The phosphate content of human fibronectin

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Fibronectin is a crucial extracellular matrix glycoprotein involved in cell adhesion, migration, and differentiation.
• The post-translational modification of fibronectin, including phosphorylation, can significantly impact its structure and function.
• Understanding the precise location and nature of fibronectin phosphorylation is essential for elucidating its biological roles.

Purpose of the Study:

• To investigate the nature and distribution of covalently bound phosphate in human fibronectin.
• To identify the specific amino acid residues and regions phosphorylated within the fibronectin molecule.
• To determine if phosphorylation is conserved across different forms of fibronectin.

Main Methods:

• Limited tryptic digestion of [32P]orthophosphate-labeled fibronectin to analyze phosphate-containing fragments.
• Cyanogen bromide (CNBr) cleavage of 32P-labeled fibronectin to map phosphorylated regions.
• Chromatographic purification of phosphopeptides.
• Analysis of molecular weight and co-purification of phosphopeptides.

Main Results:

• Phosphate was exclusively detected as phosphoserine, not on asparagine-linked oligosaccharides.
• Limited tryptic digestion revealed phosphate associated with progressively smaller molecular weight peptides, ultimately concentrating in a ~6,000 Mr phosphopeptide.
• Phosphorylation was localized to a 40,000-50,000 segment near one end of the fibronectin molecule, distinct from the COOH-terminal interchain disulfide bond region.
• CNBr cleavage produced a ~5,000 Mr phosphopeptide, consistent across plasma and fibroblast fibronectin.
• The phosphorylation site is conserved across plasma, cell surface, and secreted fibronectin.

Conclusions:

• Fibronectin phosphorylation is a site-specific event, restricted to a small region of the protein.
• The exclusive presence of phosphoserine indicates a specific enzymatic modification.
• The conserved phosphorylation site across various fibronectin forms suggests a fundamental and important biological function.
• Further research is warranted to elucidate the functional consequences of this specific phosphorylation event.