Sialyltransferase Research Articles

Glycohistochemical analysis of seborrheic keratosis in eyelids

The glycoconjugates of seborrheic keratosis in the eyelids were examined by in situ hybridization histochemistry using cRNA probes for sialyltransferase (ST) and lectin histochemistry. We considered that the cells, which expressed both cytoplasmic distribution of ST-mRNA and binding of lectins specific for sialic acids to the cell surfaces, were actively producing sialoglycans. We also considered that the cells whose surfaces were stained with the lectins without cytoplasmic distribution of ST-mRNA have completed the synthesis of sialoglycans. These viewpoints suggest that the O-linked sialoglycan, whose turnover-rate is slow, may be distributed over the cells of the thickened spinocellular layer in the tumor of seborrheic keratosis and involved in its pathomechanism. It also appears that the turnover rate of the terminal sialic acids in the N-linked glycan in the spinocellular layer may be fast.

The biochemical consequences of α2,6(N) sialyltransferase induction by dexamethasone on sialoglycoprotein expression in the rat H411e hepatoma cell line

Previous studies have demonstrated sialyltransferase (ST) enzyme activity to be induced in hepatic cells by corticosteroids. In this study, we used the H411e rat hepatoma cell line to further characterise this induction with particular reference to the subsequent changes in the pattern of sialoglycoprotein (SGP) expression. The induction of total ST activity by dexamethasone was concentration dependent with maximum induction occurring 12 h subsequent to drug addition. Western blot analysis demonstrated that the induction was associated with an increase in the expression of the α2,6(N) ST enzyme with no change in the expression levels of the α2,3(N) enzyme. While the induction resulted in an increase in the reaction velocity ( V max) of the enzyme for both the sugar donor (CMP-Neu5Ac) and the asialofetuin acceptor protein, there was no significant change in the enzyme affinity ( K m) for the substrates, suggestive of either an increase in the expression or efficiency of the existing enzyme(s) rather than an induction of novel ST enzymes. Lectin blot analysis of cellular glycoprotein expression demonstrated no change in the expression patterns of either α2,3 or α2,6-linked SGP following enzyme induction. These results suggest that the available acceptor sites for the terminal sialic acid group(s) may be fully occupied in the control cells and therefore there are no further sites onto which the sialic acid can be transferred following induction of ST enzyme activity. This may be due to the high basal enzyme levels in the control cells already exhausting endogenous acceptor sites.

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues.

Human colorectal cancers express various cancer-associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc-T) and sialyltransferase (ST) isoenzymes, including Fuc-T III, IV, V, VI and VII and ST-3N, ST-30 and ST-4, in human colorectal cancer tissues by Northern blotting and RT-PCR. Regarding fucosyltransferases, mRNA of Fuc-T III and VI was not significantly altered, and only Fuc-T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non-malignant colonic epithelia taken from the same patient (273 +/- 96%; p < 0.001). The moderate increase of Fuc-T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST-3N remained unchanged, whereas that for ST-4 decreased significantly in cancer tissues, to 32 +/- 29%, (p < 0.005). The most remarkable finding was that the message of ST-30 was prominently increased in cancer tissues compared with non-malignant colorectal mucosa. When further investigated by quantitative RT-PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST-30 was 459 +/- 200% compared with that in adjacent non-malignant epithelium (significant at p < 0.0001). The increase of ST-30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST-30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type 1 chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos-7 cells.

Altered mRNA expression of specific molecular species of fucosyl‐ and sialyl‐transferases in human colorectal cancer tissues

Human colorectal cancers express various cancer-associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc-T) and sialyltransferase (ST) isoenzymes, including Fuc-T III, IV, V, VI and VII and ST-3N, ST-30 and ST-4, in human colorectal cancer tissues by Northern blotting and RT-PCR. Regarding fucosyltransferases, mRNA of Fuc-T III and VI was not significantly altered, and only Fuc-T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non-malignant colonic epithelia taken from the same patient (273 ± 96%; p < 0.001). The moderate increase of Fuc-T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST-3N remained unchanged, whereas that for ST-4 decreased significantly in cancer tissues, to 32 ± 29%, (p < 0.005). The most remarkable finding was that the message of ST-30 was prominently increased in cancer tissues compared with non-malignant colorectal mucosa. When further investigated by quantitative RT-PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST-30 was 459 ± 200% compared with that in adjacent non-malignant epithelium (significant at p < 0.0001). The increase of ST-30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST-30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type I chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos-7 cells. Int. J. Cancer 71:556-564, 1997 © 1997 Wiley-Liss, Inc.

Flow cytometric detection of the Golgi apparatus using antibodies to glycosyltransferases.

Intracellular glycosyltransferase protein expression can be assessed by flow cytometry. We report the detectability of the Golgi associated beta1,4 galactosyltransferase (GT) and alpha2,6 sialyltransferase (ST) upon permeabilization in Jurkat and EBV-JY cells representing a T- and B-lymphoid cell line, respectively. The method employs fixation with paraformaldehyde and permeabilization with saponin. It ensures reliable internalization of the antibody and little background staining and does not cause leakage of the antigen. We first applied monoclonal antibodies to GT for establishment of this method by flow cytometry. The obtained flow cytometric signal could be localized to the Golgi apparatus by confocal laser scanning microscopy. To exclude interference from possible cell-surface staining, measurements were carried out on non-permeabilized cells. No signal was found in Jurkat cells, while low but measurable ecto-galactosyltransferase was found on EBV-JY cells. F(ab)'2 fragments of polyclonal antisera to GT and ST were generated and shown to be useful for double indirect staining with the monoclonal antibody. The method described here permits relative assessment of Golgi glycosyltransferase expression in lymphoid cells by flow cytometry.

Flow cytometric detection of the Golgi apparatus using antibodies to glycosyltransferases

Intracellular glycosyltransferase protein expression can be assessed by flow cytometry. We report the detectability of the Golgi associated beta1,4 galactosyltransferase (GT) and alpha2,6 sialyltransferase (ST) upon permeabilization in Jurkat and EBV-JY cells representing a T- and B-lymphoid cell line, respectively. The method employs fixation with paraformaldehyde and permeabilization with saponin. It ensures reliable internalization of the antibody and little background staining and does not cause leakage of the antigen. We first applied monoclonal antibodies to GT for establishment of this method by flow cytometry. The obtained flow cytometric signal could be localized to the Golgi apparatus by confocal laser scanning microscopy. To exclude interference from possible cell-surface staining, measurements were carried out on non-permeabilized cells. No signal was found in Jurkat cells, while low but measurable ecto-galactosyltransferase was found on EBV-JY cells. F(ab)'2 fragments of polyclonal antisera to GT and ST were generated and shown to be useful for double indirect staining with the monoclonal antibody. The method described here permits relative assessment of Golgi glycosyltransferase expression in lymphoid cells by flow cytometry.

Chronic Ethanol Downregulates Gal-beta-1,4GlcNAc alpha2,6- Sialyltransferase and Gal-beta-1,3GlcNAc alpha2,3-Sialyltransferase mRNAs in Rat Liver

We have previously demonstrated that chronic ethanol specifically decreases the hepatic level and rate of synthesis of 2,6-sialyltransferase (2,6-ST). To understand its mechanism of action, effects of 8 weeks of chronic ethanol feeding on the expression of sialyltransferase (ST) genes in rat liver and kidneys were determined by Northern-blot analysis of ST mRNAs. It was found that, compared with the pair-fed control rats, the percentage decreases in ST mRNAs in the ethanol-fed group were as follows: liver-Gal-beta-1,4GlcNAc alpha 2,6-ST (2,6-ST): 59% (p < 0.001); liver-Gal-beta-1,3GlcNAc alpha 2,3-ST (2,3-ST): 32% (p < 0.01); and kidneys-2,6-ST: 5% (NS). In contrast, glyceral-dehyde-3-phosphate dehydrogenase mRNA in both liver and kidneys was unaffected by the same ethanol treatment. Taken together, these results demonstrate that chronic ethanol downregulates the expression of 2,6-ST and 2,3-ST genes in rat liver.

Chronic Ethanol Downregulates Gal‐β‐l,4GlcNAc α2,6‐Sialyltransferase and Gal‐β‐l,3GlcNAc α2,3‐Sialyltransferase mRNAs in Rat Liver

We have previously demonstrated that chronic ethanol specifically decreases the hepatic level and rate of synthesis of 2,6‐sialyltrans‐ferase (2,6‐ST). To understand its mechanism of action, effects of 8 weeks of chronic ethanol feeding on the expression of sialyltrans‐ferase (ST) genes in rat liver and kidneys were determined by North‐emblot analysis of ST mRNAs. It was found that, compared with the pair‐fed control rats, the percentage decreases in ST mRNAs in the ethanol‐fed group were as follows: liver—Gal‐β‐1,4GlcNAc α2,6‐ST (2,6‐ST): 59% (p &lt; 0.001); liver—Gal‐β‐1,3GlcNAc α2,3‐ST (2,3‐ST): 32% (p &lt; 0.01); and kidneys—2,6–ST: 5% (NS). In contrast, glyceral‐dehyde‐3‐phosphate dehydrogenase mRNA in both liver and kidneys was unaffected by the same ethanol treatment. Taken together, these results demonstrate that chronic ethanol downregulates the expression of 2,6‐ST and 2,3‐ST genes in rat liver.

Elongation of N-acetyllactosamine repeats in diantennary oligosaccharides.

Glycosylated [Asn22]lysozyme has been shown to contain N-acetyllactosamine repeats when expressed in chinese hamster ovary (CHO) cells. We find that the major portion of N-acetyllactosamine repeats are associated with diantennary oligosaccharides. In Lec2 CHO cells, which are deficient in sialylation, glycosylated lysozyme is synthesized with increased contents of N-acetyllactosamine repeats terminating in beta-galactosyl residues. In the Lec2 cells and the parental CHO cell line, Pro 5, only a minor portion of the oligosaccharides in lysozyme are of the triantennary type. Previously, it has been shown that the synthesis of N-acetyllactosamine repeats in Asn-linked oligosaccharides is enhanced by an increase in the activity of the elongating beta-N-acetylglucosaminyl transferase and by the synthesis of beta-1,6-linked antennae. The results with glycosylated lysozyme suggest that glycoproteins bearing diantennary oligosaccharides can contain several N-acetyllactosamine repeats and that the number of the latter can be increased by decreasing the activity of the capping sialyl transferases.

Developmentally regulated expression of peanut agglutinin (PNA)-specific glycans on murine thymocytes.

Intrathymic maturation of T lymphocytes is characterized by variable expression of O-linked Gal beta 1,3GalNAc glycans reactive with peanut agglutinin (PNA) lectin. Recent studies on human thymocytes show that conversion from PNA+ to PNA- phenotype is correlated with increased expression of alpha 2,3 O-linked sialyltransferase (ST), which sialylates Gal beta 1,3GalNAc glycans, masking their binding sites for PNA. Interestingly, alpha 2,3 O-linked ST expression is highest within the regions of the thymus containing the most immature and most mature thymocyte subsets, suggesting that PNA-specific glycans are intermittently masked by sialylation during thymic selection processes. Here, we studied expression of PNA receptors on developing thymocytes in the murine system using thymocytes from both normal mice and transgenic mice that are genetically arrested at the early phases of T cell development. Our results confirm and extend recent findings in the human system by showing that murine T cells sequentially progress from PNAlo-->PNAhi-->PNAlo stages during their differentiation within the thymus. In addition, our data demonstrate that a similar set of polypeptides is variably masked by sialylation throughout T cell development.

Invasive phenotype of Trypanosoma cruzi restricted to a population expressing trans-sialidase.

Trypanosoma cruzi expresses a developmentally regulated trans-sialidase implicated in the pathogenesis of Chagas' disease. On inhabitation of the extracellular milieu of cultured cells by infective trypomastigotes, the enzyme is restricted to a small (20 to 30%) population of parasites. The biological significance of trans-sialidase expression on this subset, termed TS+, and not on the majority (70 to 80%) of morphologically similar trypanosomes, named TS-, is unknown. To determine the roles of the TS+ and TS- subsets in T. cruzi invasion, we prepared pure populations of TS- and TS+ trypanosomes using magnetic beads coated with a monoclonal antibody specific for the tandem repeat unit of the trans-sialidase C terminus. After removal of nonadherent TS- trypomastigotes, the TS+ trypomastigotes were isolated from the beads by specific elusion with a synthetic peptide epitope of the trans-sialidase monoclonal antibody. Confirmation of TS+ and TS- phenotypes was obtained by immunofluorescence, immunoblotting, and sialidase or sialyl transferase activity measurements. The TS+ trypanosomes were highly invasive, as they attached to, penetrated, and thrived in cultured mammalian cells much more efficiently than did unfractionated parasites. The critical role of the trans-sialidase in invasion was underscored by the observation that infection was neutralized by human antibodies to transsialidase. What's more, the TS- parasites, in sharp contrast to their TS+ counterparts, were extremely inefficient in invading epithelial cells and fibroblasts. Further, introduction of small amounts of exogenous trans-sialidase into suspensions of nonpenetrating TS- parasites converted them to a highly invasive phenotype indistinguishable from that of the TS+ population. Rescue of the invasive phenotype was specific for the T. cruzi enzyme, for it didn't happen with bacterial and viral sialidases. The in vitro results were confirmed in the murine model of Chagas' disease, as TS- trypomastigotes were relatively avirulent while TS+ trypomastigotes were more virulent than unfractionated parasites.

Molecular cloning and functional analysis of sialyltransferases.

To elucidate the regulatory mechanism for carbohydrate expression and to understand the meaning of the carbohydrate-structural diversity, we started to clone sialyltransferase (ST) genes based on two different strategies, i.e. expression and homology cloning. So far, 13 STs have been cloned in our laboratory, 7 of which turned out to be new ones. The primary enzyme structures deduced from all the cloned ST genes suggest a putative domain structure with a type II transmembrane topology. There are no significant amino acid sequence similarities among these cloned STs, except for in two sialyl motifs, L and S, which are proposed to be the CMP-sialic acid recognition and/or catalytic sites. Northern blot analysis revealed the developmental stage-dependent and/or tissue-specific expression of most of the cloned STs. The cloned STs are classified into four families according to the carbohydrate linkages they synthesize, i.e. the ST3Gal-, ST6Gal-, ST6GalNAc-, and ST8Sia-families. Generally, enzymes in these families exhibit strong activity toward certain acceptor groups but show very weak activity toward other acceptor groups, and the substrate specificities of the enzymes overlap one another, as indicated by in vitro experiments. Enzymes in the ST3Gal-family are expressed mainly in a tissue-specific manner. However, those in the ST6GalNAc- and ST8Sia-families are expressed in a tissue-as well as developmental stage-specific manner. In vivo conditions are supposed to be more complex. Therefore, it is quite important to examine their substrate specificities in vivo and the mechanism of their expression to elucidate the physiological role of each enzyme and the meaning of the diversity in carbohydrate structure. Using cloned cDNAs and expressed enzymes, we have been studying how sialylcarbohydrate expression is regulated and what the functions of sialylcarbohydrate chains are. Recently, we found that transfection of the GD3 synthase, an alpha 2,8-ST (ST8Sia I), gene triggers cholinergic neuritogenesis in Neuro2a cells through the de novo expression of GD3, suggesting that the GD3 synthase gene behaves as a neural differentiation inducer.

Decidual sialylation shows species-specific differences in the pregnant mouse and rat

Biotinylated lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA), which bind to alpha 2,6-linked and alpha 2,3-linked sialyl residues, respectively, were used as probes to study glycan terminal modifications associated with decidualization in the uterine stroma of pregnant rats and mice. Binding of lectins from Erythrina cristagalli (ECA), Phaseolus vulgaris (leukoagglutinin, L-PHA), Triticum vulgaris (WGA) and Bandeiraea simplicifolia (BSA-1B4) was also examined. Tissues from rats between day 5 and day 8 of gestation and mice between day 5 and day 7 of gestation were fixed in Bouin's solution and embedded in wax prior to lectin histochemistry. On day 7 in rats and day 6 in mice, there was a marked reduction in the binding of SNA in the subluminal decidua surrounding the implantation site. In rats, MAA binding to enlarged decidual cells around the implantation chamber was increased markedly, but there was no change in mice. In both species there was de novo binding of ECA in the SNA-negative area, suggesting that the loss of alpha 2,6-linked sialyl residues unmasks terminal N-acetyl lactosamine. These findings are consistent with previous evidence of a close structural and functional similarity between the artificially induced deciduoma and true decidua of rats and show identical changes to the glycosylation patterns previously found in differentiating rat deciduoma. In both species, therefore, decidua exhibits regionally specific terminal glycosylation. However, the species-specific expression of alpha 2,3-linked sialyl residues suggests distinct patterns of steroidally modulated sialyl transferase expression.

Serum carbohydrate-deficient transferrin

Carbohydrate-deficient transferrin (CDT) is now considered to be the most sensitive and specific biological marker of alcohol abuse. However, the mechanism by which chronic alcohol consumption causes an elevation of CDT levels in serum is still not understood. Therefore, we fed eight pairs of male rats a nutritionally adequate liquid diet containing either alcohol (36% of energy) or isocaloric dextrose (control) for 4 weeks, after which blood and liver samples were obtained. Serum CDT content in alcohol-treated rats increased by 45% (P < .05) in ethanol-fed animals compared with their corresponding controls. In contrast, in rats fed ethanol, the activities of sialyltransferase (ST), galactosyltransferase (GT), and N-acetylglucosamine transferase (N-AGT), which are glycosyltransferases involved in transferrin carbohydrate side chain synthesis, were diminished by 24% and 40% (P < .05), 23% and 51% (P < .05, .001), and 20% and 26% (P < .05) in total liver homogenates and Golgi fraction (GF) 1, respectively, when expressed as units/100 g body weight. These enzymes were also significantly less active in hepatic GFs 2 and 3. The depression of the transferase activities in ethanol-fed rats appeared to be due, at least in part, to enzyme inactivation by acetaldehyde, whereas ethanol itself was without effect. Similar results were obtained in humans: five alcohol abusers were found to exhibit a 23% decrease in hepatic sialyltransferase and a 41% increase in sialidase activities, respectively, when compared with three nondrinking subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Neisseria sialyltransferases and their role in pathogenesis

Neisseria sialyltransferases and their role in pathogenesis

Interferon alpha-2b modulates beta-galactoside alpha-2,6-sialyltransferase gene expression in rat testes.

Sperm surface glycoproteins are modified during passage through the epididymis, a process believed to be important in the production of functionally mature spermatozoa. The effect of various cytokines on reproductive events has recently been investigated, with conflicting results. In the present investigation, the effect of interferon-alpha-2b (IFN alpha 2b) on sialyltransferase (ST) activity and beta-galactoside alpha-2,6-sialyltransferase (Gal 2,6-ST) mRNA expression was studied in rat testicular tissue. The results revealed the presence of Gal 2,6-ST mRNA in rat testicular tissue, similar in molecular size to that found previously in rat spleen, lung, ovary, kidney, heart, and brain. In addition, we observed that IFN alpha 2b reduced Gal 2,6-ST mRNA and ST activity in rat testes by a comparable magnitude. These findings provide insight into an additional mechanism by which cytokines may affect the reproductive system.

Serum carbohydrate-deficient transferrin: Mechanism of increase after chronic alcohol intake

Carbohydrate-deficient transferrin (CDT) is now considered to be the most sensitive and specific biological marker of alcohol abuse. However, the mechanism by which chronic alcohol consumption causes an elevation of CDT levels in serum is still not understood. Therefore, we fed eight pairs of male rats a nutritionally adequate liquid diet containing either alcohol (36% of energy) or isocaloric dextrose (control) for 4 weeks, after which blood and liver samples were obtained. Serum CDT content in alcohol-treated rats increased by 45% (P < .05) in ethanol-fed animals compared with their corresponding controls. In contrast, in rats fed ethanol, the activities of sialyltransferase (ST), galactosyltransferase (GT), and N-acetylglucosamine transferase (N-AGT), which are glycosyltransferases involved in transferrin carbohydrate side chain synthesis, were diminished by 24% and 40% (P < .05), 23% and 51% (P < .05, .001), and 20% and 26% (P < .05) in total liver homogenates and Golgi fraction (GF) 1, respectively, when expressed as units/100 g body weight. These enzymes were also significantly less active in hepatic GFs 2 and 3. The depression of the transferase activities in ethanol-fed rats appeared to be due, at least in part, to enzyme inactivation by acetaldehyde, whereas ethanol itself was without effect. Similar results were obtained in humans: five alcohol abusers were found to exhibit a 23% decrease in hepatic sialyltransferase and a 41% increase in sialidase activities, respectively, when compared with three nondrinking subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum carbohydrate-deficient transferrin: Mechanism of increase after chronic alcohol intake

Carbohydrate-deficient transferrin (CDT) is now considered to be the most sensitive and specific biological marker of alcohol abuse. However, the mechanism by which chronic alcohol consumption causes an elevation of CDT levels in serum is still not understood. Therefore, we fed eight pairs of male rats a nutritionally adequate liquid diet containing either alcohol (36% of energy) or isocaloric dextrose (control) for 4 weeks, after which blood and liver samples were obtained. Serum CDT content in alcohol-treated rats increased by 45% ( P < .05) as assessed with an isoelectric focusing/Western blot method (IEF/WB) thereby mimicking the clinical condition. In particulate fractions (plasma membranes) prepared from the liver samples, the activity of sialidase was increased 1.62-fold ( P < .05) in ethanol-fed animals compared with their corresponding controls. In contrast, in rats fed ethanol, the activities of sialyltransferase (ST), galactosyltransferase (GT), and N-acetylglucosamine transferase ( N-AGT), which are glycosyltransferases involved in transferrin carbohydrate side chain synthesis, were diminished by 24% and 40% ( P < .05), 23% and 51% ( P < .05, .001), and 20% and 26% ( P < .05) in total liver homogenates and Golgi fraction (GF) 1, respectively, when expressed as units/100 g body weight. These enzymes were also significantly less active in hepatic GFs 2 and 3. The depression of the transferase activities in ethanol-fed rats appeared to be due, at least in part, to enzyme inactivation by acetaldehyde, whereas ethanol itself was without effect. Similar results were obtained in humans: five alcohol abusers were found to exhibit a 23% decrease in hepatic sialyltransferase and a 41% increase in sialidase activities, respectively, when compared with three nondrinking subjects. In conclusion, the increased activity of sialidase in liver particulate fractions, combined with the decrease in transferrin glycosyltransferases in GFs, mediated by ethanol (or acetaldehyde derived from ethanol) most likely accounts for the enhanced serum CDT in response to chronic ethanol consumption in rats as well as in humans.

An investigation of the role of transmembrane domains in Golgi protein retention.

The single transmembrane domains (TMDs) of the resident glycosylation enzymes of the Golgi apparatus are involved in preventing these proteins moving beyond the Golgi. It has been proposed that either the TMDs associate, resulting in the formation of large oligomers of Golgi enzymes, or that they mediate the lateral segregation of the enzymes between lipid microdomains. Evidence for either type of interaction has been sought by examining the retention of sialyltransferase (ST), an enzyme of the mammalian trans Golgi. No evidence could be obtained for specific interactions or 'kin recognition' between ST and other proteins of the trans Golgi. Moreover, it is shown that the previously described kin recognition between enzymes of the medial Golgi involves the lumenal portions of these proteins rather than their TMDs. To investigate further the role of the ST TMD, the effects on Golgi retention of various alterations in the TMD were examined. The addition or removal of residues showed that the efficiency of retention of ST is related to TMD length. Moreover, when a type I plasma membrane protein was expressed with a synthetic TMD of 23 leucines it appeared on the cell surface, but when the TMD was shortened to 17 leucines accumulation in the Golgi was observed. These observations are more consistent with lipid-based sorting of ST TMD, but they also allow for reconciliation with the kin recognition model which appears to act on sequences outside of the TMD.

Differential Ontogenic Regulation of Basolateral and Canalicular Bile Acid Transport Proteins in Rat Liver

The hepatic transport systems mediating bile acid uptake and excretion undergo independent, stage-specific expression during development in the rat. In this study, the mechanisms underlying ontogenic regulation of both the Na(+)-dependent basolateral bile acid transporter and canalicular bile acid transporter/ecto-ATPase were examined. Steady state mRNA levels for the basolateral transporter were less than 20% of adult values prior to birth, increased to 35% on the first postnatal day, and reached adult levels by 1 week of age. This was paralleled by transcription rates, which were low prior to birth, reached 47% by day 1, and were maximal by 1 week of age. Steady state mRNA levels for ecto-ATPase were 12% of adult values prior to birth and showed a 2-fold increase by the first day of life. Thereafter, there was a gradual increase in mRNA for this transporter, with adult levels being reached at 4 weeks of age. Transcription rates paralleled this increment, although adult levels were reached earlier. Surprisingly, for both transporters, the full complement of protein was present well before adult levels of mRNA were reached. The basolateral protein was expressed at 82% of adult levels on the first day of life but was of lower apparent molecular mass (39 kDa), a difference that persisted until 4 weeks of age. N-Glycanase digestion suggested that this difference could be fully accounted for by N-linked glycosylation. The ecto-ATPase protein was present at 33% of adult levels prior to birth, 77% by 1 day, and 84% of adult levels by 1 week of age. Unlike the basolateral transporter, the apparent molecular weight of this protein did not change during development. In summary, the ontogeny of bile acid transporters on the plasma membrane of the hepatocyte is complex and appears to be regulated at transcriptional, translational, and post-translational levels.