title: The B16F10 cell receptor for a metastasis-promoting site on laminin-1 is a heparan sulfate/chondroitin sulfate-containing proteoglycan.
abstract: Exposure to AG73, a synthetic peptide (LQVQLSIR) from the COOH-terminal region of the laminin alpha1 chain, induces a malignant phenotype in B16F10 melanoma cells. Coinjection of this peptide with the cells results in an increase of lung tumors and also the formation of liver tumors in approximately 50% of the mice (W. H. Kim et al., Int. J. Cancer, 77: 632-639, 1998). Here we have characterized the cell surface receptor and its functional groups on B16F10 cells. Peptide affinity chromatography identified a cell surface protein eluting with 1 M NaCl, which ran in SDS gels as a broad band of M(r) approximately 150,000-200,000. Digestion with heparitinase and chondroitinase produced a core protein of lower molecular weight (M(r) approximately 90,000). Involvement of the glycosaminoglycan (GAG) side chains was demonstrated by inhibition of cell binding to the peptide by heparin, heparan sulfate, and chondroitin sulfate B, but not by chondroitin sulfates A or C, or hyaluronic acid. The IC(50) for heparin was the lowest, followed by heparan sulfate, then chondroitin sulfate B, suggesting that the overall sulfation of the GAG side chain is critical. This was confirmed by inhibition of attachment with chemically modified heparin and heparan sulfate, which also showed that N or O linkages were not important for function. Using sized heparin fragments to inhibit cell binding to the peptide demonstrated that 16-mer is the minimum length required. B16F10 cells form a network when grown on Matrigel, and this is prevented by addition of the AG73 peptide. The GAGs alone did not affect network formation, but heparin, heparan sulfate, and chondroitin sulfate B reversed the inhibitory effect of the peptide, whereas other GAGs were inactive. Furthermore, removal of cell surface GAGs inhibited cell attachment to the peptide. Cells treated with glycosidases and coinjected with the peptide formed liver tumors equal to the control group receiving no peptide, suggesting that the GAGs play an early role in peptide-mediated tumor metastasis. These data indicate that the B16F10 cell receptor for a laminin metastasis-promoting sequence is a heparan sulfate/chondroitin sulfate-containing proteoglycan, and these GAG side chains are functionally important in the cell-peptide interaction.
authors.affiliation: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland 20892-4370, USA.
authors.forename: Jean A
authors.initials: JA
authors.lastname: Engbring
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Hoffman
authors.forename: Arezo J
authors.initials: AJ
authors.lastname: Karmand
authors.forename: Hynda K
authors.initials: HK
authors.lastname: Kleinman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002809:Chondroitin Sulfates / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000556:secondary; D008175:Lung Neoplasms / Q000378:metabolism / Q000556:secondary; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D007797:Laminin; D010446:Peptide Fragments; D011509:Proteoglycans; D011956:Receptors, Cell Surface; C105786:AG 73; C121346:laminin 1