ABSTRACT
Assessment of biological potency and its comparison with clinical effects are important in the quality control of therapeutic glycoproteins. Animal models are usually used for evaluating bioactivity of these compounds. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with animal studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of recombinant human erythropoietin (rhEPO). In this study, we used capillary zone electrophoresis, a charge-based separation method, to estimate the sialic acid content for predicting in vivo bioactivity of rhEPO. In vivo bioactivities of rhEPO subfractions were measured and compared with sialylation levels. The results obtained indicated that in vivo bioactivity of rhEPO is not simply correlated with the sialylation level, which suggests that it is difficult to predict biological potency from the sialic acid content alone. N-Glycan moieties as well as sialic acid residues may have a significant impact on in vivo bioactivity of rhEPO.
Subject(s)
Erythropoietin/analysis , Erythropoietin/metabolism , N-Acetylneuraminic Acid/analysis , N-Acetylneuraminic Acid/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Electrophoresis, Capillary , Glycosylation , Humans , Recombinant ProteinsABSTRACT
Two distinct extracellular bifunctional proteins with beta-L-arabinopyranosidase/alpha-D-galactopyranosidase activities were purified from the culture filtrate of Fusarium oxysporum 12S. The molecular masses of the enzymes were estimated to be 55 (Fo/AP1) and 73 kDa (Fo/AP2) by SDS-PAGE. They hydrolyzed both p-nitrophenyl beta-L-arabinopyranoside and p-nitrophenyl alpha-D-galactopyranoside with different specificities. Fo/AP1 also showed low activity towards alpha-D-galactopyranosyl oligosaccharides such as raffinose. Interestingly, both enzymes hydrolyzed larch wood arabinogalactan (releasing arabinose) but not carob galactomannan, which has alpha-D-galactopyranosyl side chains. When larch wood arabinogalactan was incubated with excess Fo/AP1 or Fo/AP2, both enzymes released approximately 10% of the total arabinose in the substrate. cDNAs encoding Fo/AP1 and Fo/AP2 (Foap1 and Foap2) were isolated by in vitro cloning. The coding sequences of Foap1 and Foap2 genes were 1,647 and 1,620 bp in length and encode polypeptides of 549 and 540 amino acids, respectively. The N-terminal halves of both proteins had high similarity to putative conserved domains of the melibiase superfamily (Pfam account number 02065). The deduced amino acid sequences of the two enzymes indicate that they belong to glycosyl hydrolase family 27. Moreover, the C-terminal regions of both proteins contain a putative family 35 carbohydrate-binding module.
Subject(s)
Arabinose/metabolism , Enzymes/genetics , Enzymes/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fusarium/enzymology , Galactose/metabolism , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Fungal/chemistry , DNA, Fungal/genetics , Enzymes/chemistry , Enzymes/isolation & purification , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Fusarium/genetics , Molecular Sequence Data , Molecular Weight , Oligosaccharides/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a beta-1,6-galactanase that preferentially debranches beta-1,6-galactobiose from the substrate.
