J. Biol. Chem., Vol. 259, Issue 11, 6773-6782, 06, 1984
Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. Affinity fractionation on columns of platelet factor 4, plasma fibronectin, and octyl-sepharose
MW Lark and LA Culp
Both newly formed and long-term culture-generated substratum adhesion
sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were
extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt
and several protease inhibitors under conditions which result in maximal
solubilization of the sulfate-radiolabeled proteoglycans. Because of the
functional importance of heparan sulfate proteoglycans in the
fibronectin-dependent cell-substratum adhesion processes of these cells,
these proteoglycans were fractionated on affinity columns of
octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor
4 or plasma fibronectin. These affinity matrices resolved a number of both
binding and nonbinding classes of heparan sulfate proteoglycan from both
types of adhesion sites. In particular, the platelet factor 4 column could
resolve several proteoglycans with differing binding affinities.
Approximately twice as much heparan sulfate proteoglycan from newly formed
sites bound to all three matrices as proteoglycan from longterm sites. The
proteoglycan which bound to one matrix was then tested for binding to a
second matrix; this approach resolved a number of biochemically distinct
species. For example, one-half of the fibronectin-Sepharose-binding
fraction from the long-term sites could also bind to platelet factor
4-Sepharose; however, over 90% of the fibronectin-binding fraction from
newly formed sites could bind to platelet factor 4. A major portion of the
octyl- Sepharose-binding fractions of the original extracts could bind to
fibronectin-Sepharose. These studies indicate that some of these
proteoglycans have overlapping affinities for fibronectin, platelet factor
4, and octyl-Sepharose and that a portion of the heparan sulfate
proteoglycan from these adhesion sites cannot bind to any of these affinity
matrices. These results are discussed with regard to the functional
significance of these various heparan sulfate proteoglycans in mediating
adhesion to extracellular matrices containing fibronectin or platelet
factor 4.
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