Digestion resulted in key products of about 46 and 25 kDa (Fig. four) but only the full-length uncleaved protein along with the 25-kDa product reacted with the polyhistidine MAb (data not shown), indicating that the 46-kDa band represented the Nterminal fragment. These apparent Protocadherin-10 Proteins Molecular Weight masses are larger thanXIANG AND MOSSJ. VIROL.FIG. 4. In vitro cleavage of MC54L with recombinant furin. MC54L proteins that had been full length or had an internal deletion of (142-173) or (140-235) had been expressed individually in BS-C-1 cells by recombinant vaccinia viruses and purified by metal affinity chromatography. Recombinant MC54L proteins have been incubated with or without the need of recombinant furin and with or without having decRVKR-cmk and after that resolved by SDS-PAGE and detected by Coomassie staining. The values around the left indicate the mobilities and masses in kilodaltons of marker proteins.these predicted around the basis from the amino acid sequence due to N-glycosylation (24). The specificity of furin cleavage was demonstrated by the comprehensive inhibition created by the furin inhibitor dec-RVKR-cmk (Fig. four). The MC54L proteins with deletions (140-235) and (142-173) lack the 5 arginines comprising the predicted cleavage web site (Fig. 1). As shown in Fig. four, these proteins were completely resistant to furin digestion. In addition, when the latter proteins were expressed in 293T cells by a nonviral expression vector, only the uncleaved types, which bound IL-18 with higher affinity, have been detected (22). The full-length MC54L protein binds to glycosaminoglycans with higher affinity by means of the C-terminal tail. About half of the amino acids from residue 190 to the C terminus of MC54L are basic (Fig. 1), suggesting that this region may possibly bind negatively charged biomolecules for instance glycosaminoglycans. Fulllength MC54L bound to heparin-agarose quite tightly, as the CCL13 Proteins Accession binding was prevented only by salt concentrations of 0.55 M (Fig. 5A). The binding was specific, as it was inhibited by excess free of charge heparin (Fig. 5A) and no binding involving MC54L and control protein A-agarose was observed (data not shown). The heparin binding internet site was localized for the C terminus of MC54L, as the MC54L (140-235) protein failed to bind to heparinagarose whereas the MC54L (142-173) protein bound to heparin-agarose like full-length MC54L (Fig. 5A). As furin cleavage solutions of MC54L, as well as full-length MC54L, are released from infected cells, their abilities to bind to heparin had been also tested. The furin digestion merchandise were developed by in vitro cleavage of purified full-length MC54L and incubated with heparin-agarose. As predicted, the C-terminal furin cleavage products of MC54L have been capable to bind to heparinagarose although the N-terminal furin cleavage solution failed to bind to heparin (Fig. 5B). The binding affinity of MC54L for heparin was measured by surface plasmon resonance assay with a BIAcore apparatus. The artificial proteoglycan albumin-heparin and control albumin had been immobilized on two distinct flow cells of a BIAcore sensor chip. Several concentrations of full-length MC54L were then injected more than the chip, as well as the sensorgrams had been globallyFIG. five. Heparin binding properties of full-length and mutated forms of MC54L. MC54L proteins that were complete length or lacked amino acids 142 to 173 or 140 to 235 had been expressed individually in BS-C-1 cells by recombinant vaccinia viruses and purified by metal affinity chromatography. (A) Except for the manage lanes, recombinant MC54L proteins were incubat.