Department of Cell Biology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06510, USA
Received on June 1, 1999; revised on October 19, 1999; accepted on November 7, 1999.
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Abstract |
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Key words: ganglioside/Golgi apparatus/immunohistochemical localization/nervous system/sialyltransferase
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Introduction |
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We isolated a clone encoding the mouse sialyltransferase, mST3GalV (CMP-NeuAc: lactosylceramide 2,3-sialyltransferase), in a screen for a vertebrate homologue of Gliolectin, a Drosophila carbohydrate binding protein. mST3GalV synthesizes ganglioside GM3, the precursor for simple and complex a- and b-series gangliosides (van Echten and Sandoff, 1993
; Ishii et al., 1998
; Kono et al., 1998
; Fukumoto et al., 1999
). Therefore, the expression and regulated activity of mST3GalV/GM3 synthase is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in such diverse cellular processes as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cellcell interactions (Goldenring et al., 1985
; Blackburn et al., 1986
; Bremer et al., 1986
; Kreutter et al., 1987
; Nojiri et al., 1988
; Tsuji et al., 1988
; Hakomori and Igarishi, 1993
, 1995; Yamamura et al., 1997
). While greatly enriched in both neurons and glial cells, gangliosides are also found in non-neural tissue. Importantly, the first phenotypes described in transgenic knock-out mice lacking a key ganglioside synthetic enzyme, GM2/GD2 synthase, were manifest in non-neural tissue (Takamiya et al., 1998
; Liu et al., 1999
). Therefore, establishing the normal expression pattern of ganglioside biosynthetic enzymes should assist in evaluating future loss-of-function mutants.
The necessity for mST3GalV activity to act early in the pathway leading to synthesis of complex gangliosides has previously suggested that this enzyme should be localized to the cis-Golgi, analogous to the sequential topographical distribution of glycoprotein processing enzymes. However, characterization of the Golgi distribution of GM3 synthase activity by subcellular fractionation or in vitro pharmacologic manipulation has yielded reports of coenrichment with both proximal and distal Golgi markers (van Echten et al., 1990; Young et al., 1990
; Iber et al., 1992
; Lannert et al., 1998
; Maccioni et al., 1999
). Confocal immunohistochemical analysis of the subcellular localization of ganglioside biosynthetic enzymes, as we report here, provides an alternative to biochemical fractionation and allows spatial colocalization with defined Golgi markers in a minimally disrupted cell.
We have investigated the expression pattern and subcellular distribution of mST3GalV in neural and non-neural tissue. Immunohistochemistry and in situ hybridization reveal that mST3GalV is broadly expressed in neurons and glial cells throughout the nervous system. Robust expression of mST3GalV is evident in efferent neurons that project in myelinated pathways while oligodendrocytes express relatively low levels of the enzyme in comparison to neurons or Schwann cells. ST3GalV is highly expressed in distinct cell types in both liver and testis. Furthermore, the large and spatially distributed Golgi apparatus found in several neuronal types provides a favorable architecture for the colocalization of mST3GalV with markers that define Golgi subcompartments. By confocal analysis, mST3GalV fails to colocalize with a cis-Golgi marker but exhibits complete colocalization with a medial/trans-Golgi marker.
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Results |
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Similarity between Drosophila Gliolectin, ST3GalV, and other sialyltransferases
Mouse ST3GalV possesses similarity to Drosophila Gliolectin across a 27 amino acid stretch that flanks the amino- and carboxy-terminal borders of the ST3GalV S-motif, displaying 26% amino acid identity and 59% similarity (81% weak similarity) (Figure 1A). Across this region, overall nucleotide identity between mST3GalV and Drosophila Gliolectin is 47% (excluding the S-motif) while the 25 nucleotide stretches 5' and 3' to the S-motif display, respectively, 64% and 72% identity between the two sequences. Upon optimal alignment, overall nucleotide identity between mST3GalV and Drosophila Gliolectin is 41%.
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Distribution of mST3GalV expression in neural cells
By in situ hybridization, mST3GalV mRNA was readily detectable in several populations of large, efferent neurons of the central nervous system (CNS). In particular, cerebellar Purkinje cells, spinal motorneurons and olfactory bulb mitral cells demonstrate significant mST3GalV mRNA expression (Figure 2). Some interneuron pools, such as spinal neurons within the dorsal horn, are also visualized by in situ hybridization. Consistent with the observed distribution of mRNA, antiserum (designated CS2) raised against an mST3GalV peptide (amino acids K227-I272) reveals an extensive and intricately arrayed Golgi apparatus within populations of large efferent neurons. Preimmune sera did not stain any of the tissues examined.
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Discussion |
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Amino acid alignment of Drosophila Gliolectin with mST3GalV reveals two regions of similarity, one on either side of the sialyltransferase S-motif. Each of these regions in mST3GalV is similar to the other, possessing a motif of appropriately spaced amino acids (hDxxQxxPxxxh). Drosophila Gliolectin, a type II transmembrane protein like mST3GalV, was originally identified based on its ability to bind a subset of GlcNAc-terminated glycosphingolipids isolated from Drosophila embryos (Tiemeyer and Goodman, 1996). Although mST3GalV and Drosophila Gliolectin exhibit divergent saccharide-binding specificities, (Galß4Glc- for ST3GalV and GlcNAcß3/4Hex- for Gliolectin) both proteins recognize carbohydrate in the context of a glycosphingolipid. In fact, mST3GalV appears to have at least 100-fold lower activity towards lactose than towards lactosylceramide (Kono et al., 1998
). It has been suggested that the sialyltransferase S-motif may facilitate acceptor carbohydrate recognition (Datta and Paulson, 1995
). Perhaps the extended S-motif consensus highlighted by alignment with Drosophila Gliolectin reflects the conservation of residues that accommodate glycosphingolipid within a binding site. Alternatively, this motif may reveal the signature of an ancestral carbohydrate recognition domain that has acquired new binding specificities relevant to the divergent glycan profiles of evolving organisms.
While Drosophila Gliolectin has not yet been demonstrated to possess enzymatic activity, the reaction catalyzed by ST3GalV defines the mouse sialyltransferase as a key component in the regulation of ganglioside biosynthesis. Synthesis of ganglioside GM3 is the first committed step in the formation of ganglioside. If vectorial elaboration, from cis- to trans-Golgi compartments, underlies ganglioside biosynthesis, the sub-Golgi localization of mST3GalV should identify the site where synthesis is initiated. Analysis of the sub-Golgi distribution of GM3 synthase activity by biochemical fractionation has indicated that the enzyme is enriched in the cis-compartment (Trinchera and Ghidoni, 1989; Trinchera et al., 1991
; Iber et al., 1992
). Consistent with this observation, pharmacologic manipulation of Golgi integrity by Brefeldin A, a fungal toxin that blocks intra-Golgi transport causing collapse of the Golgi (cis- through trans-compartments but not TGN) into the endoplasmic reticulum, has been reported to dissociate GM3 synthesis from later-acting ganglioside glycosyltransferases (van Echten et al., 1990
; Young et al., 1990
). However, other fractionation studies have demonstrated that synthesis of GM3 and its precursor (lactosylceramide) occurs in the trans-Golgi and TGN compartments where it is functionally coupled to other ganglioside glycosyltransferases (Lannert et al., 1998
).
Reported differences in sub-Golgi localization of GM3 synthase activity may reflect Golgi organization that is specific to cell-type or cell maturity and may also be complicated by methodological considerations (Maccioni et al., 1999). Confocal immunohistochemical localization provides an alternate approach that allows assessment of enzyme distribution in a non-disrupted cell located in its normal tissue environment. Consistent with reports placing GM3 synthesis in later Golgi compartments, we find that anti-mST3GalV immunoreactivity fails to colocalize with a cis-Golgi marker, partially colocalizes with a broadly distributed Golgi marker and completely colocalizes with a medial/trans-Golgi marker in spinal motorneurons. The concentration of multiple ganglioside glycosyltransferases in distal Golgi compartments suggests that, compared to glycoprotein oligosaccharide processing, the control of ganglioside synthesis may be less reliant on topographical enzyme distribution or targeted vesicular traffic.
While all neural cell types (neuron and glia) synthesize ganglioside and should, therefore, express ST3GalV/GM3 synthase, determining the relative ganglioside content of neuronal or glial subsets is hampered by the difficulty inherent in obtaining appropriately enriched cell populations. However, cell-specific ganglioside expression has been demonstrated through analysis of glycolipids in mouse neurological mutants, in which defined neuronal populations are missing or degenerate in the perinatal period, and through biochemical characterization of ganglioside content in brain subcellular fractions (Suzuki et al., 1967; DeVries and Zmachinski, 1980
; Seyfried et al., 1983
; O'Gorman and Sidman, 1985
). Immunohistochemical localization has provided another means of discerning the distribution of ganglioside expression (Kotani et al., 1993
, 1994; Kotani et al., 1995
). Anti-ganglioside antibodies reveal that a subset of the a- and b-series gangliosides, specifically GD1a and GT1b, are diffusely enriched throughout the adult rat cerebellar molecular layer, whose neuropil contains both the extensive Purkinje cell dendritic arbor and the parallel fibers of granule cells. In addition, GM1 and GM3 are enriched in cerebellar white matter which consists of myelinated Purkinje cell efferents and cerebellar afferent processes. Interestingly, however, the only characterized sialylated glycolipid reported to be enriched at the Purkinje cell somata, O-acetyl-LD1 (a disialylated neolactotetraosylceramide derivative), does not require the synthesis of GM3 as precursor. Therefore, Purkinje cells, which clearly express significant amounts of ST3GalV, demonstrate a robust ability to target the transport or sequestration of glycolipid to specific cell surface domains.
Immunohistochemical characterization of mST3GalV in mouse brain demonstrates that the enzyme is broadly but differentially expressed in neurons and glia. The common trait shared by neurons exhibiting robust anti-mST3GalV immunostaining is that their axonal projections form myelinated efferent pathways; projection lengths range from the relatively short pathway between cerebellar cortical Purkinje cells and cerebellar deep nuclei to the long-distance relay between ventral motor neurons and peripheral muscle. In contrast, small neurons, such as the granule cell populations of the cerebellar cortex, the olfactory bulb and the hippocampus, which give rise to non-myelinated local circuit projections, present lower levels of CS2 immunostaining. While differential staining between neuronal populations could arise from variations in Golgi architecture that generate alternate spatial distributions of a relatively constant amount of enzyme (Torre and Steward, 1996), the enrichment of mRNA for mST3GalV within large efferent neurons, revealed by in situ hybridization, is consistent with quantitative differences in enzyme expression.
Myelinated efferent projections that express high levels of ST3GalV differ from other neuronal pathways in at least one major respect. The interaction of the projecting neuron with an oligodendrocyte results in the formation of a myelin sheath. If axon caliber exceeds a threshold, neuron-glial communication commits the oligodendrocyte to myelination (Friede, 1972; Trapp et al., 1988
). While ganglioside function in the initiation of myelination has yet to be conclusively demonstrated, axonal gangliosides recognized by Myelin Associated Glycoprotein, a member of the SIG-Lec family, contribute to the stabilization of formed myelin (Collins et al., 1997
; Yin et al., 1998
; Sheikh et al., 1999
).
Although enriched in neural tissue, gangliosides are a normal constituent of non-neural cells as well. Previous analysis of hepatic ganglioside expression has shown that Kupffer cells and sinusoidal endothelial cells are enriched in gangliosides (primarily GM3 and other simple species) relative to hepatocytes, the only liver cells capable of expressing complex gangliosides (Gabellec et al., 1983; Senn et al., 1990
). Furthermore, Kupffer cells and sinusoidal endothelial cells possess 7-fold higher GM3 synthase activity than hepatocytes (Gabellec et al., 1983
; Senn et al., 1990
). Consistent with the biochemical characterization of liver ganglioside expression, immunostaining for ST3GalV with CS2 antiserum is below detectable limits in hepatocytes but clearly evident in Kupffer cells and a spatially restricted subset of sinusoidal endothelial cells.
Hepatocytes and sinusoidal endothelial cells are born at the portal triad and progress during their life toward the central vein and senescence (Arber et al., 1988; Zajicek et al., 1988
). Therefore, a centripetally graded increase in hepatocyte and non-parenchymal cell maturation exists along the hepatic cords. Furthermore, zonal subsegments of hepatic cords exhibit differential drug toxicity and pathophysiologic susceptibility that correlates with functional specialization (Jungermann and Katz, 1982
; Tsukamoto et al., 1986
). The distribution of ST3GalV suggests that regional differences in sinusoidal endothelial cells mirror hepatocyte specialization.
Analysis of mice lacking GM2/GD2 synthase, the enzyme acting after ST3GalV in the pathway from lactosylceramide to complex gangliosides, has implicated ganglioside function in spermatogenesis (Takamiya et al., 1998; Liu et al., 1999
). In particular, testosterone accumulation in interstitial Leydig cells results in a decrease in circulating androgen levels in GM2/GD2 synthase null mice and a concomitant deficiency in germ cell maturation. The loss of complex ganglioside is accompanied by an increase in GM3 and GD3, primarily in non-germline cells, and altered Leydig cell morphology. Consistent with this reported shift in non-germline ganglioside distribution, we observe that ST3GalV immunoreactivity is high in Leydig cells. The reported absence of the stem cell population in some seminiferous tubules of GM2/GD2 synthase null mice and the expression of ST3GalV and GM2/GD2 synthase in a broad range of testicular cell types, including germ cells and epididymal epithelial cells, indicates that gangliosides function at multiple steps in spermatogenesis and spermiogenesis (Kawai et al., 1998
).
With the cloning of ST3GalV by multiple approaches, at least one transferase has now been identified for each step in ganglioside oligosaccharide biosynthesis (Furukawa, 1998). Characterizing the wild-type distribution of synthetic enzymes is important for interpreting loss-of-function phenotypes arising from transgenic manipulation and will indicate which tissues may warrant closer examination. For instance, while global deletion of ST3GalV expression may be expected to present early-embryonic lethality, conditional loss-of-function in endothelial cells may reveal important aspects of liver organization while loss of ST3GalV expression in Purkinje cells would allow the assessment of ganglioside function in a myelinated efferent projection. Furthermore, the availability of appropriate reagents, targeting a complete ensemble of ganglioside biosynthetic enzymes, will reveal the subcellular spatial organization of glycosphingolipid synthesis and will allow evaluation of the topological interrelation between processing pathways for multiple glycoconjugate classes.
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Materials and methods |
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Cloning of mouse ST3GalV/GM3 synthase
Plaques (1 x 106) of a mouse embryonic brain cDNA library were screened at reduced stringency with a random-primed, 32P-dCTP labeled cDNA probe complementary to the full-length Drosophila Gliolectin coding sequence. Nylon filters bearing phage DNA were hybridized and washed at 55°C as described by Church and Gilbert (Church and Gilbert, 1984). Positive phage clones were plaque purified and inserts were recovered in Bluescript by in vivo excision with ExAssist helper phage. Nucleotide sequencing was performed by the dideoxy chain-termination method at the Keck Sequencing Facility (Yale University School of Medicine). The full-length sequence was subsequently completed by multiple approaches, including 5'RACE (Frohman, 1993
), a genomic library screen (Lambda Fix II mouse genomic library) and RT-PCR (utilizing primers against Human EST AA386324). DNA sequence was obtained on both strands from at least three clones for the entire length of mST3GalV and was compiled, analyzed and aligned with the University of Wisconsin Genetics Computer Group software (GCG) and sequence analysis tools available through the National Center for Biotechnology Information (NCBI).
In situ hybridization
In situ hybridization was performed on 20 µm transverse sections of central nervous system regions taken from postnatal day 30 mice. Cyrostat sections were thaw mounted onto 3-aminopropyltriethoxysilane coated slides (2% AAS in acetone) and postfixed in 4% paraformaldehyde for 20 min. Solutions and subsequent treatment of slides were as previously described (Giger et al., 1996). Prehybridization and hybridization were performed in 0.1% w/v Ficoll, 0.1% w/v polyvinylpyrolidone, 0.1% bovine serum albumin, 0.025% w/v yeast tRNA, 0.02% w/v salmon sperm DNA, 50% formamide v/v in 5 x SSC. After prehybridization for 2 h at ambient temperature, the solution was removed and replaced with hybridization solution containing one-third of a T7 polymerase probe preparation (~50 ng of probe, see below). Following incubation at 55°C for 15 h in a humidified chamber, sections were washed sequentially in 5x SSC at 55°C for 5 min, 2x SSC at 55°C for 1 min, 1x SSC in 50% v/v formamide at 55°C for 30 min, and finally in 1x SSC at ambient temperature for 5 min. Hybridization of labeled probe was detected by incubation with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments diluted 1:5000 following the manufacturers recommended protocol. Alkaline phosphatase-induced chromophore precipitation (5-bromo-4-chloro-3-indolyl phosphate with nitroblue tetrazolium) was performed in the presence of 1 mM levamisole and tissue sections were subsequently dehydrated and mounted in Entellan.
To generate template for the incorporation of digoxigenin-UTP into RNA probes by T7 RNA polymerase, a 295 bp fragment of mST3GalV (from nt 727 to nt 1022) was generated by PCR and subcloned in both orientations into the TA cloning vector pCR2.1. A 1013 bp SpeI/AflIII fragment was then released from two plasmid clones containing insert in opposite orientation, sense or anti-sense relative to the T7 primer site. Following gel purification, 300 ng of fragment was used as template for probe synthesis as previously described (Giger et al., 1996). An aliquot of each T7 RNA polymerase product was spotted onto nylon membrane at multiple 2-fold dilutions and then probed with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments to verify that sense and anti-sense probe preparations achieved equivalent digoxigenin incorporation.
mST3GalV antibody production
An antibody to ST3GalV was raised against a TrpE fusion protein in rabbit. Nucleotides 709847 (K227-I272) were subcloned into the pATH11 vector and expression of the fusion protein was induced in E.coli XL-1 Blue as described previously (Koerner et al., 1991). Fusion protein was purified by preparative SDSPAGE followed by electroelution from the gel (Smith, 1992
). To generate antigen for injection, the gel purified fusion protein was separated from excess free SDS by precipitation of the detergent as its potassium salt (Zaman and Verwilghen, 1979
). One rabbit was initially injected with 200 µg protein of the resulting antigen preparation in complete Freunds adjuvant and was subsequently boosted with the 100 µg of protein in incomplete Freunds adjuvant every 3 weeks. The harvested serum was designated CS2 and a portion was affinity purified by binding to and eluting from an Affigel 15 column previously coupled with mST3GalV-TrpE fusion protein. All protein determinations were by the bicinchoninic acid assay (Smith et al., 1985
).
Western blot analysis
Enriched Golgi membrane fractions were prepared from adult mouse brain, liver, and testis tissue as described previously (Fleischer and Kervina, 1974; Ma and Colley, 1996
). Maximal recovery of immunoreactive mST3GalV required complete reduction and aggressive alkylation before SDSPAGE; optimal reduction was determined to require 50 mM dithiothreitol followed by sulfhydral alkylation with 250 mM iodoacetamide. For SDSPAGE, 1 µg membrane protein was electrophoresed and transferred to nitrocellulose (Harlow and Lane, 1988
). Nitrocellulose blots were blocked in 5% dry milk, 0.2% Tween-20 in PBS. The primary antibody, anti-mST3GalV antisera (CS2 antisera), was diluted 1:5000 and the secondary antibody, HRP-conjugated goat anti-rabbit, was used at 1:5000 dilution. Antibody binding was detected by ECL Plus with the manufacturers recommended conditions. No difference was detected between blots probed with affinity-purified or nonpurified serum.
Immunohistochemistry and Immunofluorescence
Cortex, cerebellum, spinal cord, olfactory bulb, liver, and testis were harvested from three adult mice following perfusion with 4% paraformaldehyde in PBS. Dissected tissues were postfixed in 4% paraformaldehyde in PBS for an additional 4 h at 4°C and then equilibrated in sucrose solutions of increasing concentration to 18% final; 10 µm cyrostat sections were mounted onto Superfrost Plus slides (VWR) and stored dry at 20°C until use. Before immunostaining, slides were thawed to room temperature under dessication and the sections were then overlaid with wash buffer (0.3% Triton X-100, 0.45 M NaCl in 0.02 M Na2HPO4 pH 7.4). Tissue sections were blocked for 45 min in 15% normal goat serum, 0.3% Triton X-100, 0.3 M NaCl in 0.04 M Na2HPO4 pH 7.4. Primary antibody dilutions were 1:100 for CS2 and anti-Golgi antisera. Incubation with primary antisera was for 2 h in a humidified chamber at ambient temperature. Slides were then washed in Wash Buffer, reblocked for 15 min and incubated with secondary antibody (Goat anti-rabbit) conjugated either to HRP (1:500 dilution) or to alkaline phosphatase (1:1000 dilution). For HRP-conjugated secondary, antibody binding was visualized by precipitation of diaminobenzidine (DAB) in the presence of hydrogen peroxide. 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium were used as substrate for alkaline phosphatase conjugated secondaries. All slides were coverslipped under 50% glycerol in PBS. Immunohistochemical staining was identical in tissues probed with affinity-purified or nonpurified serum.
For immunofluorescent staining, primary antibody dilutions were 1:50 for CS2 and anti-GM130, 1:100 for anti-Giantin, and 1:30 for anti-Golgi antisera. Secondary antibodies, fluorescein-conjugated goat anti-rabbit, Texas-red goat anti-mouse, and Texas-red goat anti-rabbit, were used at a 1:250 dilution. Tissue sections were mounted in 4% w/v 1,4-diazabicyclo[2.2.2]octane (DABCO) in 50% glycerol in PBS and confocal images were acquired on a Zeiss Axiovert 100 microscope equipped with an argon/krypton laser. The microscope operates under control of the Zeiss LSM510 version 2.3 software package which was also used to acquire images. Optical sections were taken every 1.0 µm parallel to the coverslip. Acquired images were prepared for publication with Adobe software by minimally adjusting background fluorescence levels equally across all color channels. The relative fluorescence between red and green channels was unaltered from the original confocal scan.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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