferative activity, and it did not antagonize the antiviral activity of rHuIFN gamma-A.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 98%

mentioning

confidence: 82%

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Evidence for a polypeptide segment at the carboxyl terminus of recombinant human .gamma. interferon involved in expression of biological activity

Seelig¹,

Wijdenes²,

Nagabhushan³

et al. 1988

Biochemistry

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“…A number of studies investigating structure-function relationships have indicated that certain synthetic peptides of IFN-y produced by recombinant techniques or peptides derived by chemical or proteolytic cleavage of intact rIFN-y exhibit modified bioactivity in vitro (1,16,40). Studies by Arakawa et al (1) and, more recently, Leinikki et al (16) have demonstrated that cleavage of approximately 10% of the C-terminal portion of human rIFN-y by trypsin, pronase, or other proteases with broad substrate specificities markedly inhibits antiviral activity. Although our results are consistent with these reports, we have not, as yet, identified the site at which AP cleaves IFN--y.…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity

Horvat

Parmely

1988

Infect Immun

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This study was performed to determine the effect of Pseudomonas aeruginosa on gamma interferon (IFN-gamma) production by antigen-stimulated human T-cell clones. Crude bacterial filtrates prepared from certain strains of P. aeruginosa inhibited IFN-gamma production by T cells and reduced the antiviral activity of preformed IFN-gamma. Bacterial filtrates prepared from mutant strains that did not produce the exoenzyme alkaline protease (AP) did not inhibit IFN-gamma activity. The inhibitory activity of bacterial filtrates was heat and trypsin sensitive and was neutralized by an antiserum to AP. Crystalline AP mimicked the effects of the bacterial filtrates, and an inactive filtrate from a protease-deficient mutant strain was reconstituted by the addition of AP. AP-treated recombinant IFN-gamma showed altered migration on Western blots (immunoblots) of polyacrylamide gels, and this modification correlated with a dose-dependent loss of antiviral activity. The ability of recombinant IFN-gamma to elevate the expression of Fc receptors on cells of the U-937 histiocytic cell line was also diminished by AP treatment. These results indicate that the Pseudomonas protease AP can inhibit the antiviral and immunomodulatory activities of IFN-gamma.

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“…Although the dimeric structure was established with the recombinant E. coli derived IFN-, it is very likely that the natural, glycosylated protein with C-terminal processing is also a dimer. This speculation is based on the observations that neither glycosylation nor C-terminal processing apparently alters the protein conformation (Arakawa et al, 1986b) and that the self-association of E. coli derived IFNoccurs without 13 C-terminal residues (Arakawa et al, 1986a). These results suggest that the binding site to form the dimer is identical in recombinant E. coli derived and natural IFNpreparations.…”

Section: Discussionmentioning

confidence: 99%

Sedimentation equilibrium measurements of recombinant DNA-derived human interferon .gamma.

Yphantis

Arakawa²

1987

Biochemistry

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Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.

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Role of polycationic C-terminal portion in the structure and activity of recombinant human interferon-gamma.

Cited by 64 publications

References 12 publications

Evidence for a polypeptide segment at the carboxyl terminus of recombinant human .gamma. interferon involved in expression of biological activity

Evidence for a polypeptide segment at the carboxyl terminus of recombinant human .gamma. interferon involved in expression of biological activity

Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity

Sedimentation equilibrium measurements of recombinant DNA-derived human interferon .gamma.

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