Purification and characterization of heparan sulfate from human primary osteoblasts.
Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts-soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non-identical by a number of parameters, and that these differences have significant ramifications for their ligand-binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS-dependent factors between the ECM and the cell surface.
Murali
Sadasivam
S
Stem Cells and Tissue Repair Group, Institute of Medical Biology, A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.
Manton
Kerry J
KJ
Tjong
Vinalia
V
Su
Xiaodi
X
Haupt
Larisa M
LM
Cool
Simon M
SM
Nurcombe
Victor
V
D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D005109:Extracellular Matrix / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D010006:Osteoblasts / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding
D055396:Bone Morphogenetic Protein 2; D017077:Culture Media, Conditioned; D016222:Fibroblast Growth Factor 2; D006497:Heparan Sulfate