Generation of artificial proteoglycans containing glycosaminoglycan-modified CD44 - Demonstration of the interaction between RANTES and chondroitin sulfate

All CD44 isoforms are modified with chondroitin sulfate (CS), while only th ose containing variably spliced exon V3 are modified with both CS and hepar an sulfate (HS). The CS is added to a serine-glycine (SG) site in CD44 exon E5, while NS and CS are added to the SGSG site in exon V3, Site-directed m utagenesis and other molecular biology techniques were used to determine th e minimal motifs responsible for the addition of CS and HS to CD44 (see acc ompanying paper (Greenfield, B,, Wang, W,-C,, Marquardt, W., Piepkorn, RI., Wolff, E. A., Aruffo, A., and Bennett, K. L, (1999) J, Biol. Chem. 274, 25 11-2517)). We have used this information to generate artificial proteoglyca ns containing the extracellular domain of the cell adhesion protein lymphoc yte function-associated antigen-3 (LFA-3)(CD58) and CD44 motifs modified wi th CS or a combination of CS and HS. Analysis of the CD44-modified LFA-3 pr otein showed that it retains the ability to engage and trigger the function of its natural ligand CD2, resulting in T cell activation. In addition, th e glycosaminoglycan-modified artificial proteoglycan is capable of binding the chemokine RANTES (regulated upon activation, normally T cell expressed and Secreted) and delivering it to human T cells, resulting in enhanced T c ell activation. These data demonstrate that artificial proteoglycans can be ;engineered with functional domains that have enhanced activity by codelive ring glyaosaminoglycan-binding molecules. The artificial proteoglycans were also used as a model system to explore the glycosaminoglycan binding prope rties of basic-fibroblast growth factor and the chemokine RANTES. While bas ic-fibroblast growth factor was shown to bind BS alone, this model revealed that RANTES binds not only HS, as has been demonstrated in the past, but a lso CS. Thus, artificial proteoglycans can be used for studying the glycosa minoglycan binding patterns of growth factors and chemokines and provide a means to manipulate the levels, types, and activity of glycosaminoglycan-bi nding proteins in vitro and in vivo.