Liver Golgi Research Articles

A monomeric protein in the Golgi membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate.

Recent studies have shown that the rat liver heparan sulfate N-deacetylase/N-sulfotransferase is a glycoprotein encoded by a single polypeptide chain of 882 amino acids. Using radiation inactivation analyses, we have now determined that in rat liver Golgi vesicles the target size for the N-deacetylase is 88 +/- 14 kDa, whereas that of the N-sulfotransferase is 92 +/- 8 kDa. These results, together with previous biochemical and molecular cloning approaches, demonstrate that 1) in rat liver Golgi membranes there exists only on population of molecules expressing both activities, 2) the active protein in the Golgi membrane functions as a monomer, and 3) there is no evidence that a large independent protein acts as a regulator of either activity.

Isolation of a matrix that binds medial Golgi enzymes.

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.

Ligand affinity chromatographic purification of rat liver Golgi endomannosidase.

In order to achieve isolation of endo-alpha-D-mannosidase, a Golgi-located processing enzyme that accomplishes deglucosylation of glycoproteins with N-linked carbohydrate units by cleaving the linkage between the glucose-substituted mannose residue and the remainder of the oligosaccharide, we have prepared an affinity matrix (Glc alpha 1-->3Man-O-(CH2)8CONH-Affi-Gel 102) containing the derivative of the characteristic disaccharide product of this enzyme. Chromatography of a Triton extract of rat liver Golgi membranes on a column of this gel in the presence of castanospermine to prevent binding of alpha-glucosidases permitted a rapid purification of the endomannosidase (70,000-fold over the homogenate) with a 12% yield. This purified enzyme was free of other processing glycosidases and was completely inhibited by Glc alpha 1-->3(1-deoxy)mannojirimycin. Examination of the endomannosidase by SDS-polyacrylamide gel electrophoresis revealed a doublet (M(r) 60,000 and 56,000) with the bands being of approximately equal density. Gel permeation high performance liquid chromatography indicated that in its native form the enzyme has an oligomeric structure (M(r) approximately 560,000) consisting of eight to ten subunits.

Impairment of glucagon-induced hepatic system a activity by short-term ethanol administration in the rat

Background/Aims: System A is a membrane-bound, hormonally regulated carrier of amino acids that is induced by liver regeneration and impaired by ethanol. The mechanism of ethanol inhibition of system A is unknown; this study examines the effects of ethanol on the subcellular expression of system A activity following hormonal induction. Methods: Following hormonal treatment and short-term ethanol administration to rats, isolated liver Golgi and plasma membrane vesicles were examined for system A transport, and the kinetic parameters were determined. Results: Four hours after ethanol administration, the initial rate of system A activity was depressed 30% ± 9% and 19% ± 7% into Golgi and plasma membrane vesicles, respectively. The affinity constant of 2-(methylamino)-isobutyric acid uptake was unchanged between control and ethanol-treated vesicles, regardless of their subcellular origin. However, the maximal velocity of system A transport decreased from 1030 to 850 pmol ·mg−1 protein ·10 s−1 in Golgi vesicles and from 740 to 355 pmol ·mg−1 protein 10 s−1 in plasma membrane vesicles. Conclusions: Ethanol impairs hormonally induced system A activity in Golgi as well as in the plasma membrane vesicles. Ethanol potentially reduces glucagon induction of system A activity through an impairment of carrier biosynthesis or expression.

Sialylation of n-alkyllactosides, galactosides and glucosides by sialyltransferases from rat liver Golgi vesicles.

n-Alkyl alpha- and beta-lactosides, galactosides and glucosides with different alkyl chain lengths (C2, C8, C14 and C20) were synthesized and used as acceptors for sialyltransferases from rat liver Golgi vesicles. The beta-galactosides, beta-glucosides, and both alpha- and beta-lactosides, were sialylated. Keeping the acceptor concentration constant, sialylation rates reached a maximum for the n-octyl alpha- and beta-lactosides, n-octyl beta-galactoside and n-octyl beta-glucoside, respectively. n-Octyl alpha-galactoside and n-octyl alpha-glucoside were not sialylated. The reaction products were characterized by TLC. With n-octyl lactoside and galactoside as acceptors, two major sialylation products were formed. They could be separated by preparative TLC, and their structures were identified as 2-3 and 2-6 sialylated acceptors, respectively, by a combination of periodate oxidation, NaBD4 reduction, permethylation and subsequent analysis by fast atom bombardment mass spectrometry (FAB-MS). The structure of the single product obtained from n-octyl beta-glucoside was determined to be the 2-6 sialylated glucoside. Competition experiments with n-octyl lactoside and lactosylceramide and ganglioside Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer (GM1) as acceptors for sialyltransferases suggested that SAT-I [NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer (GM3) synthase] was at least in part responsible for the 2-3 sialylation of n-octyl lactoside.

Binding of the cytosolic p200 protein to Golgi membranes is regulated by heterotrimeric G proteins.

The formation of vesicles for protein trafficking requires the dynamic binding of cytosolic coat proteins onto Golgi membranes and this binding is regulated by a variety of GTPases, including heterotrimeric G proteins. We have previously shown the presence of the pertussis toxin-sensitive G alpha i-3 protein on Golgi membranes and demonstrated a functional role for G alpha i-3 in the trafficking of secretory proteins through the Golgi complex. We have also described a brefeldin A-sensitive phosphoprotein, p200, which is found in the cytoplasm and on Golgi membranes. The present study investigates the role of heterotrimeric G proteins in the regulation of p200 binding to Golgi membranes. An in vitro binding assay was used to measure the binding of cytosolic p200 to LLC-PK1 cell microsomal membranes and to purified rat liver Golgi membranes in the presence of specific activators of G proteins. The binding of p200 to Golgi membranes was compared to that of the coatomer protein beta-COP, for which G protein-dependent membrane binding has previously been established. Membrane binding of both p200 and beta-COP was induced maximally by activation of all G proteins in the presence of GTP gamma S. More selective activation of the heterotrimeric G proteins, with AlFn or mastoparan, also induced membrane binding of p200 and beta-COP. Pertussis toxin pretreatment of Golgi membranes, to selectively inactivate G alpha i-3, reduced the AlFn and mastoparan-induced binding of p200 to Golgi membranes, whereas no significant effect of pertussis toxin on beta-COP binding was found in this assay. The effect of pertussis toxin thus implicates G alpha i-3, as one component of a regulatory pathway, in the binding of cytosolic p200 to Golgi membranes. The effects of AlFn and pertussis toxin on p200 membrane binding were also shown in intact cells by immunofluorescence staining. AlFn treatment of cells induced translocation of p200 from the cytoplasm onto the Golgi complex, resulting in a conformational change in some Golgi membranes. The translocation of p200 was blocked by pretreatment of intact NRK cells with pertussis toxin. The data presented here support the conclusion that the binding of the p200 protein to Golgi membranes involves regulation by the pertussis toxin-sensitive heterotrimeric G proteins, specifically the G alpha i-3 protein.

Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]galactose, CMP-[9-3H]N-acetylneuraminic acid, and [acetyl-3H]acetyl-coenzyme A

When a rat liver Golgi apparatus-enriched subcellular fraction is incubated with UDP-[3H]Gal, CMP-[3H] Neu5Ac, or [acetyl-3H]acetyl (Ac)-CoA, label is efficiently transferred to endogenous acceptors, which are resistant to added proteases, unless detergent is added at a sufficiently high concentration. Thus, the acceptors are within the lumen of intact compartments of correct topological orientation, which are likely to be similar to those of the Golgi apparatus in the intact cell. In each case, approximately 90% of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase digestion, as labeled N-linked oligosaccharides. Label from UDP-[3H]Gal is transferred to several distinct N-linked oligosaccharides, and many of these carry sialic acid (Sia) residues. This amount increases if the transfer reaction is chased with CMP-Neu5Ac. A major fraction of the [3H]Gal is directly "covered" with Sia residues, indicating that at least a portion of the beta-galactosyltransferase(s) are co-localized with one or more sialyltransferases. The majority of the [3H]Gal is found in a beta 1,3-linkage, rather than the more common beta 1,4-linkage. The N-linked oligosaccharides labeled by CMP-[3H] Neu5Ac carry labeled Sia residues in either alpha 2,3 or alpha 2,6 linkage, and showed a range of charge distribution. The transferred [3H]Neu5Ac is not O-acetylated even when Ac-CoA is added at saturating concentrations, implying that the sialyltransferases and the O-acetyltransferase(s) are not functionally co-localized. However, approximately 20% of label released from N-linked oligosaccharides by sialidase does not co-migrate with authentic Neu5Ac in high performance liquid chromatography analysis, indicating that transferred [3H] Neu5Ac is modified by unknown enzymes in the Golgi. Most of the [3H]acetate transferred from [acetyl-3H] Ac-CoA to N-linked oligosaccharides is on Sia residues that are exclusively alpha 2,6-linked, and is enriched on tri- and tetra-antennary chains that do not appear to carry any 2,3-linked Sia residues. These data indicate a restricted substrate preference of the O-acetyltransferase(s). About one-quarter of the [3H]acetate transferred is sialidase-resistant, indicating either transfer to monosaccharides other than sialic acid, or to sialidase-resistant sialic acids. While most of these sialidase-resistant oligosaccharides remain negatively charged, about 10% are neutralized by sialidase, confirming transfer of [3H]acetate to monosaccharides other than sialic acid.

The biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked and O-linked glycans labeled by UDP-[6-3H]N-acetylgalactosamine

Endogenous acceptors in a Golgi apparatus-enriched subcellular fraction from rat liver were labeled with UDP-[3H]GalNAc. The great majority of these acceptors were protected from protease degradation in the absence of detergent. These molecules are therefore present in intact vesicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-[3H]GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl)asparagine amidase releases label from a few specific proteins, indicating that [3H]GalNAc is transferred to N-linked oligosaccharides. Both neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the [3H]GalNAc found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and phosphate. A major portion of the [3H] GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a phosphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structures. O-linked oligosaccharide biosynthesis was also studied in the same labeled rat liver Golgi apparatus preparations. beta-Elimination releases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively [3H] GalNAcitol. If other unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phosphosulfate are added during the chase period two anionic O-linked oligosaccharides are synthesized, indicating that the UDP-GalNAc:peptide-N-acetylgalactosaminyltransferase is at least in part functionally co-localized with enzymes that extend and modify O-linked oligosaccharides.

Flow Cytometric Analysis of Concanavalin A Binding to Isolated Golgi Fractions from Rat Liver

Flow cytometry (FCM) has been used repeatedly to study lectin binding to whole cells. However, there are very few attempts to analyze glycoconjugates in isolated subcellular organelles. We have applied FCM to quantitate the specific binding of fluorescein-conjugated concanavalin A (FITC-Con A) to isolated cis and trans fractions of rat liver Golgi complex, the cell compartment involved in glycoprotein maturation and sorting. Our results show similar intensities of Con A-specific binding in the two fractions. Using this method we show a decreased FITC-Con A binding to both Golgi fractions in ethanol-treated rats, which is consistent to previous work on alcoholic effects on galactosyltransferase. The possible applications of this technique are discussed.

Effect of membrane lipids on the lactosylceramide molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide sialyltransferase

It has previously been shown that when the molecular species specificity of rat liver Golgi CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase was determined, using as the substrate lactosylceramide (LacCer) incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a pronounced variation in activity towards the various molecular species of LacCer (J. Lipid Res. 1989. 30: 1789-1797). In this paper, -the LacCer molecular species specificity of sialyltransferase from neuroblastoma NB2a cells was examined using five naturally occurring and three synthetic molecular species of LacCer. The enzyme activity was determined by following the formation of [14C]GM3 from CMP-[14C]neuraminic acid and individual molecular species of LacCer incorporated into liposomes. Nonspecific lipid transfer protein was included in the enzyme assay to facilitate the transfer of LacCer and other lipids between the liposomes and the membrane where sialyltransferase is located. In these enzyme assays the liposomes contained approximately 10 times more lipid phosphorus than either the microsomal fraction of NB2a cells or the Golgi fraction of rat liver. Thus, in the presence of nonspecific lipid transfer protein, the lipid composition of the membrane where sialyltransferase is located was modified to resemble the lipid composition of the liposomes. When the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the enzyme showed no specificity towards the various molecular species of LacCer. However, when the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a variation in activity towards the various LacCer molecular species similar to that observed with the liver Golgi enzyme using liposomes prepared with liver Golgi lipids. Likewise, when the molecular species specificity of rat liver Golgi sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the liver enzyme then showed no specificity towards the various molecular species of LacCer. These results indicate that the lipid environment of the membrane can alter the molecular species specificity of sialyltransferase towards its lipid substrate, LacCer.

A single protein catalyzes both N-deacetylation and N-sulfation during the biosynthesis of heparan sulfate.

Heparan sulfate is a highly sulfated carbohydrate polymer that binds to and modulates the activities of numerous proteins. The formation of these protein-binding domains in heparan sulfate is dependent on a series of biosynthetic reactions that modify the polysaccharide backbone; the initiating and rate-limiting steps of this process are the N-deacetylation and N-sulfation of N-acetylglucosamine residues in the polymer. We now report that in the rat liver, biosynthesis of heparan sulfate utilizes a single protein that possesses both N-deacetylase and N-sulfotransferase activities. This was accomplished by demonstrating that both activities resided in a purified soluble fusion protein containing the Golgi-lumenal portion of the enzyme. We propose that this protein be renamed the rat liver Golgi heparan sulfate N-deacetylase/N-sulfotransferase.

Evidence for the role of a cathepsin D-like activity in the release of Galβ1-4GlcNAcα2-6sialyltransferase from rat and mouse liver whole-cell systems

1. 1. Sialyltransferase is a liver Golgi membrane-bound enzyme that is released from the liver under conditions of experimental inflammation. Previous work showed that the action of a cathepsin D-like proteinase was responsible for release of the enzyme from isolated Golgi membranes. This study shows that the same enzyme is responsible for release of sialyltransferase in whole-cell systems. 2. 2. Galβ1-4GlcNAcα2-6sialyltransferase (EC 2.4.99.1) was secreted from slices of rat and mouse liver into the incubation medium with larger amounts of activity being secreted from slices of liver from animals suffering from experimental inflammation. 3. 3. The presence in the incubation medium of the cathepsin D proteinase inhibitor, pepstatin A, at 10 −4 M was sufficient to inhibit the release of sialyltransferase into the medium by about 60% after a 6 hr incubation. 4. 4. The release of albumin and α 1 glycoprotein from rat liver slices, was not affected by the presence of pepstatin A, indicating that the proteinase inhibitor did not affect the synthesis and secretion of typical secretable proteins by the liver. 5. 5. Intraperitoneal injections of pepstatin A into mice prior to preparation of liver slices also resulted in a significant reduction of the secretion of sialyltransferase into the incubation medium. 6. 6. The results from these studies support the idea that a cathepsin D-like proteinase is responsible for the release of sialyltransferase into the extracellular space in whole cells in the rat and the mouse.

Chronic ethanol consumption induces accumulation of proteins in the liver Golgi apparatus and decreases galactosyltransferase activity.

The effect of chronic ethanol consumption on labeled glycoprotein secretion and galactosyltransferase activity has been analyzed in cis- and trans-Golgi apparatus fractions isolated from rat liver. Rats were injected intraperitoneally with 3H-leucine, after different chase periods (30, 60, 180 min), and the radioactivity of the different subcellular fractions as well as of the isolated Golgi apparatus was measured. Chronic alcohol treatment induces an increase in liver weight as well as an enhancement of total liver protein. Ethanol treatment produces a significant accumulation of labeled proteins in isolated Golgi apparatus fractions after a 60- and 180-min chase. An accumulation of labeled proteins in the cytosolic fraction was observed only after 180 min. The alcohol treatment also induces a significant decrease in the activity of galactosyltransferase in both liver homogenate and Golgi apparatus fractions. These results suggest that an impairment of Golgi apparatus functions, including glycosylation and glycoprotein trafficking, could be one of the mechanisms involved in the accumulation of hepatic protein and thus in the pathogenesis of alcohol-induced injury in the liver of chronic ethanol-consuming animals.

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes.

A mAb AD7, raised against canine liver Golgi membranes, recognizes a novel, 200-kD protein (p200) which is found in a wide variety of cultured cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Golgi complex and more diffuse staining of p200 in the cytoplasm. The p200 protein in the Golgi complex was colocalized with other Golgi proteins, including mannosidase II and beta-COP, a coatomer protein. Localization of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi cisternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutions and is phosphorylated. The p200 protein is located on the cytoplasmic face of membranes, since it was accessible to trypsin digestion in microsomal preparations, and is recovered in approximately equal amounts in membrane pellets and in the cytosol of homogenized cells. Immunofluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of this drug. The effect of BFA was reversible, since upon removal of the toxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resistant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with the coatomer protein, beta-COP.

1‐Deoxy‐1‐phosphatidylethanolamino‐lactitol‐type neoglycolipids serve as acceptors for sialyltransferases from rat liver golgi vesicles

1-Deoxy-1-phosphatidylethanolamino-lactitols (LacPtdEtns), 1-deoxy-1-phosphatidylethanolamino-sialyllactitols (NeuAcLacPtdEtns) and their corresponding N-acetylated derivatives were synthesized and characterized by fast-atom-bombardment mass spectrometry (FAB MS). The neoglycolipids were used as acceptors for sialyltransferases from rat liver Golgi vesicles. Sialylation rates were as good as or even better than those obtained with the corresponding authentic acceptors lactosylceramide (LacCer) and ganglioside GM3. The sialylation of LacPtdEtns and NeuAcLacPtdEtns yielded sialyl and disialyl compounds, respectively, as shown by FAB MS analysis of the reaction products. The results of competition experiments indicate that the neoglycolipids and the authentic acceptors are sialylated by the same sialyltransferases.

Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, bothat 1.5 μM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP-or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatate-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal changes in sialylation of glycoproteins in rat liver

Glycoproteins containing N-linked oligosaccharides were prepared from plasma and liver microsomes of rats aged 0-5 weeks, and galactose and sialic acid content were determined. The sialic acid/galactose ratios in plasma membrane N-glycans remained at about 1 throughout the postnatal period, suggesting that most of the galactose residues are sialylated. In the same way, it was suggested that most of the galactose residues of microsomal N-glycans were sialylated at 0, 4 and 5 weeks of age, but that the degree of sialylation was lower at the other ages, with a minimum at 2 weeks. When the activities of sialyltransferase and galactosyltransferase in liver Golgi membranes were determined, age-dependent changes were found, not only in the specific activities of the enzymes, but also in the Golgi membrane content per g of liver. The activity of galactosyltransferase per g of liver increased immediately after birth, whereas that of sialyltransferase remained at a low level for 2 weeks and then increased to a constant level at 4 weeks. It is probable that this delayed increase in the activity of sialyltransferase results in the decreased sialylation of microsomal N-glycans at 1, 2 and 3 weeks. Sialyltransferase was solubilized from the liver microsomes of rats aged 2, 3 and 4 weeks and characterized. Phosphocellulose column chromatography separated the activity into two subfractions, designated transferase I and transferase II in the order of elution. The increase in total sialyltransferase activity during this period was caused mainly by an increase in transferase I. Rechromatography of each transferase from 3-week-old rats after neuraminidase treatment showed that transferase I but not transferase II contained sialic acid residue(s) and that desialylated transferase I was eluted in a similar way as transferase II. Although the apparent Km value for CMP-N-acetylneuraminic acid and the heat stability of transferase I were different from those of transferase II, the difference was abolished by treating transferase I with neuraminidase, suggesting that transferase II may be a desialylated form of transferase I. These changes in the sialylation of membrane glycoproteins, including sialyltransferase, may be related to the control of liver growth during postnatal development.

Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes.

A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle-mediated transport/secretion.