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Hyung Keun Lee, Joon H Lee, Wungrak Choi, Insil song, Eung Kweon Kim, SEED: Study group for Environmental Eye Disease; Expression of tyrosine sulfated decoy receptors mimicking traps and their effect on preventing chemokine induced cell migration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5442. doi: https://doi.org/.
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Tyrosine sulfation is an important post-translational modification that occurs in higher eukaryotes and is involved in cell-cell communication, adhesion, and chemotaxis. The purpose of this study is to generate tyrosine sulfated human decoy receptor D6 mimicking trap and determine their effect on the inhibiting chemokine induced cell migration for reducing ocular inflammation.
To generate tyrosine sulfated decoy receptor D6 mimicking trap (sD6MT), human D6 was cloned and cDNAs encoding fragments of D6 were amplified by PCR. Then, the D6 fragments cDNAs were co-translationally inserted pSUPAR6-L3-3SY, which were response to the amber nonsence codon, TAG. After genetically insertion of TAG codon at the site to desired location of tyrosine sulfation, co-transformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an E. coli stain. The overexpression of the site-specifically sulfated protein was confirmed with chromatography. LPS induced THP-1 cell migration with or without sD6MT was determined with Boyden chamber assay.<br />
We successfully generated 4 types of overexpressed sD6MT and confirmed the tyrosine sulfation by negative shift of SDS-PAGE gel running. Additionally, the sulfated site and molecular weight of each protein traps were confirmed by MALDI-TOF. By the activation of LPS (10uM), THP-1 cell migration was significantly increased at 8 hours after Boyden chamber assay. However, by treating sD6MT treatment, cell migration was significantly reduced with dose dependent manner until 1mg/ml. Surprisingly, compared between non-sulfated D6MT and sD6MT on CCL2 induced THP-1 migration, sD6MT inhibit CCL2 induced THP-1 chemotaxis more effectively. Among the four types of sD6MT, we successfully selected most effective form for CCL2 induced THP-1 migration.<br />
Most of ocular diseases are related with immune or inflammatory cell migration. By the D6 expression vectors cloning and using pSUPAR6-L3-3SY, we successfully generated various types of sD6MT. Also, by treating sD6MT, LPS induced cell migration was more effectively inhibited compared with nonsulfated form. Conclusively, tyrosine sulfation in D6 decoy receptor may be an important step for enhancing its chemokine decoying activity. And sD6MT may apply to prevent chemokine induced cell migration in ocular immune or inflammatory diseases. <br />
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