Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Development of liver regenerative therapy using glycoside-modified bone marrow cells

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration.     

Comparison of desialylation of rat transferrin by cellular and non-cellular methods.

We have previously shown that the liver endothelium can desialylate the glycoprotein transferrin (Tf). In the present work we provide evidence that asialotransferrin obtained by this means behaves differently on Ricinus communis agglutinin (RCA120) lectin affinity chromatography from asialotransferrin obtained by either neuraminidase treatment or acid hydrolysis. Purified rat transferrin was radiolabelled either with 125I (protein moiety) or with 3H (sialyl residues), and subsequently saturated with iron. It was then passed through an RCA120-agarose column to isolate the fully sialylated component. Sialylated Tf was then desialylated either by incubation with purified rat liver endothelium or, in vitro, by neuraminidase treatment or by acid hydrolysis. The protein was again subjected to RCA120 column chromatography. Although both neuraminidase treatment and acid hydrolysis almost completely desialylated the glycoprotein (as evidenced by near absence of 3H label), the glycoprotein was not retained by the RCA120-agarose column. By contrast, liver endothelium partially desialylated the glycoprotein, but this desialylated fraction was retained by the RCA120-agarose column. These results suggest that desialylation with neuraminidase or acid hydrolysis may be inadequate for functional studies of asialotransferrin.     

The effect of calmodulin on the active calcium-ion transport and (Ca2+ + Mg2+)-dependent ATPase in microsomal fractions of smooth muscle compared with that in erythrocytes and cardiac muscle.

Kupffer cells isolated from the rat liver are able to bind neuraminidase-treated rat erythrocytes via a D-galactose-specific receptor on the cell surface. Binding of desialylated erythrocytes was inhibited by several mono- and oligo-saccharides related to D-galactose, but not by unrelated sugars. However, after phosphorylation at position 6 D-glucose was as good an inhibitor as D-galactose. Two synthetic glycoproteins, D-galactosyl-albumin and, at a higher concentration, D-glucosyl-albumin, strongly inhibit cell contacts. Lectin-mediated binding of desialylated erythrocytes is dependent on the presence of Ca2'ons, but independent of ATP formation and cell motility. It is concluded that binding of desialylated erythrocytes by rat Kupffer cells is mediated by a Ca2-dependent D-galactosyl/D-glucosyl-recognition system.     

Kupffer cell receptor CLEC4F is important for the destruction of desialylated platelets in mice

The liver has recently been identified as a major organ for destruction of desialylated platelets. However, the underlying mechanism remains unclear. Kupffer cells, which are professional phagocytic cells in the liver, comprise the largest population of resident tissue macrophages in the body. Kupffer cells express a C-type lectin receptor, CLEC4F, that recognizes desialylated glycans with an unclear in vivo role in mediating platelet destruction. In this study, we generated a CLEC4F-deficient mouse model (Clec4f^−/−) and found that CLEC4F was specifically expressed by Kupffer cells. Using the Clec4f^−/− mice and a newly generated platelet-specific reporter mouse line, we revealed a critical role for CLEC4F on Kupffer cells in mediating destruction of desialylated platelets in the liver in vivo. Platelet clearance experiments and ultrastructural analysis revealed that desialylated platelets were phagocytized predominantly by Kupffer cells in a CLEC4F-dependent manner in mice. Collectively, these findings identify CLEC4F as a Kupffer cell receptor important for the destruction of desialylated platelets induced by bacteria-derived neuraminidases, which provide new insights into the pathogenesis of thrombocytopenia in disease conditions such as sepsis.Subject terms: Glycobiology, Cell death and immune response, Haematological diseases     

Desialylation of transferrin by liver endothelium is selective for its triantennary chain.

Liver endothelium can remove and transport the glycoprotein transferrin (TF). During this process the molecules are desialylated; however, in contrast with other such glycoproteins, for example caeruloplasmin, only half of transported TF is desialylated. To explore which component of TF is desialylated, we double-labelled fully sialylated TF with [3H]sialic acid residues and a 125I-protein moiety. This was then 'chased' through purified liver endothelium in pulse-chase experiments. Endothelium-conditioned TF was fractionated on an RCA120 affinity column into sialylated and desialylated components. Each component was then re-fractionated on a concanavalin A affinity column, which separates the glycoprotein according to the branching pattern of its glycan chain. The desialylated fraction was eluted only as a triantennary component, whereas the non-desialylated fraction consisted only of bi- and tetra-antennary chains. The significance of this selective desialylation of triantennary chain of TF in the subsequent metabolism of its iron content and its possible role in the pathogenesis of alcohol-induced hepatic siderosis are discussed.     

Asialoglycoprotein clearance in the chicken: evidence for the lack of a hepatic asialoglycoprotein receptor.

The behaviour of desialylated human and chicken acid alpha1-glycoproteins is reported in chickens. Although desialylation resulted in accelerated disappearance rates from the plasma of both proteins, nevertheless the asialoproteins were eliminated much more slowly than expected on the basis of earlier observations in mammals. Analysis of tissue radioactivities, including kidney, liver, lung and spleen, failed to demonstrate any accumulation of the labeled asialoproteins in the liver, which is contrary to the situation in mammals. The main pathway for the elimination of desialylated human acid alpha1-glycoprotein in the chicken is via the kidney (tubular catabolism and/or urinary excretion). The clearance of desialylated chicken acid alpha1-glycoprotein is more complex as it involves the kidney as well as the reticuloendothelial system. These findings indicate that, unlike mammals, chickens do not possess a hepatic plasma membrane receptor for asialoglycoproteins. This raises the possibility that the presence or absence of this specific receptor may constitute a fundamental biological difference between mammals and birds.     

Metabolism of the human serum beta-hexosaminidase isoenzymes in the rat: an in vivo and in vitro study

Plasma clearance of purified human serum beta-hexosaminidase isoenzymes was studied in the rat. The serum beta-hexosaminidase isoenzymes (A, B and P) showed a slow clearance from circulation compared to their tissue counterparts. After desialylation, the clearance rate of all serum isoenzymes was markedly enhanced. The uptake of native as well as desialylated serum beta-hexosaminidase isoenzymes was studied in rat liver nonparenchymal cells and hepatocytes. No detectable uptake of any native serum isoenzyme was noticed in either cell type. However, when these isoenzymes were desialylated by neuraminidase treatment, isoenzymes A and B were taken up by the nonparenchymal cells. No uptake was observed for the P form. None of the desialylated serum forms was taken up by hepatocytes.     

Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse.

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.     

Specificity of sialytransferase: structure of alpha1-acid glycoprotein sialylated in vitro. A new methodology for the determination of the structure of the linkage formed by glycosyltransferase action on galactosyl-oligosaccharide-protein acceptors.

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.     

A sialylation-sensitive cell-based in vitro bioassay for erythropoietin; incorporation of the galactose-binding Erythrina crista-galli lectin.

The in vivo biological activity of erythropoietin (Epo) is dependent on its being adequately sialylated. Current in vitro bioassays for Epo do not correlate with the in vivo bioassays as the former do not take into account the role the liver plays in clearing desialylated glycoproteins from the circulation. Here we describe a sialylation-sensitive cell-based Epo bioassay. In the first instance, Epo activity in vitro was measured using proliferation of AS-E2 cells, and in vivo using the polycythaemic mouse bioassay. Activity in vivo was progressively abolished by controlled desialylation, whereas activity in vitro was essentially unaffected. Incorporation of an incubation step with a solid-phase galactose-binding lectin (Erythrina crista-galli), effectively mimicking passage through the liver in vivo, renders the in vitro bioassay sensitive to desialylation, such that Epo desialylated almost to completion had <10% of the activity of untreated Epo. These studies offer proof of principle, that rational manipulation of in vitro bioassays can allow prediction of activity in vivo without the use of live animals.     

Study of the interaction between desialylated erythrocytes and Kupffer cells using perfusion of liver in situ

Desialylated erythrocytes, when perfused in mice liver in situ, were shown to be sequestered. This sequestration was inhibited by dihydrocytochalasin B and a neoglycoprotein: N-Acetylgalactosaminyl-albumin. Our results confirm that, under physiological conditions, the recognition of desialylated erythrocytes appear to be related to the presence of N-Acetylgalactosamine or galactose receptor on the surface of Küpffer cells.     

Relationship of the glycan structure of glycoproteins to the desialylation process by rat liver endothelium

To investigate the variations in desialylation of glycoproteins by liver endothelium, we compared endothelial desialylation for 3 glycoproteins, human ceruloplasmin, human and rat transferrin. Radiolabeled glycoproteins were chased through purified rat liver endothelium and then fractionated by lectin affinity chromatography. Endothelium processed glycoproteins were fractionated by RCA120 chromatography into sialylated and desialylated components. The latter was then studied by Con A chromatography. Desialylation occurred only when the molecule contained at least a single triantennary chain of glycan. Desialylation was minimal in the case of human transferrin which contains mostly biantennary branching pattern. Thus, it appears that a single triantennary glycan chain is necessary and sufficient to trigger desialylation of glycoproteins by liver endothelium and this process is an all-or-none phenomenon.     

Possible mechanism of entrapment of neuraminidase-treated lymphocytes in the liver

Neuraminidase-treated rat lymphocytes adhere strongly to rat hepatocytes in vitro. Binding between cells is due to stereo-specific interactions between a mammalian hepatic membrane lectin and galactosyl residues which are exposed on the lymphocyte surface after removal of sialic acid residues. The hepatic galactose specific lectin may play a role in the trapping of recirculating desialylated lymphocytes in the liver.     

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

The role of endosomal traffic in the transendothelial transport of ceruloplasmin in the liver

Through a process resembling receptor-mediated endocytosis, liver endothelium binds and internalizes the plasma glycoprotein ceruloplasmin (CP) on the luminal side. The protein is then transported via a vesicular system to the albuminal side where it is externalized to the space of Disse. In its path, the glycoprotein is fully desialylated. To determine if the endosomal compartment is involved in this transport, we used endosomal inhibitors NH4Cl, ethylamine as well as monensin to quantitatively measure the magnitude of radiolabeled CP transport across purified liver endothelial cells. All three reagents inhibited the transport of CP and its discharge by endothelium. The magnitude of inhibition was dose-related for all three reagents. We conclude that the endosomal compartment is involved in the transendothelial transport of CP across the liver endothelium.     

alpha3-galactosylated glycoproteins can bind to the hepatic asialoglycoprotein receptor.

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose-specific hepatic lectin, the 'asialoglycoprotein receptor'. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galalpha1-->3Galbeta1-->4R is present on glycoprotein glycans. To investigate if alpha3-galactosylated glycoproteins, just like desialylated glycoproteins, could be cleared by the hepatic lectin, the affinities of alpha3-galactosylated compounds towards this lectin were determined using an in vitro inhibition assay, and were compared with those of the parent compounds terminating in Galbeta1-->4R. Diantennary, triantennary and tetraantennary oligosaccharides that form part of N-glycans were alpha3-galactosylated to completion by use of recombinant bovine alpha3-galactosyltransferase. Similarly, desialylated alpha1-acid glycoprotein (orosomucoid) was alpha3-galactosylated in vitro. The alpha3-galactosylation of a branched, Galbeta1-->4-terminated oligosaccharide lowered its affinity for the membrane-bound lectin on whole rat hepatocytes 50-250-fold, and for the detergent-solubilized hepatic lectin 7-50-fold. In contrast, alpha3-galactosylation of asialo-alpha1-acid glycoprotein caused only a minor decrease in affinity, increasing the IC50 from 5 to 15 nM. Fully alpha3-galactosylated alpha1-acid glycoprotein, intravenously injected into the mouse, was rapidly cleared from the circulation, with a clearance rate close to that of asialo-alpha1-acid glycoprotein (t1/2 of 0.42 min vs. 0.95 min). Its uptake was efficiently inhibited by pre-injection of an excess asialo-fetuin. Organ distribution analysis showed that the injected alpha1-acid glycoprotein accumulated predominantly in the liver. Taken together, these observations suggest that serum glycoproteins that are heavily alpha3-galactosylated will be rapidly cleared from the bloodstream via the hepatic lectin. It is suggested that glycosyltransferase expression in murine hepatocytes is tightly regulated in order to prevent undesired uptake of hepatocyte-derived, circulating glycoproteins.     

Cell surface changes in diabetic rats. Studies of lectin binding to liver cell plasma membranes.

We have previously reported changes in the chemical composition of cell surface membranes in diabetic rats (Chandramoulis, V. and Carter, Jr., J. R. (1975) Diabetes 24, 257-262 [1]). To examine the possible implications of these changes for cell surface structures, we have measured the binding of labeled lectins and desialylated glycoproteins to plasma membranes prepared from the livers of streptozotocin--diabetic and control rats. Lectins were chosen which have affinities for different carbohydrate moieties. The binding of ricin and concanavalin A to liver cell membranes from the diabetic rats was significantly reduced, but no change in the binding of wheat germ agglutinin was noted. Binding of desialylated thyrozine--binding globulin, previously shown to be dependent on membrane sialic acid residues, ws strongly suggest that insulin deficiency leads to generalized changes in cell surfaced glycoproteins, at least in this animal model of diabetes.     

Size and stability to sodium dodecyl sulfate of alkaline phosphatases from their three established human genes

Subunit molecular weights of human alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) were dependent upon acrylamide concentration, a reflection of their glycoprotein nature. Molecular weights at a concentration of 7% (w/w) or greater were 68300, 80800 and 79400 for the enzymes from placenta, liver and mucosa of small intestine, respectively. All enzymes were dimers, the respective native Mr values determined by gradient gel electrophoresis being 138000, 186000 and 180000. None of the molecular weights was altered by desialylation. Stability of the catalytic activity of the purified enzymes to SDS varied and was very dependent on pH. SDS at 1% (w/v) rapidly denatured both native and desialylated alkaline phosphatase from placenta at pH 7.5 but had little effect on these at pH 10.3. Compared with placenta, the native enzyme from liver had greater stability at pH 7.5 and both native and desialylated forms had lower stability at pH 10.3. The enzyme from intestinal mucosa was sharply different from the other two isoenzymes: SDS had little effect at pH 7.5 but very rapidly denatured the enzyme at pH 10.3. The size of alkaline phosphatases and their stability to SDS can be used to identify gene products and to recognize heterodimers formed between products of more than one gene.     

小鼠肝部分切除模型

小鼠肝部分切除模型是在经典的大鼠模型基础上发展起来的。随着显微外科技术的发展和小鼠腹部手术围手术期管理的完善,快速、可靠、重复性高的小鼠模型得以建立。因为成本较低,且存在大量为研究肝切除后肝再生的转基因小鼠,小鼠已经成为研究肝再生的重要动物模型。肝再生的调控相当复杂,且不是由单一因素控制的,因此需要多种类的转基因小鼠进行肝切除后肝再生研究来明确各因子在肝再生过程中的确切作用与机制。通过小鼠肝切除后肝再生的研究,目前已证实的参与调控肝再生的细胞因子和生长因子有:肝再生增强因子(ALR)、肿瘤坏死因子α(TNFα)、白介素6(IL-6)、白介素1(IL-1)、肝细胞生长因子(HGF)、去甲肾上腺素(NE)、卵泡抑素(FS)、转化生长因子α(TGF-α)、转化生长因子β-1(TGF-β-1)等。