Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes

Ak, Rao; Garver, FA; Mendicino, Joseph

doi:10.1021/bi00668a009

Cited by 50 publications

(20 citation statements)

References 57 publications

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“…It was further assumed that the volume of each Golgi compartment (V) was equal to 10 -13 ml, as estimated using electron microscopy of Golgi cisternae [33]. In the cases that multiple glycan reactants are substrates for the same enzyme, the values were assumed to be equal for each glycan (Table 2) [16], [21], [23], [25]-[27], [29], [34]-[36].…”

Section: Methodsmentioning

confidence: 99%

Systems Analysis of N-Glycan Processing in Mammalian Cells

2007

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N-glycosylation plays a key role in the quality of many therapeutic glycoprotein biologics. The biosynthesis reactions of these oligosaccharides are a type of network in which a relatively small number of enzymes give rise to a large number of N-glycans as the reaction intermediates and terminal products. Multiple glycans appear on the glycoprotein molecules and give rise to a heterogeneous product. Controlling the glycan distribution is critical to the quality control of the product. Understanding N-glycan biosynthesis and the etiology of microheterogeneity would provide physiological insights, and facilitate cellular engineering to enhance glycoprotein quality. We developed a mathematical model of glycan biosynthesis in the Golgi and analyzed the various reaction variables on the resulting glycan distribution. The Golgi model was modeled as four compartments in series. The mechanism of protein transport across the Golgi is still controversial. From the viewpoint of their holding time distribution characteristics, the two main hypothesized mechanisms, vesicular transport and Golgi maturation models, resemble four continuous mixing-tanks (4CSTR) and four plug-flow reactors (4PFR) in series, respectively. The two hypotheses were modeled accordingly and compared. The intrinsic reaction kinetics were first evaluated using a batch (or single PFR) reactor. A sufficient holding time is needed to produce terminally-processed glycans. Altering enzyme concentrations has a complex effect on the final glycan distribution, as the changes often affect many reaction steps in the network. Comparison of the glycan profiles predicted by the 4CSTR and 4PFR models points to the 4PFR system as more likely to be the true mechanism. To assess whether glycan heterogeneity can be eliminated in the biosynthesis of biotherapeutics the 4PFR model was further used to assess whether a homogeneous glycan profile can be created through metabolic engineering. We demonstrate by the spatial localization of enzymes to specific compartments all terminally processed N-glycans can be synthesized as homogeneous products with a sufficient holding time in the Golgi compartments. The model developed may serve as a guide to future engineering of glycoproteins.

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Section: Methodsmentioning

confidence: 99%

Systems Analysis of N-Glycan Processing in Mammalian Cells

2007

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“…It was reported that the lactose synthetase and other p1-4GalTs are activated by Mn2+, which is involved in maintaining the structural integrity of the enzyme, and the optimal Mn2+ concentrations of these enzymes were in the range of 10-15 mM [2][3][4][5][6][7][8][9][10][11]241. The pCMGTl-directed P1-4GalT in COS-1 cells was also activated by Mn2' and its optimal Mn2+ concentration was about 10 mM.…”

Section: Activation Q F U Fli-4gnlt H-v Mn2' Ionmentioning

confidence: 99%

Characterization of a murine β1‐4galactosyltransferase expressed in COS‐1 cells

Nakazawa

Furukawa

Kobata

et al. 1991

European Journal of Biochemistry

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We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGTl -directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGTl -transfected cells was approximately eightfold higher than that in mock-or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGTl -transfected cells and mock-or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal j-galactosidase, most of the linkage created by this enzyme was in the Galbl-4GlcNAc group.The acceptor specificity of the pCMGTl -directed enzyme was changed from N-acetylglucosamine to glucose by adding a-lactalbumin in the reaction mixture. a-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent K, values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 pM and 24 pM, respectively.These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a j1-4 linkage The carbohydrate moieties of glycoproteins, glycolipids and proteoglycans are synthesized through a series of sugar chain elongation reactions which are catalyzed by specific glycosyltransferases [l]. A fl1-4galactosyltransferase (UDPGal :N-acetylglucosamine /?1-4galactosyltransferase ; PI -4-GalT is a member of the glycosyltransferase family. This enzyme catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine (GlcNAc) with the specific intersugar linkage, Galpl-4GlcNAc. During the last two decades, a number of investigators have reported the purification and characterization of jl-4GalTs from various sources such as human and bovine milk [2, 31, swine [ll]. Among these /31-4GalTs, a milk b1-4GalT (lactose synthetase A protein) is capable of recognizing glucose as an acceptor in the presence of a-lactalbumin (lactose synthetase B protein) [12]. The fll-4GalT activities purified from other tissues toward N-acetylglucosamine were also reported to be inhibited by a-lactalbumin, and the acceptor specificities of these b1-4GalTs were analogous to thatCorrespondence to H. Narimatsu,

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“…TaMe 1. Degradation rates (t 89 of brush border (BBM) and basolateral membrane (BLM) phospholipids determined using 2-3H acetate and uniformly labeled 3H-glycerota carried out using standard kinetic assays as previously reported from our laboratory [16,17] and galactosyltransferase was assayed according to the method of Rad et al [19]. Lipids from approximately 1 mg of membrane protein were extracted in 6 ml of chloroform/methanol (1 : 2 vol/vol) isolated and quantitated as we have previously described [16,17].…”

Section: Preparation and Characterization Of Membranesmentioning

confidence: 99%

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Molitoris

Simon

1986

J. Membrain Biol.

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Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using 32P and either [3H]-glycerol or [2-3H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies, again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 X faster than into the brush border membrane and its relative composition was 2.5 X greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.

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Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes

Cited by 50 publications

References 57 publications

Systems Analysis of N-Glycan Processing in Mammalian Cells

Systems Analysis of N-Glycan Processing in Mammalian Cells

Characterization of a murine β1‐4galactosyltransferase expressed in COS‐1 cells

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

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