owed significantly 44%, 28% and 61% lower cell binding and thus decreasing internalization by 52%, 22223206 30% and 66%. However, C-terminally curtailed mutants, ECP3239 and ECP3238 showed similar cellular binding as ECP32 41 but lower internalization than wild type ECP3241. Most importantly, only 946128-88-7 supplier ECP3241 showed the highest cellular X: amino-n-butyric acid. ND: not 
determined. The result is expressed as the mean 6 S.D., n = 4. P,0.001; P,0.01; P,0.05. a Beas-2B cells were incubated with 5 mM FITC-peptides at 4uC for 1 h, washed twice with PBS, and subjected to ELISA. The amount of FITC-ECP3241 bound to Beas-2B cells was normalized to 100%. b Beas-2B cells were incubated with 5 mM FITC-peptides at 37uC for 1 h. The cells were washed twice with 500 ml PBS, trypsinized at 37uC for 15 min, suspended in 500 ml PBS, and then subjected to flow cytometry. The fluorescence of cells treated with ECP3241 was set as 100%. doi:10.1371/journal.pone.0057318.t002 uptake, strongly 19239230 suggesting that the length of our ECP-derived CPP was critical for internalization and residues from 32 to 41 were required. Effects of GAG on ECP3241 Binding Cell-membrane GAGs including HS, CS/DS, and HA are necessary for CPP internalization. To assess the effect of GAGs on ECP3241 cellular binding ability, soluble GAGs including LMWH, CSC, and HA were used as competitors to inhibit the attachment of ECP3241 to Beas-2B cells. At concentrations between 0.01 and 1 mg/ml, LMWH, CSC, and HA inhibited ECP3241 binding, with LMWH being the most effective and HA being the least. At concentrations exceeding 50 mg/ml, LMWH and CSC prevented approximately 70% of the normal ECP3241 binding, whereas 53% inhibition was observed for 100 mg/ml HA. Hence HS and CS might be involved in ECP3241 binding to Beas-2B cells. To clarify the roles of cell-surface HS and CS, the binding of ECP3241 to wild-type and two mutant strains of Chinese hamster ovary cells was assessed by ELISA. CHO-pgsD677 cells do not express Nacetylglucosaminyltransferase and glucuronyltransferase, and therefore lack HS, but produce three times more CS than wildtype CHO-K1 cells. CHO-pgsA745 cells are deficient in xylosyltransferase so that no GAG was present on the surface. The amount of ECP3241 bound to CHO-pgsA745 cells was found to be 52% less than that bound to CHO-K1 cells, suggesting that GAG was required for binding. HS, instead of CS, is therefore the major ECP3241 receptor. To further confirm that cell-surface HS, rather than CS, was involved in ECP3241 internalization, Beas-2B cells were treated with heparinase I, heparinase III or chondroitinase ABC for 2 h, and then with FITC-ECP3241 for 1 h prior to measuring the cell fluorescence signal. Pre-treatment of Beas-2B cells with heparinase I or heparinase III decreased ECP3241 internalization by 43% and 51%, respectively. CS depletion had little effect on ECP3241 internalization, thus heparinase, but not chondroitinase ABC, inhibits ECP3241 internalization. To verify that selective cell-surface polysaccharides had been enzymatically removed, the treated cells were subsequently incubated with anti-HS or anti-CS monoclonal antibodies. As expected, heparinase I and heparinase III had decreased the amount of HS on Beas-2B cell surfaces by 48% and 61%, respectively, and chondroitinase ABC removed 72% of the cell-surface CS. The HS that remained on Beas-2B cell surface accounted for the observed ECP3241 internalization into heparinase-treated cells. Conversely, even though 70% of
