Heparan Sulfate Proteoglycans Retain Noggin at the Cell Surface A POTENTIAL MECHANISM FOR SHAPING BONE MORPHOGENETIC PROTEIN GRADIENTS*

Bone morphogenetic proteins (BMPs) are expressed broadly and regulate a diverse array of developmental events in vivo . Essential to many of these functions is the establishment of activity gradients of BMP, which pro-vide positional information that influences cell fates. Secreted polypeptides, such as Noggin, bind BMPs and inhibit their function by preventing interaction with receptors on the cell surface. These BMP antagonists are assumed to be diffusible and therefore potentially important in the establishment of BMP activity gradients in vivo . Nothing is known, however, about the potential interactions between Noggin and components of the cell surface or extracellular matrix that might limit its diffusion. We have found that Noggin binds strongly to heparin in vitro and to heparan sulfate proteoglycans on the surface of cultured cells. Noggin is detected only on the surface of cells that express heparan sulfate, can be specifically displaced from cells by heparin, and can be directly cross-linked to a cell surface proteoglycan in culture. Heparan sulfate-bound Noggin remains functional and can bind BMP4 at the plasma membrane. A Noggin mutant with a deletion in a putative heparin binding domain has reduced binding to heparin and does not bind to the cell surface but has (cid:1) B2, encoding expression of full-length human Noggin (hNog) and a genetically engineered deletion mutant were a gift of Regeneron. For the generation of stably transfected cell lines, cells were co-transfected with pSVZeo from Invitrogen. Cell Culture and Transfection— Parental Chinese Hamster ovary cells (CHOK1) as well as mutant lines derived from these same cells but which are defective in either heparan sulfate (PGSD-677) or both heparan and chondroitin sulfate (PGSA-745) biosynthesis were a gift of Jeff Esko (University of California, San Diego). Cells were maintained in DMEM/F-12 medium (BioWhittaker) containing 10% fetal bovine se- rum (Hyclone). Liposome-mediated transfection was performed using Geneporter (Gene Therapy Systems) according to the manufacturer’s recommendations. Cells were cotransfected with pSVZeo (Invitrogen, San Diego, CA) and selected with 0.5 mg/ml Zeocin (Invitrogen, San Diego, CA) in DMEM/F-12 medium containing 10% fetal bovine serum, and individual clones were harvested and subcultured. Western blot- ting with RP57–16 antibody identified positive clones expressing similar levels of Noggin. Metabolic Labeling, Pulse-Chase, and Immunoprecipitation— For metabolic labeling of Noggin-expressing cultures, cells were incubated in methionine- and cysteine-free DMEM (Invitrogen) for 40 min. cells cells ( ( of (cid:3) g/ml heparin during the resulted rapid ( this within the ( In contrast, treatment of had no effect on the fate of Noggin ( C and G suggesting that the effect of heparin is specific and represents competition sulfate binding on cell Consistent with these observations, the hNog (cid:1) B2 deletion mutant of Noggin, with reduced binding to heparin, is rapidly released from CHOK1 cells ( D ) without heparin treatment and shows stable accumulation within the medium ( H ). essential requirement for heparan sulfate as a coreceptor for BMP.