title: Neural crest cell interaction with type VI collagen is mediated by multiple cooperative binding sites within triple-helix and globular domains.
abstract: Collagen type VI (Col VI) is a primary constituent of the extracellular matrix encountered by migrating avian neural crest cells in situ and is effective in promoting attachment and motility of these cells in vitro. In this study, we have explored the molecular mechanisms of neural crest-Col VI interaction by using quantitative assays for cell attachment and migration in vitro, proteolytic fragments of the collagen, and a panel of domain-specific monoclonal antibodies. Removal of the predominant portion of the amino-terminal globular domains of Col VI tetramers by pepsin digestion (P6 fragment) resulted in a > fivefold decrease in their cell adhesion and motility-promoting activity. Further digestion of P6 with bacterial collagenase, which causes a complete loss of the amino-terminal domains plus an adjacent triple-helical segment, did not affect adhesion but reduced migration down to 40% of that seen on undigested P6. Untreated and pepsin-digested Col VI monomers were significantly less effective than their tetrameric counterparts and a M(r) 200,000 fragment, generated from pepsin-digested monomers by a second pepsin treatment, only retained 40% of the motility-promoting activity while preserving the adhesive capacity. A mixture of amino- and carboxyl-terminal globular domains supported both cell attachment and migration. While neural crest cells adhered equally well to the individual intact alpha 1 (VI)/alpha 2(VI) and alpha 3(VI) chains, they migrated most extensively on the alpha 3(VI) chain. Conversely, pepsin-digested individual alpha chains were significantly less effective in promoting cell adhesion and locomotion. Selective preincubation of Col VI microfilaments and isolated tetramers with a panel of monoclonal antibodies against triple helix, carboxyl-terminal, and amino-terminal epitopes of the different constituent chains differentially perturbed neural crest cell attachment and migration. Sites differentially involved in neural crest cell attachment and migration seemed to be present at the carboxyl termini of the alpha 1(VI) and alpha 2(VI) chains and at the amino-terminus of the alpha 3(VI) chain. The results suggest that neural crest cells interact with Col VI through multiple and cooperative binding sites present within its triple-helical and globular domains. The differential involvement and efficiency of these sites in stimulating neural crest cell adhesion and migration is strongly determined by the supramolecular organization of the collagen and requires inter- and intramolecular structural integrity. Since neural crest cell attachment and migration on Col VI was completely inhibited by anti-beta 1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell--Col VI interaction.
authors.affiliation: Reference Center for Oncology, Experimental Division 2, Aviano (PN) Italy.
authors.forename: R
authors.initials: R
authors.lastname: Perris
authors.forename: H J
authors.initials: HJ
authors.lastname: Kuo
authors.forename: R W
authors.initials: RW
authors.lastname: Glanville
authors.forename: S
authors.initials: S
authors.lastname: Leibold
authors.forename: M
authors.initials: M
authors.lastname: Bronner-Fraser
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry*; D017364:Collagenases / Q000494:pharmacology; D003370:Coturnix; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015151:Immunoblotting; D016023:Integrins / Q000276:immunology; D009432:Neural Crest / Q000737:chemistry / Q000502:physiology*; D010434:Pepsin A / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D016023:Integrins; D010446:Peptide Fragments; D003094:Collagen; D010434:Pepsin A; D017364:Collagenases