• BMB Reports
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• v.29 no.3
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• pp.205-209
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• 1996

To investigate the role of heparin in keratinocyte growth factor (KGF) and acidic fibroblast growth factor (aFGF) high affinity binding to the KGF receptor (KGFR), a cell free system was established which utilized a secreted chimeric molecule between the KGFR extracellular domain and the immunoglobulin heavy chain Fc domain (KGFR-HFc). KGFR-HFc was purified from NIH 3T3 cells and demonstrated the binding of $[^3H]-heparin$ as well as heparin Sepharose. Scatchard analysis showed that the dissociation constant for heparin binding to KGFR-HFc was 140 nM. High affinity KGF and aFGF binding to KGFR-HFc remained unchanged after treatment with 0.6 M NaCl, which is the concentration sufficient to release any bound heparin to the KGFR-HFc. These results strongly suggest that although the KGFR interacts with heparin, the presence of heparin is not absolutely required for high affinity binding of either KGF or aFGF to the KGFR.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.107-113
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• 1995

Basement membrane laminin is a multidomain glycoprotein that interacts with itself, heparin and cells. The distal long arm plays major cell and heparin interactive roles. The long arm consists of three subunits (A, B1, B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). The globule is in turn subdivided into five subdomains (Gl-5). In order to analyze the functions of this region, recombinant G domains (rG, rAiG, rG5, rGΔ2980-3028) were expressed in Sf9 insect cells using a baculovirus expression vector. A hybrid molecule (B-rAiG), consisting of recombinant A chain(rAiG) and the authentic B chains (E8-B)was assembled in vitro. The intercalation of rAiG into E8-B chains suppressed a heparin binding activity identified in subdomain Gl-2. By the peptide napping and ligand blotting, the relative affinity of each subeomain to heparin was assigned as Gl> G2= G4> G5> G3, such that G1 bound strongly and G3 not at all. The active heparin binding site of G domain in intact laminin appears to be located in G4 and proximal G5. Cell binding was examined using fibrosarcoma Cells. Cells adhered to E8, B-rAiG, rAiG and rG, did not bind on denatured substrates, poorly bound to the mixture of E8-B and rG. Anti-${\alpha}$6 and anti-${\beta}$1 integrin subunit separately blocked cell adhesion on E8 and B-rAiG, but not on rAiG. Heparin inhibited cell adhesion on rAiG, partially on B-rAiG, and not on E8. In conclusion, 1) There are active and cryptic cell and heparin binding activities in G domain. 2) Triple-helix assembly inactivates cell and heparin binding activities and restores u6131 dependent cell binding activities.

• BMB Reports
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• v.50 no.2
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• pp.85-90
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• 2017

Recently, we demonstrated that superoxide dismutase 3 (SOD3) is a strong candidate for biomedicine. Anti-oxidant function of SOD3 was accomplished without cell penetration, and it inhibited the inflammatory responses via non-enzymatic functions. SOD3 has the heparin binding domain associating cell surface. Interestingly, we found that $Zn^{2+}$ promotes transduction effects of recombinant human SOD3 (rhSOD3) by increasing uptake via the heparin binding domain (HBD). We demonstrated an uptake of rhSOD3 from media to cell lysate via HBD, resulting in an accumulation of rhSOD3 in the nucleus, which was promoted by the presence of $Zn^{2+}$. This resulted in increased inhibitory effects of rhSOD3 on NF-{\kappa}B and STAT3 signals in the presence of $Zn^{2+}$, which shows elevated association of rhSOD3 into the cells. These results suggest that an optimized procedure can help to enhance the inflammatory efficacy of rhSOD3, as a novel biomedicine.

• The Korean Journal of Zoology
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• v.36 no.4
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• pp.514-521
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• 1993

Monocyte interaction with fibronectin (FN) mediates specific cell surface receptors and results in cell attachment and differentiation. Several cell-mediated activities for various fragments of FN have been documented. To investigate the regulatory mechanisms of monocyte differentiation by cell binding domains of FN and their receptors, cell attachment-, cell migration-, and its respective inhibition assay were carried out. Monocyte recognizes 38-kDa domain distinctively from its recognition of 85-kDa domain, and the heparin-binding site of the 38-kDa fragment is not involved in monocyte adhesion. Based on these experimental results, it can be suggested that monocvte/macrophase interacts with at least two different sites in FN, which is critical step in cell adhesion and (or) migration.

• KSBB Journal
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• v.17 no.2
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• pp.153-157
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• 2002

A fusion protein, consisting of a human epidermal growth factor as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as an inclusion body in recombinant E. coli, yet when the conventional solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably because of the opposite surface charge resulting from the vastly different pl values of each domain. Accordingly the solid-phase refolding process, which exploits the ionic interactions between a solid matrix and the protein, was tried, however the ionic binding yield was also very low regardless of the resins and pH conditions used. Therefore, to provide a higher affinity toward the solid matrix, six Iysine residues were tagged to the N-terminus of the hEGF domain. When cation exchange resins, such as heparin- or CM-Sepharose, were used as the matrix, the adsorption capacity increased 2.5~3-fold and the subsequent refolding yield increased nearly 15-fold compared to the conventional process. A similat result was also obtained when an Ni-NTA metal affinity resin was used.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.197-203
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• 2001

A fusion protein, consisting of human epidermal growth factor as a recognition domain and human angiogenin as a toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably due to the opposite surface charge due to vastly different pI values of each domain. Solid-phase refolding process exploiting ionic interactions between the solid matrix and the protein was tried, but the ionic binding yield was very low regardless of the resins and pH conditions used. To provide higher affinity toward the solid matrix, six lysine residues were tagged to the N -terminus of the hEGF domain When the cation exchange resins such as heparin- or CM-Sepharose were used as the matrix, the adsorption capacity increased 2.5-3 times and the subsequent refolding yield increased nearly IS times compared to the conventional process.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.57-62
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• 1999

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.7-18
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• 1985

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2571-2576
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• 2012

Breast cancer is the first of the most common ten cancers in Iraq. Its etiology is multifactorial, oxidative stress and lipid peroxidation being suggested to play important roles in carcinogenesis. The purpose of this study was to investigate the oxidant-antioxidant status in breast cancer patients, by measuring SOD isoenzyme activities (total SOD, CuZn-SOD, Mn-SOD and EC-SOD) in plasma and breast tumors, and by estimating thiobarbituric reactive substances (TBRS) in tissue homogenates. General increase in total SOD activity was observed in plasma and tissue samples of breast tumors, greater in the malignant when compared to benign group (p<0.05). Mn-SOD showed a significant decrease in tissue malignant samples (p<0.05), and insignificant decrease in plasma malignant samples compared with control and benign samples. Plasma EC-SOD activity in both patient benign and malignant breast tumors demonstrated 3.5% and 22.8% increase, respectively. However, there was a decrease in tissue EC-SOD activity in malignant breast tumors when compared with benign. A similar tendency was noted for TBRS. We suggest that elevated total SOD might reflect a response to oxidative stress, and then may predict a state of excess reactive oxygen species in the carcinogenesis process. If there is proteolytic removal of the heparin binding domain, EC-SOD will lose its affinity for the extracellular matrix and diffuse out of the tissue. This will result in a decreased EC-SOD activity, thus leading to an increase in the steady-state concentration of $O^{2-}$ in this domain, and increase in EC-SOD activity in the extracellular fluid. This might explain the results recorded here concerning the decrease in tissue EC-SOD activity and increase in plasma of breast cancer patients.