Gelatin binding to the 8F19F1 module pair of human fibronectin requires site-specific N-glycosylation

The gelatin (denatured collagen) binding domain of the extracellular matrix protein fibronectin contains three potential N-glycosylation sites. Complete deglycosylation of this domain is known to reduce the thermal stability of the eighth type 1 (⁸F1) module. We have conducted a site-specific analysis of the structural and functional consequences of N-linked glycosylation in the ⁸F1⁹F1 module pair. Three glycoforms have been identified by mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical shift differences between the glycoforms have revealed an intimate interaction between one N-linked sugar and the polypeptide that is critical for gelatin binding, as shown by affinity chromatography.