Human placental fibronectin: demonstration of structural differences between the A and B chains in the extra domain-A region.
Fibronectins are a class of cell adhesion proteins produced from a single gene. Soluble plasma fibronectin plays a role in wound healing and the insoluble cellular fibronectin form anchors cells to the substrata. The proteins possess multiple macromolecular binding domains including collagen, fibrin, and heparin. Alternative RNA splicing in at least three regions (ED-A, ED-B, and III CS) is responsible for this fibronectin polymorphism. We have been studying this polymorphism at the protein level in placental fibronectin, a poorly soluble form of cellular fibronectin. Cathepsin D-digested placental fibronectin applied to a heparin-agarose column and eluted with a NaCl stepwise gradient (0.1, 0.3, 0.5 M) gave two polypeptides (80-100 and 65 kDa) in the 0.3 M NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the carboxy-terminal heparin-binding domain) and IST-9 (specific for the ED-A portion of fibronectin) suggest that both peptides contain the carboxy-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the ED-A segment. The two peptides were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to Polybrene-coated polyvinyl difluoride membranes, and characterized by microsequence analysis. This analysis confirmed that both fragments start with the same amino acid sequence, 17 amino acids before the start of ED-A. These results demonstrate that placental fibronectin is a heterodimer, structurally distinct from plasma fibronectin due to the presence of a unique domain modification that is not seen in the plasma form.
Tressel
T
T
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Shively
J E
JE
Pande
H
H
D001483:Base Sequence; D002402:Cathepsin D / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis; D005260:Female; D005353:Fibronectins / Q000302:isolation & purification*; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D012326:RNA Splicing; D011956:Receptors, Cell Surface / Q000032:analysis
D004220:Disulfides; D005353:Fibronectins; D010446:Peptide Fragments; D010455:Peptides; D011956:Receptors, Cell Surface; C037680:heparin receptor; D002402:Cathepsin D