Influence of Carbohydrate on Structure, Stability, and Function of Gelatin-Binding Fragments of Fibronectin

The gelatin-binding region of fibronectin contains three Asn-linked carbohydrate moieties, one on the second type II module and two on the eighth type I "finger" module. Carbohydrate groups were enzymatically removed from two nonoverlapping gelatin-binding fragments (GBFs), 21-kDa GBF (modular composition I8-I9) and, with much greater difficulty, 30-kDa GBF (modular composition I6-II1-II2-I7). The gelatin-binding properties of these fragments were affected only slightly or not at all. Fluorescence and calorimetric analyses indicated that module I8 was strongly destabilized by deglycosylation such that the apo form melts near physiological temperature. A similar effect was caused by decreasing the pH of the holo form to 6.0, suggesting that one or more histidines are important for stability of module I8. The 21-kDa fragment exhibited an acid-induced change in fluorescence that occurred at higher pH in the deglycosylated derivative, providing further evidence of a stabilizing role for one or both carbohydrate moieties. By contrast, the stability of module II2 was unaffected by removal of its single carbohydrate. The effects of carbohydrate on the stability of module I8 may be important in efforts to express it or fragments containing it in bacteria.