Role of N-Glycan Chain in Factor VIII - C Biological Activity

Katiyar, Vishwa

Abstract

Factor VIII:C is a component of the blood coagulation cascade and its deficiency leads to hemophilia A. It is a bleeding disorder that affects 10-20 per 100,000 males. The disease is transmitted by X-chromosomal inheritance and causes the blood to clot slowly. Prothrombin is converted to thrombin at an abnormally slow rate and a re-bleeding phenomena is frequently seen. Cloning and expression of human Factor VIII:C has suggested the presence of 25 asparagine-linked (N-linked) glycosylation sites in a 270 kDa protein. The N-glycan structure of the recombinant Factor VIII:C expressed in baby hamster kidney (BHK) cells revealed mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. In addition, it contains Gal alpha1-3Gal group as 3 percent of the total sugar chain, and Gal alpha1-4(Fuc alpha1-3) GlcNAc beta1-4(gal alpha1-4 GlcNAc beta1-2)Man group. The presence of high levels of Gal alpha 1 -3Gal group makes the recombinant Factor VIII:C highly antigenic, and as such, is unfit for use as a drug. Our long-term objective is to develop Factor VIII:C from a naturally occurring cell type that is free from side effects and that can be used to combat hemophilia A.
The specific aim of our proposal is to test the hypothesis that N-glycosylation is essential for a naturally expressed Factor VIII:C blood clotting activity, and discrete changes in its N-glycan structure affects the ability to activate Factor X. Using a capillary endothelial cell line as a source for naturally occurring active Factor VIII:C, we propose to (i) identification and determination the sequence of N-glycan chains by the FACE OLIGO Profiling and Sequencing systems; (ii) determine the N-glycan structure by Electrospray Ionization Mass Spectrometry; and (iii) develop Factor VIII:C variants defective in N-glycan chain using exo- and endo-glycosidases in vitro as well as using glycosylation and/or oligosaccharide processing inhibitors in vivo to study its biological activity. At the conclusion of the project we expect to have the complete N-glycan structure of endothelial cells expressed Factor VIII:C. In addition, we will also obtain critical information on the role of N-glycans in the Factor VIII:C biological activity, i.e. its ability to convert Factor X to Factor Xa.