Heparan sulfate proteoglycans of human neuroblastoma cells: affinity fractionation on columns of platelet factor-4+.
Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca2+ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C18 cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of "single-chain" heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.
D002118:Calcium; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006025:Glycosaminoglycans / Q000302:isolation & purification*; D006497:Heparan Sulfate / Q000302:isolation & purification*; D006801:Humans; D009447:Neuroblastoma / Q000032:analysis*; D009474:Neurons / Q000032:analysis*; D011509:Proteoglycans / Q000302:isolation & purification*
D002118:Calcium; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans