Structural Study of the Sugar Chains of Porcine Factor VIII - Tissue- and Species-Specific Glycosylation of Factor VIII

Hatori, Takashi; Furukawa, Kiyoshi; Esmon, P C; Yokota, Tsuyoshi; Brown, Joseph; Sawada, Shuzo; Fournel, Michael A.; Kato, Masakatsu; Minaga, Takeyoshi; Kobata, Akira

doi:10.1006/abbi.1993.1595

Cited by 25 publications

(19 citation statements)

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“…These findings provide an enzymatic basis for the down-regulation of the ␣-galactosyl epitope by GnT-III but also suggest that the bisecting GlcNAc plays an in vivo role in regulating the biosynthesis of the terminal structures on the N-glycans as well as the core structures. In fact, many structural analyses of N-glycans from various species have reported that the ␣-galactosylated bisected oligosaccharides are very limited (49,50), whereas sialylated bisected ones are more abundant (26,28,29,(31)(32)(33), and this evidence would reasonably be accounted for by the findings reported herein.…”

Section: Discussionsupporting

confidence: 69%

Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene

Koyota¹,

Ikeda²,

Miyagawa³

et al. 2001

Journal of Biological Chemistry

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The down-regulation of the ␣-Gal epitope (Gal␣1,3Gal␤-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pigto-human xenotransplantation. In an earlier study, we reported that the introduction of the ␤1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the ␣-Gal epitope. In this study, we report on the mechanism for this down-regulation of the ␣-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of Nlinked oligosaccharides bearing the ␣-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than ␣-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on ␣-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

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Section: Discussionsupporting

confidence: 69%

Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene

Koyota¹,

Ikeda²,

Miyagawa³

et al. 2001

Journal of Biological Chemistry

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“…This suggests that a heatlabile component in NHS is responsible for promoting the greater effect of nanA in this serum source. Therefore, the difference between NHS and BRS could be due to increased exoglycosidase substrate specificity, since S. pneumoniae is a pathogen adapted to humans and glycosylation patterns can vary between species (17,29,40). Therefore, we believe that antibodies in NHS contribute to the deposition of complement on the pneumococcal surface but are not being acted upon by pneumococcal exoglycosidases.…”

Section: Discussionmentioning

confidence: 99%

Three Surface Exoglycosidases fromStreptococcus pneumoniae, NanA, BgaA, and StrH, Promote Resistance to Opsonophagocytic Killing by Human Neutrophils

Dalia¹,

Standish²,

2010

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Streptococcus pneumoniae (the pneumococcus) is a major human pathogen and a leading cause of inflammatory infections such as pneumonia and otitis media. An important mechanism for host defense against S. pneumoniae is opsonophagocytic killing by neutrophils. To persist in the human host, the pneumococcus has developed strategies to evade opsonization and subsequent neutrophil-mediated killing. Utilizing a genomic approach, we identified NanA, the major pneumococcal neuraminidase, as a factor important for resistance to opsonophagocytic killing in ex vivo killing assays using human neutrophils. The effect of NanA was shown using both type 4 (TIGR4) and type 6A clinical isolates. NanA promotes this resistance by acting in conjunction with two other surface-associated exoglycosidases, BgaA, a ␤-galactosidase, and StrH, an N-acetylglucosaminidase. Experiments using human serum showed that these exoglycosidases reduced deposition of complement component C3 on the pneumococcal surface, providing a mechanism for this resistance. Additionally, we have shown that antibodies in human serum do not contribute to this phenotype. These results demonstrate that deglycosylation of a human serum glycoconjugate(s) by the combined effects of NanA, BgaA, and StrH, is important for resistance to complement deposition and subsequent phagocytic killing of S. pneumoniae.

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“…Differences in the size and/or composition of N-linked glycans are frequently observed in the comparison of a recombinant protein and its naturally occurring counterpart (25,(27)(28)(29), which reflect the cell-, tissue-, and species-specificity of glycosylation (30). In some cases such differences have consequences for the functional properties of the recombinant protein.…”

Section: Figmentioning

confidence: 99%

“…However, in other cases differences in glycosylation have no apparent effect on the functional behavior of the recombinant protein. Human factor VIII expressed in BHK cells contains differences in the composition of its N-linked glycans relative to plasma-derived factor VIII (28), yet the two factor VIII preparations are similar with respect to cleavage by thrombin, factor Xa, and activated protein C (24), subunit association and dissociation (24), and pharmacokinetic parameters in baboons (28). Although we did not observe any functional differences between rTAFI and pTAFI attributable to differences in glycosylation, differences in, for example, the pharmacokinetics of rTAFI and pTAFI or in their binding to as yet undescribed substrates cannot be ruled out.…”

Section: Figmentioning

confidence: 99%

Plasma and Recombinant Thrombin-activable Fibrinolysis Inhibitor (TAFI) and Activated TAFI Compared with Respect to Glycosylation, Thrombin/Thrombomodulin-dependent Activation, Thermal Stability, and Enzymatic Properties

Boffa¹,

Wang

Bajzar

et al. 1998

Journal of Biological Chemistry

179

135

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Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B. Cleavage of the zymogen by thrombin/ thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo. In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with k cat ‫؍‬ 0.55 s ؊1 , K m ‫؍‬ 0.54 M, and K d ‫؍‬ 6.0 nM for rTAFI and k cat ‫؍‬ 0.61 s ؊1 , K m ‫؍‬ 0.55 M, and K d ‫؍‬ 6.6 nM for pTAFI. The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis. The half-life of both enzymes at 37°C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to ϳ62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme. Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (⌬H o ‡ Х 45 kcal/mol and ⌬S o ‡ Х 80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and ⑀-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.The balance between the activities of the coagulation and fibrinolytic cascades is essential to protect the organism from excessive blood loss upon injury as well as to maintain the fluidity of blood within the vasculature. Imbalances lead to a tendency toward either bleeding or thrombosis, the latter of which is manifested as heart attacks and strokes.The coagulation and fibrinolytic cascades consist of a series of zymogen to enzyme conversions, terminating in the proteolytic enzymes thrombin and plasmin, which, respectively, catalyze the deposition and removal of fibrin. However, when in a complex with the endothelial cell-surface cofactor thrombomodulin, the specificity of thrombin is changed from fibrinogen to protein C (1), thus changing thrombin to an anticoagulant rather than a procoagulant enzyme.When formed in the context of a fibrin clot, activated protein C was fou...

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Structural Study of the Sugar Chains of Porcine Factor VIII - Tissue- and Species-Specific Glycosylation of Factor VIII

Cited by 25 publications

References 0 publications

Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene

Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene

Three Surface Exoglycosidases fromStreptococcus pneumoniae, NanA, BgaA, and StrH, Promote Resistance to Opsonophagocytic Killing by Human Neutrophils

Plasma and Recombinant Thrombin-activable Fibrinolysis Inhibitor (TAFI) and Activated TAFI Compared with Respect to Glycosylation, Thrombin/Thrombomodulin-dependent Activation, Thermal Stability, and Enzymatic Properties

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