The Release of a Soluble Glycosylated Protein from Glycogen by Recombinant Lysosomal α-Glucosidase (rhGAA) In Vitro and Its Presence in Serum In Vivo

Abstract

In studies on the degradation of glycogen by rhGAA, a glycosylated protein core material was found which consists of about 5–6% of the total starting glycogen. There was an additional 25% of the glycogen unaccounted for based on glucose released. After incubation of glycogen with rhGAA until no more glucose was released, no other carbohydrate was detected on HPAEC-PAD. Several oligosaccharides are then detectable if the medium is first boiled in 0.1 N HCl or incubated with trypsin. It is present in serum either in an HCl extract or in a trypsin digest. The characteristics of the in vivo serum material are identical to the material in the in vitro incubation medium. One oligosaccharide cannot be further degraded by rhGAA, from the incubation medium as well as from serum co-elute on HPAEC-PAD. Several masked oligosaccharides in serum contain m-inositol, e-inositol, and sorbitol as the major carbohydrates. The presence of this glycosylated protein in serum is a fraction of glycogen that is degraded outside the lysosome and the cell. The glycosylated protein in the serum is not present in the serum of Pompe mice not on ERT, but it is present in the serum of Pompe disease patients who are on ERT, so it is a biomarker of GAA degradation of lysosomal glycogen.

The Action of Recombinant Human Lysosomal α-Glucosidase (rhGAA) on Human Liver Glycogen: Pathway to Complete Degradation

“…The significance of these results of the glucose released is that whereas rhGAA incubation resulted in the release of increasing amounts of glucose with time, it did not release the minor constituents [24]. In a time course of TFA hydrolysis, the minor constituents are not released until well after the glucans are hydrolyzed [25].…”

“…Figure 2 shows glucose released from Control 2 glycogen and the calculated daily remaining glycogen substrate, glucose released per day and the content of rhGAA remaining. The significance of these results of the glucose released is that whereas rhGAA incubation resulted in the release of increasing amounts of glucose with time, it did not release the minor constituents [24]. In a time course of TFA hydrolysis, the minor constituents are not released until well after the glucans are hydrolyzed [25].…”

“…Clearly, rhGAA has greater access to the glucose containing structures in Pompe and KOH-treated Control 2 glycogens. The residue material is not a purified fraction but rather a mixture of the residues from all of the oligosaccharides of a glycogen which are shown in Figure 5 and discussed further in Murray et al [24]. At this time, the nature of the association of the protein and carbohydrate components with the oligosaccharide components of glycogen is not known.…”

“…The average interior chain length for the Pompe glycogen was 3.7. The average exterior chain lengths were 15.9, 15.2 and 8.9 for Control 1, Control 2 and Pompe glycogens, respectively [23][24][25][26]. The protein content was less than one per cent for all but one sample.…”

Lysosomal Storage Disorders: Molecular Basis and Therapeutic Approaches

“…Regarding the same topic, Murray described his findings on glycogen-containing carbohydrates masked by an unknown protein derived from the recombinant GAA (rhGAA)-dependent glycogen breakdown outside of the lysosome and the cell. The author proposes the use of these new detected terminal degradation products of rhGAA in the serum as biomarkers for follow-up and treatment protocols [7]. Gragnaniello and collaborators presented their long-term experience on a wide newborn screening for Fabry disease and proposed lyso-Gb3 as a useful biomarker for diagnostic and follow-up protocols [8].…”