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 those containing variably spliced exon V3 are modified with both CS and heparan sulfate (HS). The CS is added to a serine-glycine (SG) site in CD44 exon E5, while HS and CS are added to the SGSG site in exon V3. Site-directed mutagenesis and other molecular biology techniques were used to determine the minimal motifs responsible for the addition of CS and HS to CD44 (see accompanying paper (Greenfield, B., Wang, W.-C., Marquardt, H., Piepkorn, M., Wolff, E. A., Aruffo, A., and Bennett, K. L. (1999) J. Biol. Chem. 274, 2511-2517)). We have used this information to generate artificial proteoglycans containing the extracellular domain of the cell adhesion protein lymphocyte function-associated antigen-3 (LFA-3) (CD58) and CD44 motifs modified with CS or a combination of CS and HS. Analysis of the CD44-modified LFA-3 protein showed that it retains the ability to engage and trigger the function of its natural ligand CD2, resulting in T cell activation. In addition, the 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 cell activation. These data demonstrate that artificial proteoglycans can be engineered with functional domains that have enhanced activity by codelivering glycosaminoglycan-binding molecules. The artificial proteoglycans were also used as a model system to explore the glycosaminoglycan binding properties of basic-fibroblast growth factor and the chemokine RANTES. While basic-fibroblast growth factor was shown to bind HS alone, this model revealed that RANTES binds not only HS, as has been demonstrated in the past, but also CS. Thus, artificial proteoglycans can be used for studying the glycosaminoglycan binding patterns of growth factors and chemokines and provide a means to manipulate the levels, types, and activity of glycosaminoglycan-binding proteins in vitro and in vivo.
Wolff
E A
EA
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA. wolffe@bms.com
Greenfield
B
B
Taub
D D
DD
Murphy
W J
WJ
Bennett
K L
KL
Aruffo
A
A
D001483:Base Sequence; D018968:CD58 Antigens / Q000378:metabolism; D002455:Cell Division; D018946:Chemokine CCL5 / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism*; D017931:DNA Primers; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry*; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000378:metabolism*; D008213:Lymphocyte Activation; D016297:Mutagenesis, Site-Directed; D011509:Proteoglycans / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D018417:Th1 Cells / Q000166:cytology / Q000378:metabolism; D018418:Th2 Cells / Q000166:cytology / Q000378:metabolism
D018968:CD58 Antigens; D018946:Chemokine CCL5; D017931:DNA Primers; D006025:Glycosaminoglycans; D018960:Hyaluronan Receptors; D011509:Proteoglycans; D016222:Fibroblast Growth Factor 2; D002809:Chondroitin Sulfates