Spermidine-induced glycoprotein fucosylation in immature rat intestine.

In rat small intestine, during postnatal development, the glycoprotein fucosylation is markedly increased at weaning. At the same time, a rise in the intestinal spermidine level was observed, partly due to the increase in the spermidine content of solid food given to animals at this period as compared to the spermidine content of milk. In order to mimic the spermidine increase observed in weanling rat intestines, we had treated suckling rats with spermidine by oral ingestion to study its role as maturation factor of the small intestine. In spermidine-treated suckling rats, the spermidine and N-acetyl-spermidine contents were highly increased. Spermidine treatment induced the rise in alpha-1,2-fucosyltransferase activity and the precocious appearance in the brush-border membrane of some alpha-1,2-fucoproteins in weaned rats. Such results indicate that spermidine could be a maturation factor implicated in the appearance of alpha-1,2-fucoproteins naturally observed at weaning time.     

Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation

Monoclonal antibodies are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute that has ramifications for patient safety and physiological efficacy—one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-F-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from Days 4 to 7 in a batch process used for IgG1 production from Chinese hamster ovary cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by ∼30%. The supplements caused significant changes in gene expression by Day 4 of the cultures where 2FP addition significantly reduced fucosyltransferase 8 and nucleotide sugar transporter gene expression (by ∼2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1–6 genes (by 1.5–3-fold). These gene expression data alongside glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes.     

Epidermal fucosylation of cell surface glycoprotein

The formation of covalently linked, high molecular weight protein aggregates has been thought to play an important role in opacification of the human lens. Antisera were used in Western blot analysis to demonstrate the involvement of all major classes of lens proteins (alpha, beta and gamma crystallin; the major intrinsic membrane polypeptide) in covalent aggregation. Of these classes, aggregation of gamma and beta crystallins via intermolecular disulfide bonding and aggregation of the major intrinsic membrane polypeptide via intermolecular, non-disulfide bonding were more pronounced in cataractous as compared with normal lenses.     

The role of cernitins in cadmium effect on the absorption processes in rat small intestine

The effect of long-term intoxication of cadmium (administered subcutaneously in a dose of 1 mg Cd/kg of body weight once a week for one month) on the absorption of water, sodium, potassium, glucose, glycine and thiamine in the small intestine of rats was investigated. In addition, the influence of cernitins (special pollen extract) on the action of cadmium intoxication was tested. The cernitins were given by stomach pump in the form of an aqueous solution of Pollitabs Sport tablets in a dose of 1.5 mg of cernitin T-60 and 0.075 mg of cernitin GBX per individual twice a week for two weeks or four weeks according to the group of animals tested. The results indicated that long-term administration of cadmium increased the intestinal absorption of all tested substances. Meanwhile, the application of cernitins reduced the effects of cadmium intoxication upon intestinal absorption and the processes of absorption was close to normal.     

Slow-waves in rat small intestine

Rat small intestine generates rhythmic slow-wave activity. The slow-waves are not eliminated by ouabain application or incubation in potassium free solution. Exposure to low sodium or calcium free solution decreases slow-wave activity. Incubation in sodium and calcium free solution eliminates activity. It is concluded that rat small intestinal slow-waves may not result from the same mechanism as in the cat.     

A shift in microvillus membrane fucosylation to sialylation by ethanol ingestion in rat intestine

The luminal surface of enterocytes is covered with glycocalyx which is rich in glycoproteins. Ethanol ingestion is shown to induce morphological and biochemical changes in the intestine. In this study, the effect of ethanol ingestion on membrane glycoproteins has been investigated. Chemical analysis of microvillus membranes revealed an increase in hexose and sialic acid contents, but a reduction in fucose levels in ethanol-fed rats compared with controls. The observed changes were apparent in animals fed with ethanol for 35–56 days compared with controls. Lectin-binding assay indicated an increase in Wheat germ agglutinin (affinity for GlcNAc/sialic acid) and a decrease in Aleuria aurantia (affinity for α-l-fucose) reactivity of brush borders in ethanol-fed animals for 4–8 weeks. Western blot analysis using biotin-labeled Wheat germ agglutinin revealed increased binding to proteins of M_r 66–205 kDa in ethanol-fed rats compared with controls. The binding of Aleuria aurantia to membrane proteins of M_r 97–185 kDa was reduced in ethanol-fed animals. These findings suggest that long-term ethanol feeding modulates the sialylation and fucosylation processes of microvillus membrane proteins in rat intestine. This could affect the intestinal digestive and absorptive functions in chronic alcoholism.     

The role of bile and digestive juices in cadmium effect on the absorption processes in rat small intestine

The effects of bile and digestive juices was studied on the intestinal absorption of water, sodium and glucose in the small intestine of rats after their intoxication with one dose of cadmium 1.33 mg/kg of body weight injected intravenously. The investigations were carried out on 60 rats by the method of intestinal perfusion. The obtained results showed that cadmium inhibited the intestinal absorption of these substances. Bile and digestive juices abolished partially this effect during their physiological secretion. After administration of cholagogues no protective role of bile and digestive juices was observed alleviating the toxic effects of cadmium, and the intestinal absorption was even more reduced.     

Glucocorticoid-regulated glycoprotein maturation in wild-type and mutant rat cell lines

Glucocorticoid hormones can regulate the posttranslational maturation of mouse mammary tumor virus (MTV) precursor polyproteins in M1.54, a stably infected rat hepatoma cell line. We have used complement- mediated cytolysis to recover variants of M1.54 that fail to express MTV cell surface glycoproteins in a hormone-regulated manner (Firestone, G.L., and K.R. Yamamoto, 1983, Mol. Cell. Biol., 3:149- 160). One such clonal isolate, CR4, is similar to wild-type with respect to synthesis of MTV mRNAs, production of the MTV glycoprotein precursor (gPr74env) and a glycosylated maturation product (gp51), and hormone-induced processing of two MTV phosphoproteins. In contrast, three viral cell surface glycoproteins (gp78, gp70, and gp32) and one extracellular species (gp70s), which derive from gPr74env in glucocorticoid-treated wild-type cells, fail to appear in CR4. CR4 showed no apparent alterations in proliferation rate, cell shape, or expression of total functional mRNA and bulk glycoproteins. We conclude that the genetic lesion in CR4 defines a highly selective hormone- regulated glycoprotein maturation pathway that alters the fate of a restricted subset of precursor species.     

Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells

Glycosylation of recombinant therapeutics like monoclonal antibodies (mAbs) is a critical quality attribute. N-glycans in mAbs are known to affect various effector functions, and thereby therapeutic use of such glycoproteins can depend on a particular glycoform profile to achieve desired efficacy. However, there are currently limited options for modulating the glycoform profile, which depend mainly on over-expression or knock-out of glycosyltransferase enzymes that can introduce or eliminate specific glycans but do not allow predictable glycoform modulation over a range of values. In this study, we demonstrate the ability to predictably modulate the glycoform profile of recombinant IgG. Using CRISPR/Cas9, we have engineered nucleotide sugar synthesis pathways in CHO cells expressing recombinant IgG for combinatorial modulation of galactosylation and fucosylation. Knocking out the enzymes UDP-galactose 4′-epimerase (Gale) and GDP-L-fucose synthase (Fx) resulted in ablation of de novo synthesis of UDP-Gal and GDP-Fuc. With Gale knock-out, the array of N-glycans on recombinantly expressed IgG is narrowed to agalactosylated glycans, mainly A2F glycan (89%). In the Gale and Fx double knock-out cell line, agalactosylated and afucosylated A2 glycan is predominant (88%). In the double knock-out cell line, galactosylation and fucosylation was entirely dependent on the salvage pathway, which allowed for modulation of UDP-Gal and GDP-Fuc synthesis and intracellular nucleotide sugar availability by controlling the availability of extracellular galactose and fucose. We demonstrate that the glycoform profile of recombinant IgG can be modulated from containing predominantly agalactosylated and afucosylated glycans to up to 42% and 96% galactosylation and fucosylation, respectively, by extracellular feeding of sugars in a dose-dependent manner. By simply varying the availability of extracellular galactose and/or fucose, galactosylation and fucosylation levels can be simultaneously and independently modulated. In addition to achieving the production of tailored glycoforms, this engineered CHO host platform can cater to the rapid synthesis of variably glycoengineered proteins for evaluation of biological activity.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.     

Studies on inositolphosphatase in rat small intestine

The possibility that inositolphosphatase differs from other intestinal phosphatases was tested by comparing several enzymatic characteristics of phosphatase activities of rat intestinal homogenate acting on various specific substrates. Optimum pH and temperature, Km, Vmax, heat stability, inhibition and metal ion requirement studies suggest that inositolphosphatase differs from phytase and p-nitrophenylphosphatase. Furthermore, we found that inositolphosphatase activity was about 2 times higher in duodenum and jejunum than ileum. It sedimented (90-100%) with a high-speed particulate fraction of mucosal homogenate; 42% of the activity was separated with the brush border membrane isolated from mucosal homogenate. Partial separation by gel filtration on Sephadex G200 and chromatography on phenyl Sepharose CL 4B provided additional evidence to suggest that inositolphosphatase and phytase are different enzymes.     

Adaptation of the rat small intestine to ischemia

Pre-conditioned rats underwent one cycle of intestinal ischemia/reperfusion with subsequent assessment of the heart remote ischemic pre-conditioning (IPC). A local IPC exerted an obvious protective action whereas a remote IPC was ineffective. The local IPC depended partially on the nitric oxide (NO) synthesis. L-arginine administration reduced the intestinal injury. The findings suggest that the ischemic pre-conditioning of intestine depends partially on the NO synthesis and that the remote IPC in intestine does not exist.     

Protein metabolism in the small intestine of the ethanol-fed rat

The effects of chronic ethanol feeding on the small intestine were investigated in young rats. Rats were fed a nutritionally-adequate liquid diet, containing 36 per cent of total energy as ethanol (treated, n = 7), or isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose (controls, n = 7). After six weeks the wet weight and total tissue contents of protein, RNA and DNA were significantly reduced by 21 per cent, 23 per cent, 16 per cent and 28 per cent respectively, (p less than 0.014). Rates of protein synthesis were measured with L[4(3H)]phenylalanine and fractional rates (defined as the percentage of constituent tissue protein synthesised each hour, i.e. ks, % h-1) were calculated from the specific radioactivity of free phenylalanine in both tissue homogenates and plasma. Ethanol-feeding reduced ks by approx 10 per cent (p less than 0.181). The amount of protein synthesized unit-1 RNA was also reduced by approx 15 per cent (p less than 0.059) but the amount of protein synthesis unit-1 DNA was unaffected by ethanol-feeding (p less than 1.000). In contrast, the absolute rates of protein synthesis were reduced by approximately 30 per cent (p less than 0.022). It was concluded that, as the small intestine contributes to approx. 20-25 per cent of whole body synthesis these results may have an important effect on whole body nitrogen homeostasis and may have implications for the gastrointestinal effects of ethanol seen during chronic alcoholic abuse.     

Absorption and metabolism of genistin in the isolated rat small intestine

Uptake and intestinal metabolism of physiologically active genistin were studied in an ex vivo intestinal perfusion model; luminally applied concentrations were 5.9, 12.0, and 23.8 micromol/l. The intestinal absorption of genistin was 14.9% (+/-2.3, n=9), irrespective of the amounts applied. The majority of the absorbed genistin appeared as genistein glucuronide (11.6%), also recovered as the main metabolite on the luminal side (19.5%). Minor amounts of genistin (1.3%) and genistein (1.9%) were found on the vascular side, whereas 15.4% of applied genistin was luminally cleaved to yield genistein. Sulfate derivatives of genistein or genistin were not observed.