Purification and Properties of Erythropoietin.

Goldwasser, Eugene; Kung, Charles

doi:10.2172/4675513

Cited by 15 publications

(18 citation statements)

References 9 publications

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“…Asialo-rHuEPO showed a 2.5-fold increase in the in vitro activity, confirming the previous results with sheep plasma EPO [8], human urinary EPO [6], and rHuEPO [6]. EPOs further deglycosylated with P-galactosidase or with both pgalactosidase and p-N-acetylhexosaminidase also showed a similar degree of increase of the in vitro activity.…”

Section: Discussionsupporting

confidence: 88%

“…Digestion of human urinary EPO by endoglycosidase F, by which most N-linked oligosaccharides were removed, yielded EPO with no in vivo activity, while it retained the in vitro activity [5]. It has been reported, using EPO partially purified from serum of anemic sheep, that the in vivo activity was completely lost by asialylation but the in vitro activity was increased when asialo-EPO was assayed at low levels [8]. Thus, it appears from these results that terminal sialic acids and N-linked carbohydrates of EPO are not essential for manifestation of the in vitro activity, although the mechanism by which asialo-EPO gains an increased in vitvo activity is poorly understood.…”

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confidence: 99%

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The role of carbohydrate in recombinant human erythropoietin

Tsuda

Kawanishi

Ueda

et al. 1990

European Journal of Biochemistry

232

131

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1. Recombinant human erythropoietin has N-linked sugar Biochemistry 27, 5646 -56541. Here we have demonstrated the presence of 0-linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and GalP(1-3)GalNAc.2. To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activitiy of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitvo activity. Although erythropoiein from which N-linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N-linked sugar abolished the in vivo biological activity completely, indicating that the presence of N-linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin.3. Incubation of asialo-erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 "C for 15 min decreased the biological activity to 35% and 11 YO of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.Erythropoietin (EPO), a highly glycosylated 34 -38-kDa protein, regulates red blood cell levels by promoting maturation and growth of erythroid precursor cells. The molecular mass of the peptide chain deduced from its cDNA is about 18 kDa [l] and therefore about 50% of the mass of the fully glycosylated EPO is made up of Carbohydrates. Enzymes. Endoglycosidase F (EC 3.2.1.96); endo-cc-N-acetylgalactosaminidase (EC 3.2.1.97); sialidase (EC 3.2.1.18); P-galactosidase (EC 3.2.1.23); P-N-acetylhexosaminidase (EC 3.2.1.52); Nglycanase (EC 3.5.1.52); trypsin (EC 3.4.21.4); V8 protease (EC 3.4.21.29) clearance by binding of asialylated EPO to hepatic receptors recognizing exposed galactose residues. Digestion of human urinary EPO by endoglycosidase F, by which most N-linked oligosaccharides were removed, yielded EPO with no in vivo activity, while it retained the in vitro activity [5]. It has been reported, using EPO partially purified from serum of anemic sheep, that the in vivo activity was completely lost by asialylation but the in vitro activity was increased when asialo-EPO was assayed at low levels [8]. Thus, it appears from these results that terminal sialic acids and N-linked carbohydrates of EPO are not essential for manifestation of the in vitro activity, although the mechanism by which asialo-EPO gains an increased in vitvo activity is poorly under...

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Section: Discussionsupporting

confidence: 88%

mentioning

confidence: 99%

The role of carbohydrate in recombinant human erythropoietin

Tsuda

Kawanishi

Ueda

et al. 1990

European Journal of Biochemistry

232

131

View full text Add to dashboard Cite

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“…It is well known that glycans play a very important role in maintaining the stability, conformation, and biological activity of glycoproteins (17,18). Oligosaccharides are essential recognition sequences in cell-mediated adhesions in inflammatory and immune responses (19).…”

Section: Discussionmentioning

confidence: 99%

“…Oligosaccharides are essential recognition sequences in cell-mediated adhesions in inflammatory and immune responses (19). The role of oligosaccharides in modulating the half-life and biopotency of biomolecules is indeed well known (17,18,20). It has been shown in the case of thyroid-stimulating hormone that the deglycosylated form is 10 times more potent in vitro, but is 10 times less active in vivo due to rapid clearance (21).…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Role of Oligosaccharides in the Apoptotic Activity of Glycodelin A

Jayachandran

Shaila

Karande

2004

Journal of Biological Chemistry

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Glycodelin A, also known as placental protein-14, is a multifunctional glycosylated protein secreted by the uterine endometrium during the early phases of pregnancy. It is a known suppressor of T cell proliferation, inducer of T cell apoptosis, and inhibitor of sperm zona binding. Unlike in contraceptive activity, where the glycans on the molecule have been shown to play a crucial role, mutagenesis of the asparagines at sites of N-linked glycosylation (Asn 28 and Asn 63 ) to glutamine shows that the apoptogenic activity of glycodelin A is executed by the protein backbone. Glycosylation at Asn 28 appears to play a role in the extracellular secretion of the molecule, as mutation of Asn 28 resulted in a significant decrease in the amount of secreted protein, and loss of both glycosylation sites reduced the secretion drastically. Our results also suggest that the loss of glycosylation does not affect the dimerization status of the molecule.Glycodelin A (GdA), 1 also known as PP14 (placental protein-14), is a glycosylated dimeric 162-amino acid protein secreted by human endometrium in the late secretory phase of the menstrual cycle and in the early phase of pregnancy under the regulation of the hormone progesterone (1). Glycodelin is glycosylated at two of the three putative glycosylation sites (Asn 28 and Asn 63 ) (2). Observations of several research groups suggest that GdA is immunosuppressive in function (3), and previous studies from our laboratory indicate that this effect is due to its capacity to induce apoptosis in T cells (4). Interestingly, glycodelin S (GdS), another isoform of glycodelin synthesized by the seminal vesicles and accumulating in the seminal plasma, is not apoptogenic (26) even though both isoforms share the same amino acid sequence. As the glycans on both isoforms are different and as glycosylation has been demonstrated to play an important role in the contraceptive activity of GdA (13), it appeared logical to investigate the relevance of glycosylation to the apoptogenic function. However, attempts to deglycosylate GdA and GdS under native conditions using enzymes were not successful. Therefore, we resorted to molecular biology approaches to mutate the glycosylation sites and to express the protein in a eukaryotic system. The functional analysis of protein thus expressed proves that the apoptogenic activity of the molecule resides in its protein backbone, with the oligosaccharides being either permissive or non-permissive in the manifestation of this observed property. Furthermore, we have demonstrated that glycosylation at Asn 28 plays a role in the secretory rate of the protein, as the mutagenesis of the same drastically reduces protein secretion. We also have evidence to suggest that lack of glycosylation does not compromise the dimerization state of the molecule. EXPERIMENTAL PROCEDURESCells and Cell Lines-Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from healthy volunteers (male and female, 25-49 years old) using HISTOPAQUE-1077 (Sigma) accor...

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“…Erythro. poietin [1][2][3][4] effects erythrocytes, thymosin [5][6][7] effects lymphocytes and the protein that we now call MGI [8] induces from single undifferentiated hematopoietic cells the formation of colonies with mature macrophages and granulocytes [8][9][10][11][12][13][14][15]. Erythropoietin [3][4] and thymosin [?…”

Section: Introductionmentioning

confidence: 99%