PRELIMINARY CHARACTERIZATION OF THE PROTEOGLYCANS IN THE SUBSTRATE ADHESION SITES OF NORMAL AND VIRUS-TRANSFORMED MURINE CELLS

Substrate-attached material consists of the remnants of adhesion sites left firmly adherent to an artificial tissue culture substrate after detachment of normal or SV40-transformed Balb/c 3T3 cells using the Ca2+ chelator EGTA. This material is enriched in glycosaminoglycans (GAG's) for which functions in mediating cell-substrate adhesion have been proposed. The GAG's in substrate-attached material have been investigated for their structural connection to protein and their ability to interact with each other. Intact proteoglycan species from this material were resolved by molecular sieve chromatography in NaDodSO4 buffer. Sizing of specific GAG's before and after Pronase treatment revealed that nearly all of the chondroitin and dermatan sulfate species and over 50% of the heparan sulfate species were present as proteoglycans. These species are present in different relative amounts in newly formed footpad adhesion sites as compared to substrate-attached material which contains footpads plus footprints (the latter material being remnants of the footpads left at the trailing end of cells during normal cellular movement). Little, if any, protein was detected in association with hyaluronic acid. Isopycnic density gradient centrifugation of substrate-attached material under associative conditions generated two bands of GAG-containing material at high densities and one band at low density. In the presence of 4.0 M guanidine hydrochloride, nearly all of the material in the higher density bands reversibly dissociated. This material consisted predominantly of heparan sulfate, along with some chondro-itinase-digestible GAG and glycopeptide. Competition with hyaluronate oligomers also dissociated part of these denser bands. This suggests the presence in substrate-attached material of heparan sulfate containing aggregates along with the possible presence of cartilage-like proteoglycan-hyaluronate aggregates. These results are discussed in terms of an adhesion model in which proteoglycan interactions with cell surface fibronectin can affect and modify adhesion. © 1979, American Chemical Society. All rights reserved.