The Preparation and Properties of Human Fibrinogen of Relatively High Solubility<sup>*</sup>

“…After centrifugation at 5000 g for 20 min, plasma was either processed immediately or stored at -26~ Blood was also taken for estimation of glucose and corporated, Akron, Ohio, USA) after removal of the labile fraction by means of dialysis against NaC1 (154 retool/l). Fibrinogen was estimated by the method of Strengers and Asberg [6] and purified by a modification of the method of Mosesson and Sherry [7] (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

Glycosylation of human fibrinogen in vivo

et al. 1985

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Summary.Fibrinogen was purified from plasma from 22 nondiabetic and 26 poorly controlled Type I (insulin-dependent) diabetic subjects. In non-diabetic subjects, 0.95+0.17mol glucose was bound per mol fibrinogen, whereas in the diabetic subjects 1.33 + 0.21 mol glucose was bound per tool fibrinogen (mean + SD, p < 0.001). Comparison of the amount of bound glucose, when estimated by two different methods, suggested that lysine is the site of glycosylation. It is currently unknown whether this increased glycosylation of fibrinogen alters its function.Key words: Fibrinogen, glycosylation, Type I diabetes.Glucose has been shown to bind non-enzymatically and irreversibly to all types of protein through Schiff-base formation and Amadori re-arrangement to a ketoamine [1]. This glycosylation appears to be dependent on the duration of exposure and glucose concentration in the surrounding medium. Glycosylation occurs either on the amino terminal end of the protein, as with haemoglobin [2], or on the free amino group of lysine. Because of this glycosylation, the tertiairy or quaternary structure, function and/or degradation of the protein molecule may be altered, as has been demonstrated with glycosylated haemoglobin (HbA0 Fibrinogen is a protein with a half-life of 3-4 days, which occupies a central position in blood clotting. It is known that the e-amino groups of lysine in the fibrinogen molecule play an important role in cross-linking fibrin monomers and in fibrinolysis. We have therefore investigated whether glycosylation of fibrinogen is increased in the blood of diabetic patients. Subjects and methods SubjectsTwenty-two healthy volunteers (aged 21-59 years, mean 33 years; mean blood glucose level at the time of sampling, 5.1 _+ 1.0 mmol/1) and 26 Type 1 (insulin-dependent) diabetic patients (aged 19-57 years, mean 36 years; taking no medication other than insulin) participated in the study. MethodsBlood was collected in 3.2% sodium citrate (1 : 10, wt/vol) for purification of fibrinogen and estimation of glycosylation. After centrifugation at 5000 g for 20 min, plasma was either processed immediately or stored at -26~ Blood was also taken for estimation of glucose and HbAv HbA1 was estimated by the microcolumn method ([solab In-

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“…Fetuin was purified from the fetal bovine serum (Sigma) using a modified procedure of Spiro (58). Porcine fibrinogen was purified from porcine plasma (Sigma) using a modified procedure (59).…”

Section: Methodsmentioning

confidence: 99%

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Giddens

Lomino

Amin

et al. 2016

Journal of Biological Chemistry

115

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Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptor substrate specificity toward ␣1,6-fucosyl-GlcNAc-Asn or ␣1, 6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi-and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.Protein glycosylation is one of the most prevalent posttranslational modifications, found in almost all living organisms ranging from bacteria to eukaryotes (1). Glycosylation can profoundly affect a protein's intrinsic properties, such as folding, stability, intracellular trafficking, and immunogenicity. In addition, the oligosaccharide components of glycoproteins can participate directly in a number of important biological recognition processes, including cell adhesion, signaling, hostpathogen interactions, and immune responses (2-8). It is well documented that subtle changes in glycosylation can lead to a significant impact on the biological functions and, in the case of therapeutic glycoproteins, such as monoclonal antibodies, the in vivo stability and therapeutic efficacy (7, 9 -12). Natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ only in the structures of pendant glycans, from which pure glycoforms are extremely difficult to isolate by current chromatographic techniques. As a result, synthetic homogeneous glycopeptides and glycoproteins emerge as indispensable tools for functional studies and for drug/vaccine discoveries. Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemic...

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The Preparation and Properties of Human Fibrinogen of Relatively High Solubility^*

Cited by 227 publications

References 21 publications

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Glycosylation of human fibrinogen in vivo

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

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The Preparation and Properties of Human Fibrinogen of Relatively High Solubility*

Cited by 227 publications

References 21 publications

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Glycosylation of human fibrinogen in vivo

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

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The Preparation and Properties of Human Fibrinogen of Relatively High Solubility^*