Inhibition of the bacterial lectins of Pseudomonas aeruginosa with monosaccharides and peptides.

Pseudomonas aeruginosa (PA) can cause infections in compromised hosts by interacting with the glycocalyx of host epithelial cells. It binds to glycostructures on mucosal surfaces via two lectins, which are carbohydrate-binding proteins, named PA-IL and PA-IIL, and blocking this interaction is, thus, an attractive anti-adhesive strategy. The aim of this study was to determine by ciliary beat frequency (CBF) analysis whether monosaccharides or peptides mimicking glycostructures represent blockers of PA lectin binding to human airway cilia. The treatment with monosaccharides and peptides alone did not change the CBF compared to controls and the tested compounds did not influence the cell morphology or survival, with the exception of peptide pOM3. PA-IL caused a decrease of the CBF within 24 h. D-galactose as well as the peptides mimicking HNK-1, polysialic acid and fucose compensated the CBF-modulating effect of PA-IL with different affinities. PA-IIL also bound to the human airway cilia in cell culture and resulted in a decrease of the CBF within 24 h. L(-)-fucose and pHNK-1 blocked the CBF-decreasing effect of PA-IIL. The HNK-1-specific glycomimetic peptide had a high affinity for binding to both PA-IL and PA-IIL, and inhibited the ciliotoxic effect of both lectins, thus, making it a strong candidate for a therapeutic anti-adhesive drug.

The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.

Among the recognition molecules that determine a neuron's interaction with other cells, L1 and CD24 have been suggested to cooperate with each other in neurite outgrowth and signal transduction. Here we report that binding of CD24 to L1 depends on alpha2,3-sialic acid on CD24, which determines the CD24 induced and cell type-specific promotion or inhibition of neurite outgrowth. Using knockout mutants, we could show that the CD24-induced effects on neurite outgrowth are mediated via L1, and not GPI-linked CD24, by trans-interaction of L1 with sialylated CD24. This glycoform is excluded together with L1 from raft microdomains, suggesting that molecular compartmentation in the surface membrane could play a role in signal transduction.

Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains.

We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.

SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.

ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.

A submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells.

The secretory lectin ZG16p mediated the binding of aggregated zymogens to the granule membrane in pancreatic acinar cells. Using a recently established in vitro condensation-sorting assay, we now show that pretreatment of zymogen granule membranes (ZGM) with either sodium bicarbonate at pH 10 or with phosphatidyl inositol-specific phospholipase C (PI-PLC) reduced the binding efficiency of zymogens to the same extent, as distinct components were liberated from ZGM. Analysis of the composition of the bicarbonate extract revealed the presence of the secretory lectin ZG16p, the serpin ZG46p and the GPI-linked glycoprotein GP-2, together with several unknown proteins, and small amounts of lipase and carboxylester lipase. The unknown proteins detected in 2-D gels represented a group of acidic and basic protein spots, which were positive in a glycan staining reaction and were soluble in methanol. One protein spot of the acidic group and several of the basic group reacted with a monoclonal antibody directed against chondroitin sulfate, indicating that the proteins represented proteoglycans. A staining pattern similar to the glycan reaction was observed in immunoblots using a polyclonal antibody directed against the whole bicarbonate extract. Immunogold electron microscopy revealed that this antibody reacted with components in the periphery of zymogen granules and strongly stained ZGM in the pellet fraction of a standard in vitro condensation-sorting assay. The amino acid composition of isolated components of both the acidic and basic group showed similarities to aggrecan, a cartilage-specific proteoglycan, and to glycine-rich glycoproteins, respectively. We therefore conclude that a submembranous matrix on the ZGM composed of proteoglycans and glycoproteins is involved in granule formation in pancreatic acinar cells.

A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat.

Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- and RER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent. The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied. Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5'end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation.

Complex formation among rat pancreatic secretory proteins under mild alkaline pH conditions.

Previous in vitro studies have demonstrated that enzyme proteins liberated from isolated zymogen granules of the rat pancreas aggregate already at neutral or slightly basic pH and form small particles which in the acidic pH range progressively condense into dense cores of about the size of zymogen granules. To characterize the protein composition of the original particles in more detail non-denaturing agarose gel electrophoresis was employed. Five major protein complexes were identified which upon separation of individual complexes in 1-D or 2-D gel electrophoresis were shown to be composed of a distinct set of known enzymes and several unknown proteins. Complexes 1-4 quickly dissociated when enzyme activation was induced by enterokinase, but complex 5 was resistant even to this treatment. All 5 complexes revealed a distinct fine structure when eluted from the gels and studied in negative staining electron microscopy. These findings suggest that pancreatic zymogens form complexes already in the lumen of the rough endoplasmic reticulum and are transported as such to the Golgi complex where they aggregate into granule cores due to the internal acidic pH. Complex formation may thus facilitate zymogen sorting within the rough endoplasmic reticulum and may prevent premature enzyme activation within cellular compartments.

The secretory lectin ZG16p mediates sorting of enzyme proteins to the zymogen granule membrane in pancreatic acinar cells.

The recently established in vitro assay of condensation-sorting of pancreatic enzymes to the zymogen granule membrane (ZGM) (Dartsch, H., R. Kleene, H. F. Kern: In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells. Eur. J. Cell Biol. 75, 211-222 (1998)) was used to study the involvement of a novel secretory lectin, ZG16p, in the binding of aggregated proteins to ZGM. In isolated zymogen granules the lectin is predominantly associated with the membrane and can be removed to a large extent by bicarbonate treatment at pH 11.5. In the in vitro assay in which secretory proteins aggregate at pH 5.9 but only those bound to ZGM are sedimented into the pellet, ZG16p is significantly enriched in this pellet fraction, shown both by biochemical and fine structural analysis. Pretreatment of ZGM with anti-ZG16p antibody before their addition to the assay inhibits binding to the membrane by about 50%. Similarly, removal of ZG16p or prevention of its interaction with glycosaminoglycans (GAGs) in the submembranous matrix of ZGM by sodium bicarbonate treatment or chondroitinase digestion of ZGM also inhibits the binding efficiency of secretory proteins to ZGM to about the same extent. We conclude that ZG16p may act as a linker molecule between the submembranous matrix on the luminal side of ZGM and aggregated secretory proteins during granule formation in the TGN.

Membrane dipeptidase and glutathione are major components of pig pancreatic zymogen granules.

Membrane proteins of highly purified porcine zymogen granules were separated by two-dimensional gel electrophoresis in order to isolate proteins which are involved in intracellular trafficking of digestive enzymes in the exocrine pancreas. A 48-kDa glycoprotein was a major component in membrane preparations washed with 0.1 M Na2CO3 and 0.5 M NaCl. By N-terminal amino acid sequencing this protein was identified as membrane dipeptidase (MDP; EC 3.4.13.19). MDP mRNA levels in rat pancreas were increased threefold by feeding rats with FOY-305, which is a known stimulus of endogenous cholecystokinin release from the gut. Cholecystokinin then stimulates secretion in pancreatic acinar cells. In another set of experiments treatment of the rat pancreatic acinar tumor cell line AR42J with dexamethasone led to an eightfold increase in the expression of MDP. Thus, the expression pattern of the MDP gene in response to hormonal stimulation in vivo and in vitro resembles those found for most of the enzymes and proteins which are involved in secretion. Since MDP has been thought to have a role in glutathione (GSH) metabolism, we also measured GSH concentration in zymogen granules and found high levels of GSH. Based on our data we propose a working model for the function of MDP. According to this model, MDP might play a pivotal role in maintaining the oxidizing conditions in the ER, which are required for the correct folding of secretory proteins.

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization.

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.

In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells.

To study the process of granule formation in pancreatic acinar cells in more detail we have established an in vitro system in which the whole complement of enzyme proteins released from isolated zymogen granules is mixed with a tracer amount of the same biosynthetically labeled proteins and is incubated at conditions prevailing in either pre-Golgi (pH 7.5) or trans-Golgi (pH 5.9) compartments. Condensation of the proteins into dense cores is assayed and quantitated after centrifugation of the mixture at 13000g and separation of the proteins in both the supernatant and the pellet by 2D-gel electrophoresis. At pH 7.5 about 1% of the total protein-bound radioactivity can be sedimented into the pellet and this increases 5-fold at pH 5.9 with similar sedimentation efficiency for individual enzyme proteins. The usual assumption that all aggregated proteins can be sedimented and thus only the pellet is representative for pH-dependent condensation has to be modified by the fine structural analysis of both the supernatant and pellet fraction at pH 7.5 and 5.9. Small particulate complexes form already in the supernatant at pH 7.5 which are not sedimented to a large extent into the pellet. At pH 5.9 aggregates of a homogeneous size of about 0.6 to 0.8 microm formed in the supernatant while the pellet is composed of sheets and vesicles of membranes studded with dense core particles of about 20 to 30nm size. The pH-dependent protein condensation is a stepwise process starting with the formation of small dense core particles already at pH 8.0/7.5 which then progressively aggregate to form larger cores at pH 6.0/5.0. These aggregates can only be sedimented employing higher centrifugal forces. In the condensation process of pancreatic enzyme proteins calcium ions exert an effect only at pH 7.5, leading to somewhat larger dense particles, while potassium ions are inhibitory both in protein condensation and in the binding of particles to membranes. The process of pH-dependent protein condensation is reversible and can be performed repetitively. The sedimentation of condensed proteins can be increased by the addition of isolated zymogen granule membranes. Thus the in vitro system allows the analysis of two related processes in granule formation: the condensation of secretory proteins into granule cores and their binding to the granule membrane.

Role of the N- and C-termini of porin in import into the outer membrane of Neurospora mitochondria.

The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, have not been clearly defined. Targeting information has been hypothesized to be contained in the N-terminus, which may form an amphipathic alpha-helix, and in the C-terminal portion of the protein. Here, the role of the extreme N- and C-termini of porin from Neurospora crassa in its import into the mitochondrial outer membrane was investigated. Deletion mutants were constructed which lacked the N-terminal 12 or 20 residues or the C-terminal 15 residues. The porins truncated at their N-termini were imported in a receptor-dependent manner into the outer membrane of isolated mitochondria. When integrated into the outer membrane, these preproteins displayed an increased sensitivity to protease as compared to wild-type porin. In contrast, mutant porin truncated at its C-terminus did not acquire protease resistance upon incubation with mitochondria. Thus, unlike most other mitochondrial preproteins, porin appears to contain important targeting and/or assembly information at its C-terminus, rather than at the N-terminus.

Immunodetection of alpha 1-3 fucosyltransferase (FucT-V).

The fucosyltransferases constitute a family of glycosyltransferases incorporating fucose residues into glycoprotein or glycolipid glycans. They afford one of the possible termination steps of glycoconjugate biosynthesis creating the sialyl Lewisx or sialyl Lewisa determinant, which play an important role in cell-cell interaction. While cDNA, chromosomal localization and kinetic properties of a number of fucosyltransferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies due to the pronounced homology of alpha 1, 3 fucolsyltransferases III, V and VI. Here we report development and characterization of monospecific polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) and their application for immunodetection in transfected cells. Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V. Polyclonal antisera were raised against each of both antigens and characterized by enzyme-linked immunosorbent assay, neutralization of activity, immunoblotting, immunofluorescence and immunoprecipitation of metabolically labeled COS cells, transiently transfected with cDNA encoding FucT-V. Both antibodies recognized only FucT-V. No cross-reactivity to FucT-III or FucT-VI was observed. FucT-V was localized mainly to the Golgi apparatus by colocalization with beta 1, 4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells. Expression of FucT-V in COS cells revealed three enzyme forms of 58, 53 and 50 kDa, respectively. These size differences arose by post-translational modifications, as shown by pulse-chase experiments. Our results indicate that alpha 1-3 fucosyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface.

High-level expression of endogenous acid phosphatase inhibits growth and vectorial secretion in Saccharomyces cerevisiae.

The secretion pathway of Saccharomyces cerevisiae was challenged by constitutively overexpressing plasmid-encoded acid phosphatase, a secreted endogenous glycoprotein. A 2-microns-based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncated variant of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter was used for expression. Selection for the promoterless dLEU2 marker leads to a growth arrest. This is not per se due to leucine starvation, but due to intracellular accumulation of highly glycosylated enzymatically active acid phosphatase. Immunofluorescence and cytological analysis indicate that intracellular accumulation of acid phosphatase occurs in a subpopulation of cells. By Ludox-AM density centrifugation, these cells can be enriched on the basis of their higher density. The dense accumulating cells have a higher average plasmid copy number and produce more acid phosphatase than non-accumulating cells of low density. These cells are defective in directed secretion and bud formation, therefore can no longer grow and show dramatic changes in cell morphology. We suggest that the secretion pathway in these cells is overloaded with the high level of acid phosphatase leading to a shutdown in vectorial secretion, subsequently to a standstill in growth and to the intracellular accumulation of further expressed acid phosphatase. We have indications that accumulation of acid phosphatase occurs in the late Golgi, suggesting a limitation of the overall secretion at this stage.

Large-scale production of a soluble human beta-1,4-galactosyltransferase using a Saccharomyces cerevisiae expression system.

We report in this communication the first large-scale heterologous expression of a glycosyltransferase in yeast. A soluble form of a human beta-1,4-galactosyltransferase (EC 2.4.1.38) was expressed using a Saccharomyces cerevisiae expression system. Fermentation technology afforded the means to increase the expression level of the beta-1,4-galactosyltransferase up to a concentration of 700 mU/liter. The enzyme was produced at a scale of 200 units. The recombinant soluble enzyme was purified 766-fold to a specific activity of approx. 2 U/mg using a purification protocol based on sequential affinity chromatography on N-acetylglucosaminyl- and alpha-lactalbumin-Sepharose, respectively. This study demonstrates that heterologous expression of a glycosyltransferase is possible on a large scale and offers an alternative to natural sources like human breast milk or bovine colostrum.

Expression of soluble active human beta 1,4 galactosyltransferase in Saccharomyces cerevisiae.

Sequences coding for the cytoplasmic and transmembrane domains were removed from the cDNA of the human Golgi resident membrane protein beta 1,4 galactosyltransferase (gal-T). The remaining sequences coding for the stem and catalytical domains of this glycosyltransferase were fused to sequences coding for the yeast invertase signal sequence. The hybrid was inserted together with a constitutive yeast promoter and a terminator into a E. coli/yeast shuttle vector. Saccharomyces cerevisiae strain BT150 transformed with this new expression vector expressed enzymically active soluble enzyme, whereas no activity was detectable in mock-transformed yeasts. The enzyme product was identified by HPLC analysis and shown to correspond to the expected product N-acetyllactosamine.

Human beta 1,4 galactosyltransferase and alpha 2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum.

Biosynthesis and intracellular transport of recombinant human full-length beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113-120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of beta-galactosidase-GT or beta-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radio-active sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-alpha 1,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus. These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.

Double immunofluorescent staining of alpha 2,6 sialyltransferase and beta 1,4 galactosyltransferase in monensin-treated cells: evidence for different Golgi compartments?

Beta 1,4 galactosyl- and alpha 2,6 sialyltransferase (gal-T EC 2.4.1.22 and sialyl-T EC 2.4.99.1) sequentially elongate and terminate complex N-glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi-disturbing agents. Polyclonal, peptide-specific antibodies to human sialyl-T expressed as a beta-galactosidase-sialyl-T fusion protein in E. coli were developed and applied together with mABs to human milk gal-T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl-T and gal-T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal-T followed by sialyl-T; in the reassembled Golgi apparatus sialyl-T and gal-T were co-localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal-T positive Golgi elements while sialyl-T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal-T and sialyl-T remained co-localized. Treatment with chloroquine affected Golgi structures less than monensin and led to condensation of gal-T positive and to slight enlargement of sialyl-T positive structures. Sequential recovery from BFA of gal-T and sialyl-T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.

Purification and characterization of recombinant human beta 1-4 galactosyltransferase expressed in Saccharomyces cerevisiae.

A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell beta-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum. Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly. This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.