Effect of Cysteine Substitutions on the Mitogenic Activity and Stability of Recombinant Human Keratinocyte Growth Factor

Bare, Lance A.; Brown, Mark T.; Goyal, Sunita; Idler, D. R.; Mansson, Per-Erik

doi:10.1006/bbrc.1994.2745

Cited by 9 publications

(3 citation statements)

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“…In spite of the high positive charge of OGP [Greenberg et al, 1993] the corresponding complexes migrate in an anodic PAGE system, indicating that both binding proteins are negatively charged. The binding activity of OGPBP-1 is resistant to boiling, suggesting a high intramolecular content of disulfide bonds [Bare et al, 1994]. The binding of both OGPBPs to OGP is specific inasmuch as the [3-125 I(Tyr 10 )]sOGP can be competed out from forming the complexes by an excess of unlabeled sOGP.…”

Section: Amino Acid Sequencingmentioning

confidence: 99%

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins

et al. 1997

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The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals in increases osteogenesis and hemopoiesis. In stromal cell cultures OGP stimulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP in high abundance is present in normal human and animal serum mainly complexed to OGP binding protein (OGPBP) or proteins. Here we show the presence of two OGPBPs, OGPBP-1, and OGPBP-2, in cultures of osteoblastic MC3T3 E1 cells. Immunoreactive OGP (irOGP) also accumulates in the medium of these cultures and in cultures of NIH 3T3 fibroblasts. A large amount of irOGP was released by heat inactivation of OGPBP-2 and purified by ultrafiltration and hydrophobic HPLC. The purified irOGP was identical to OGP obtained previously from rat regenerating bone marrow and human serum in terms of its amino acid sequence, immunoreactivity, and mitogenicity. Osteoblastic and fibroblastic cell proliferation can be arrested by anti-OGP antibodies and rescued by exogenous OGP, indicating that in the absence of serum or other exogenous growth stimulators the endogenously produced OGP is both necessary and sufficient for baseline proliferation. The OGP production is up- and down-regulated, respectively, by low and high doses of exogenous OGP in a manner consistent with an autoregulated feedback mechanism. The most effective OGP dose in MC3T3 E1 cells is at least two orders of magnitude lower than that in non-osteoblastic cell systems. This differential sensitivity of the osteoblastic cells could result in a preferential anabolic effect of OGP in bone.

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Section: Amino Acid Sequencingmentioning

confidence: 99%

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins

et al. 1997

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“…On the other hand, the cysteine-to-serine mutation resulted in a decrease in the thermostability in FGF1 [32]. In keratinocyte growth factor (KGF), a member of FGF family proteins, cysteine-free KGF did not bind to the heparin column, and it exhibited less mitogenic activity compared with wild-type KGF [20]. We considered that the roles of cysteine residues in FGF4 are similar to those in KGF, rather than in FGF1.…”

Section: Effect Of Point Mutations From Cysteine To Serine Residues Omentioning

confidence: 99%

“…EAX05222), respectively. Extensive analyses have been conducted to clarify the roles of these cysteine residues in disulphide bond formation and the mitogenic and heparin binding activities in FGFs [16][17][18][19][20][21]. Two conserved cysteine residues, Cys-84 and Cys-151 in the primary structure of mouse FGF4, are the only cysteine residues in FGF4.…”

Section: Introductionmentioning

confidence: 99%

Site-directed mutagenesis of cysteine to serine residues affects heparin binding and mitogenicity in fibroblast growth factor 4 produced inEscherichia coli

Kumagai

Kikuchi

Nonaka

et al. 2019

Biotechnology & Biotechnological Equipment

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The binding activity to heparan sulphate is crucial for the mitogenic activity of fibroblast growth factor 4 (FGF4) in developing mammalian embryos. There are two conserved cysteine residues in FGF family proteins, Cys-84 and Cys-151 in mouse FGF4, and these constitute all of the cysteine residues in FGF4. However, the relationships among the heparin binding activity, growth promoting activity, and the two conserved cysteine residues in FGF4 are still unclear. Consequently, we generated in Escherichia coli three kinds of point-mutated mouse FGF4, namely C84S, C151S, and C84S;C151S, by converting the cysteine residues to serine residues. In heparin column chromatography, the heparin binding activities of these mutants were attenuated. In particular, the activity of the double-mutated C84S;C151S was weakened considerably. The growth promoting activities of these mutants correlated well with their heparin binding activities. We also demonstrated that an octapeptide, the Leu-76 to Tyr-83 region, which contained four basic amino acid residues and flanked Cys-84 in mouse FGF4, enhanced the heparin binding activity when fused to glutathione-S-transferase as a recombinant protein. Overall, our findings imply that the two conserved cysteine residues in FGF4 are both involved in the heparin binding activity and mitogenicity likely by affecting the configuration of heparin binding sites in FGF4.

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Human Keratinocyte Growth Factor Recombinantly Expressed in Chinese Hamster Ovary Cells: Isolation of Isoforms and Characterization of Post-Translational Modifications

Hsu

Katta

et al. 1998

Protein Expression and Purification

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Effect of Cysteine Substitutions on the Mitogenic Activity and Stability of Recombinant Human Keratinocyte Growth Factor

Cited by 9 publications

References 0 publications

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins

Site-directed mutagenesis of cysteine to serine residues affects heparin binding and mitogenicity in fibroblast growth factor 4 produced inEscherichia coli

Human Keratinocyte Growth Factor Recombinantly Expressed in Chinese Hamster Ovary Cells: Isolation of Isoforms and Characterization of Post-Translational Modifications

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