Abstract
The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.
Highlights
The cell surface proteoglycan fraction isolated by addition to a cell surface heparan sulfate proteoglycan these mild trypsin treatment ofNMuMG mouse mammary cells bear a cell surface chondroitin sulfate proteoglycan
We concludethat this fraction contaainusnique hybrid An alternate explanation for the presence of heparan sulfate proteoglycan bearing both heparan sulfate and chondroitin sulfateglycosaminoglycansbecause (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) thbeehavand chondroitin sulfate in the cell surface fraction is that thesechainsare on the same core protein,constitutinga hybrid proteoglycan
The chondroitin sulfate proteoglycan isolated from cartilage contains chondroitin sulfate and keratansulfate GAG,‘ these GAG’Sare bound to different ior of the chondroitin sulfate inthese separation techniques is affected by heparan sulfate-specific probes amino acids on the core protein via distinct types of linkage [6]
Summary
$ Recipient of a Finnish Cancer Union Grant. Cell Culture-NMuMGmouse mammary epithelial cells were maintained in bicarbonate-buffered Dulbecco’s modified Eagle medium (GIBCO) as described previously [1, 17]. The experiments used cells passaged 13-22 times since establishment in culture (low passage cells). Labeling and Haruest of Cell Surface Proteoglycan-These experiments were conducted with several cell surface proteoglycan preparations. The cells were labeled for 24 h in 100 pCi/ml carrier-free. H235S0(N4ew England Nuclear), the medium was discarded and the cells were scraped from the dish in cold Ca2+,MF-free Tris-buffered (Sigma, 10 mM, pH 7.4) isotonic saline plus 0.5 mM EDTA
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