Structural change of N-glycan exposes hydrophobic surface of human transferrin.

Transferrin is an iron-transport protein which possesses N-glycans at Asn432 and Asn630 in humans. Transferrin glycoforms Tf-1 and Tf-2, previously identified in human cerebrospinal fluid, are defined as the lower and upper bands in gel electrophoresis, respectively. Importantly, the Tf-2/Tf-1 ratio is raised in idiopathic normal pressure hydrocephalus patients and is useful as a clinical marker. In order to gain insight into the relationship between transferrin glycoform and biological function, we performed comparative characterization of Tf-1, Tf-2 and serum transferrin (sTf). Mass spectrometric analyses confirmed that Tf-2 is modified with disialylated biantennary glycans at both of the two N-glycosylation sites, which are similar to the N-glycans of sTf. On the other hand, Tf-1 is site-specifically modified: Asn630 has biantennary agalacto-complex-type glycan with bisecting N-acetylglucosamine (GlcNAc) and core fucose while Asn432 is modified with complex/high mannose-type glycans and possibly single GlcNAc. Size exclusion chromatography and fluorescence correlation spectroscopy analysis revealed that the hydration volume of Tf-1 is slightly smaller than that of sTf. Our striking finding is that Tf-1 has an exposed hydrophobic surface as monitored by the fluorescence intensity and wavelength of a hydrophobic probe, 1-anilino-8-naphthalene sulfonate, whereas Tf-2 does not. These results suggest that the different N-glycan structure of Tf-1 lowers the apparent hydration volume and reveals a patch of hydrophobic surface on transferrin which is otherwise covered with sialoglycan in sTf and Tf-2. The carbohydrate deficiency in certain pathological conditions may also expose hydrophobic surface which may modulate the function and/or stability of transferrin.