CHARACTERIZATION OF GLYCOSYLATED AND CATALYTICALLY ACTIVE RECOMBINANTHUMAN ALPHA-GALACTOSIDASE-A USING A BACULOVIRUS VECTOR

Fabry disease is an X-linked inborn error of glycolipid metabolism cau sed by a deficiency of the lysosomal enzyme alpha-galactosidase A (Gal A; EC 3.2.1.22). In order to obtain large quantities of this human enz yme for physical characterization and for the development of new appro aches for enzyme therapy, we constructed derivatives of the Autographa californica nuclear polyhedrosis virus that produce the human enzyme. The recombinant GalA (re-GalA) is produced at high levels, and is act ive with both the artificial substrate, 4-methylumbelliferyl-alpha-D-g alactopyranoside, and the natural in vivo substrate, trihexosylceramid e. The purified re-GalA is glycosylated and is taken up by normal and Fabry fibroblasts in cell culture. Mass spectral analysis of total mon osaccharides released by hydrazinolysis indicates that it contains fuc ose, galactose, mannose and N-acetylglucosamine. Amino-acid sequence a nalysis of six proteolytic peptides corresponded to sequences predicte d by the cDNA. The molecular masses' of the purified enzyme, estimated by electrospray mass spectroscopy and laser desorption time-of-flight analysis are 46.85 and 46.62 kDa, respectively, approx. 10% greater t han the polypeptide portion predicted by the cDNA. The recombinant enz yme retains significant catalytic activity after modification with pol y(ethylene glycol), a treatment which decreases the immunogenicity and increases the circulation life of many proteins used therapeutically.